Biochemistry Essential Concepts

Biochemistry
Essential Concepts
Charles C. Hardin and James A. Knopp

Department of Molecular and Structural Biochemistry,
North Carolina State University
New York Oxford

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Hardin, Charles.
Biochemistry : essential concepts / Charles Hardin and James Knopp.
p. cm.
ISBN 978-0-19-976562-1 (cl : acid-free paper) 1. Biochemistry—Outlines, syllabi, etc.
I. Knopp, James. II. Title.
QP518.3.H37 2011
572—dc23
2011044803
Printing number: 9 8 7 6 5 4 3 2 1
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on acid-free paper
Preface
Using Biochemistry: Essential Concepts
Learning biochemistry is difficult for many life science students because, in essence, they are asked to learn
a very complicated language, filled with many new concepts, all within a sixteen-week time frame. This
“field manual” is a concise guide to biochemistry concepts and is intended as an efficient, pared-down aid
to help students assimilate the key ideas. It presents a self-contained sixteen-week course, at a level that
will help students proceed successfully to professional and medical school course work.
Biochemistry: Essential Concepts (BEC) has evolved over many years of teaching introductory
biochemistry. In one concise volume it contains (a) a textual summary of the essential information
distilled from a standard encyclopedic biochemistry textbook, and (b) relevant review questions and
sample tests with answers. BEC thus serves as a complete and self-contained handbook, notebook, and
study guide. Because BEC presents material in the same sequential order as most biochemistry
textbooks, it may easily be used alongside another text. The content in BEC is intended to provide a
backbone. It is not intended to replace a full textbook but, rather, through concurrent use, is designed to
assist students in the learning process by presenting to them a clear, pared-down presentation of the
basics together with problem-solving and review tools.
We have taught graduate- and undergraduate-level biochemistry and biophysics courses at North
Carolina State University for over twenty-five years. The main challenges we have experienced are: (1)
students arriving to take a biochemistry course who have not retained basic concepts from freshman and
organic chemistry; and (2) the vastness of the field of biochemistry itself. Many students have relied on
memorization in their previous science courses without grasping fundamental concepts such as pH, pKa,
nucleophilic attack, equilibrium, and thermodynamics. Reliance on rote memorization in a biochemistry
course is a very ineffective approach and does not result in a deep understanding of more subtle aspects of
biochemical processes, such as assessing the poise of a reaction equilibrium, predicting enzyme reactivity
and substrate-dependent regulation. Our goals in writing BEC were therefore twofold:
(1) To carefully extract and present the essential core concepts imbedded in a typical biochemistry
textbook,
(2) to reiterate in a variety of contexts those most fundamental chemical concepts that are essential to
understand the processes of biochemistry and related biological science, not simply memorize them.
The text contains several key features:
Textbook Flexibility. The approach used in BEC focuses on teaching the fundamental structure of the
field within a single semester time frame. It is not based on a single textbook. As a result, it is compatible
xvi Preface
for use as a study guide with any of a wide variety of much more definitive tomes, several of which are
cited in context within the text.
Integration of Concepts Into The Big Picture. BEC is a clear, concise guide to biochemical
concepts, which is readily accessible and provides a wealth of well-chosen examples. A key strength is that
one is rapidly orientated regarding a given subject, with emphasis on the big picture, and then shown how
fundamental concepts become integrated to produce more complex linked processes. These lessons are
reinforced by an extensive set of practice exercises and tests designed to reinforce key concepts and
relationships, highlighting techniques from medicine and other biotechnological fields.
Many Real-World Applications. These notes are a map so that students can continually look at the
big picture and see how the subject of the moment fits. The same fundamental principles govern many
aspects of biological processes, so once students have built a set of models into their memory, they can use
this knowledge to dissect some new, yet related, biological setting, predict the pertinent chemistry and sort
out the processes that matter. Students can then use this kind of circumspect viewpoint to realistically
understand the complexity of new situations. In the long view, this knowledge can be used to understand a
process, develop new procedures, troubleshoot methodological problems, design a new pharmaceutical, and
otherwise be applied to use in medicine, agriculture, and biotechnology.
A Concise, Clear Format. How does a professor decide what to keep and what to leave out when
faced with a twelve-hundred-page textbook and sixteen weeks to teach the course? Current textbooks tend
to be encyclopedic, requiring careful choice of the material to emphasize if one is to effectively transmit
both a working knowledge of the fundamental tools of the discipline as well as their breadth and
importance in medicine, materials science, genetics, cell biology, and so forth.
In the streamlined presentation of BEC, the focus is on concepts that govern and regulate biological
processes. Those concepts include equilibrium, pKa, Kd, KM, pH, nucleophilic attack, the relation between
bond polarity and reactivity, the enthalpic and entropic contributions to ΔG, and so forth. This emphasizes
the more difficult-to-learn physical and mechanistic concepts and leaves the more digestible, qualitative
and familiar biological foci for class discussion.
Reiteration of Core Concepts. The best way to show students that the same fundamental principles
govern many aspects of biological processes is to reiterate those principles, where applicable, throughout
the course in various contexts. If students grasp these core concepts, they can understand at a fundamental
level the biochemical processes that underlie much of biological science.
Reiteration is built into BEC at several levels. For example, the concept of pKa is reiterated a total of
fourteen times: nine times in various text sections, twice in review material, and three times in the sample
tests. The concept is first discussed on in the context of acid-base ionization and the relation to protonation
and deprotonation. It reappears again in the context of the bicarbonate blood buffering mechanism and
again a discussion of the functional groups of amino acids. It occurs twice in a table of pKa values and in a
section that defines the isoelectric point and explains how it is calculated. It appears again during the
explanation of pKa shifts at the C-terminus of amino acids on incorporation into a polypeptide, in a
Preface xvii
discussion of the allosteric control of oxygen binding to hemoglobin, in an explanation of enzyme activity
at different pH values, and in a discussion of the charge of backbone phosphates in nucleic acids. The
concept occurs in several contexts in the review sessions. Sample Exam 1 makes use of the pKa concept in a
question that requires students to draw a specified oligopeptide structure and the corresponding pH titration
curve. Sample Exam 1 also contains a multiple-choice question that focuses on the comparison of pKa with
pH and the partial pressure of oxygen (pO2). The final occurrence of the concept is in the sample Final
Exam in a question regarding factors that control the catalytic capability of an enzyme. Similar reiteration
strategies are employed for other fundamental principles.
The material in BEC produces an interwoven set of reiterated ideas that shows how analogous
chemical principles govern a large number of biological reactions. The intent of this reiteration is to help
students retain the basics, understand how they apply to a variety of biological processes, and develop the
ability to dissect new analogous situations on their own.
Reinforcement Through Review. Several review tools have been incorporated into BEC, complete
with answers, to foster integration of the textbook material with the questions. A single volume contains a
concise summary of the lecture material, the students’ own class notes, and directly linked review questions
and sample tests, with answers. This integrated format encourages students to work problems and think
about results in a well-organized and efficient way.
The first six sets of review questions do not have an answer key per se, but the answers are easily
located in the text of BEC. The expectation is that students can read the relevant portions of the text and
BEC and use the information to answer the assigned questions. However, the material covered in review
sessions 7–13 is more challenging and requires a more concept-driven approach. We’ve also found that
students begin to feel frustration and fatigue at this stage of the course. We’ve therefore provided answers
for each of the questions in these sections. In addition, four sample tests and a final exam are supplied with
complete answer keys.
These review tools provide a completely integrated curriculum that stresses the core concepts of
biochemistry and greatly facilitates student comprehension. By writing and drawing out the answers to
the practice questions in BEC, students exercise and refine their use of the tools and language of
biochemistry.
Brief Contents
Preface ………………………………………………………………………………………............. xv
Acknowledgments …………………………………………………………………………………... xviii
Chapter 1. Biochemistry: Subject Overview ……………………………………….……..……… 1

Chapter 2. Cell Biology Review ……………………………………………………………....….... 2
Chapter 3. Chemistry Review …………………………………………………..…………………. 9
Chapter 4. Amino Acids ………………………………………………………………………….... 20
Chapter 5. Proteins ……………………………………………………………………………..….. 26
Chapter 6. Protein Structure ………………………………………………………………….…... 33
Chapter 7. Ligand Binding and Functional Control ……………………………….……………. 45
Chapter 8. Enzymes ………………………………………………………………..………………. 51
Chapter 9. Metabolic Enzyme Action …………………………………………………………….. 70
Chapter 10. Coenzymes ……………………………………………………………….………….... 81
Chapter 11. Carbohydrates and Glycoconjugates ……………………………………..………… 93
Chapter 12. Lipids …………………………………………………………………..……….…….. 102
Chapter 13. Membranes ………………………………………………………..………………….. 111
Chapter 14. Transport Through Membranes ……………………………………………………. 116
Chapter 15. Signal Transduction ……………………………………………………………….… 120
Chapter 16. Nucleic Acids: DNA ………………………………………………………….…….… 125
Chapter 17. RNA ……………………………………………………………………………..……. 146
Chapter 18. Biotechnology ………………………………………………………………………… 154
Chapter 19. Metabolism ………………………………………………………………..………….. 163
Chapter 20. Bioenergetics …………………………………………………………………….…… 167
Chapter 21. Bioelectrochemistry ……………………………………………………………….…. 171
Chapter 22. Glycolysis ………………………………………………………………………….….. 174
Chapter 23. The Krebs Cycle ……………………………………………………..……………….. 180
Chapter 24. Gluconeogenesis ………………………………………………………..…………….. 184
Chapter 25. Electron Transport and Oxidative Phosphorylation …………………………….… 186
Chapter 26. The Malate-Aspartate Shuttle and Proteomics ………………………………….… 193
Chapter 27. Degradation and Synthesis of Lipids …………………………………………..…… 196
Chapter 28. Photosynthesis ………………………………………………………………………... 204
Chapter 29. The Calvin Cycle ……………………………………………………………….....….. 208
Chapter 30. The Urea Cycle ………………………………………………………………….……. 211
STUDY GUIDE ……………………………………………………………………….……….…… 215

Review Session for Chapters 1–3 ………………………………..…………….……………….… 215
Review Session for Chapter 4 ……………………………………………………………….…… 217
Review Session for Chapters 5 and 6 ……….…………………………………………………… 220
Review Session for Chapters 7–8.6 ……………………………………………….….……..…… 224
Review Sessions for Chapters 8.7–8.8 ………………………………………..………….…….… 225
Review Session for Chapter 9 ………………………………………………….………………… 226
Review Session for Chapters 10–11.4 ………………………………………..………………..…. 227
Review Session for Chapters 10–11.4: Key …………………………………………………...…. 228
Review Session for Chapters 11.5–12.5 ………………………………………………..……….... 231
Review Session for Chapters 11.5–12.5: Key ……………………………..…………..………..... 232
Review Session for Chapters 13–14.7 ……………………………..…….…………………….…. 235
Review Session for Chapters 13–14.7: Key ……………………………..….………………….… 236
Review Session for Chapters 16.2–17.6 ……………………………………………..………….... 238
Review Session for Chapters 16.2–17.6: Key …………………………………..………………... 239
Review Session for Chapters 19–20 …………………………………………………….………… 242
Review Session for Chapters 19–20: Key ………………………………………………………… 244
vi Brief Contents
Review Session for Chapters 21–25 ……………………………………………….....…………... 247

Review Session for Chapters 21–25: Key …………………………………………....…………... 249
Review Session for Chapter 27 ……………………………..………………………………..…… 253
Review Session for Chapter 27: Key ……………………………..…………………………..…... 254
Practice Exam 1, Key …………………………………..…………………………………..….…... 256
Practice Exam 2, Key …………………………………………………….………………………... 260
Practice Exam 3, Key ………………………………………………………………..…...........…... 267
Practice Exam 4, Key ………………………………………………………..…………….............. 276
Final Test Review ……….………………………………………………………………….…….... 281
Final Test Review (with answers) …………………………..……………….…………….………. 291
Index ………………………………………………………………………………………………..... 317

Contents
Preface ………………………………………………………………………………………..............… xv
Acknowledgments …………………………………………………………………………………...…. xviii
Chapter 1. Biochemistry: Subject Overview ……………………………………….……..………… 1

1.1 Central Themes ………………………………………………………………..……….…………. 1
1.2 Central Dogma of Molecular Biology ……………………………………………………………. 1
Chapter 2. Cell Biology Review ……………………………………………………………....……… 2

2.1 The Animal Cell …………………………………………………………………….........……….. 2
2.2 The Plant Cell ………………………………………………………………..……………....……. 3
2.3 Selected Organelles ………………………………………………………….……………………. 3
2.4 The Cell Cycle: Mitotic Cell Division ……………………………………………………………. 5
2.5 Viruses ………………………………………………………………..………….………………... 6
Chapter 3. Chemistry Review ……………………………………………………..………………….. 9

3.1 Organic Compounds ………………………………………………………………………….….... 9
3.2 Chirality ………………………………………………………………..…………………….……. 10
3.3 Chemical Reactions ……………………………………………………………………….……..... 12
3.4 Physical Chemistry Concepts …………………………………………………………………....... 14
3.5 Buffering of Blood: The Bicarbonate System …………………………………………..……........ 19
Chapter 4. Amino Acids ………………………………………………………………………….…... 20

4.1 Basic Structures ……………………………………………………………………………...……. 20
4.2 Amino Acid “R Groups” ………………………………………………………….……………..... 20
4.3 Ionization Properties …………………………………………………..……………...............…... 23
4.4 Drawing Peptide Titration Plots ………………………………………………………………….. 23
4.5 Factors That Influence the pKa of Protonatable/Deprotonatable Groups in Proteins …….……..... 25
Chapter 5. Proteins ……………………………………………………………………………….…... 26

5.1 Peptide Bonds ……………………………………………………………………….……….….... 26
5.2 Purification and Characterization of Proteins …………………………………………..……….... 27
Chapter 6. Protein Structure ………………………………………………………………….……... 33

6.1 Conformation ………………………………………………………………..…………………..... 33
6.2 Classification of Substructure ……………………………………………………….…………..... 34
6.3 Alpha (α) Helices ………………………………………………………..………………………... 35
6.4 Beta Sheets ………………………………………………………………………………..………. 35
6.5 U-Turns ………………………………………………………………..…………………….……. 36
6.6 Ramachandran Plot ……………………………………………………………………….………. 37
6.7 Stabilizing Factors ………………………………………………………………………….…….. 38
6.8 Thermodynamics of Protein Folding: The Hydrophobic Effect ………………………………….. 38
6.9 Chaotropes and The Hofmeister Series …………………………………………………………… 39
6.10 Sodium Dodecyl Sulfate (SDS): Chaotrope Action ………………………………………..……. 39
6.11 Visualizing the Energy Landscape ………………………………………………………………. 40
6.12 Protein Maturation ………………………………………………………………..……………… 41
Chapter 7. Ligand Binding and Functional Control …………………………….………….………. 45

7.1 Oxygen Transport in Blood ………………………………………………………..…..………….. 45
7.2 Hemoglobin Oxygen Binding: Cooperativity …………………………………………..………… 47
7.3 Antibodies: Immunological Recognition …………………………………………………………. 50
viii Contents
Chapter 8. Enzymes …………………………………………………………………..……………….... 51

8.1 Enzymes Are Biological Catalysts ………………………………………………………………..... 51
8.2 Enzyme Function: Activity Assays and Enzyme Kinetics …………………………………..……... 52
8.3 Requirements for Catalysis …………………………………………………………………….….... 56
8.4 Comparing Enzymes and Relative Efficiency of Use of Substrates ………..……………..………... 57
8.5 Drug Design ………………………………………………………………..………….…………..... 58
8.6 Enzyme Inhibitors ……………………………………………………………………….………...... 59
8.7 Allosterism ………………………………………………………………….………….………….... 65
8.8 Phosphorylation and Dephosphorylation ………………………………………………………….... 67
Chapter 9. Metabolic Enzyme Action ………………………………………………………………..... 70

9.1 Enzyme Mechanisms ……………………………………………………………….……………..... 70
9.2 Modes of Catalysis ……………………………………………………………….……….………... 71
9.3 The Reaction Coordinate ………………………………………………………….………………... 72
9.4 Induced Fit Revisited ………………………………………………………………….…………..... 73
9.5 Acid-Base Catalysis ………………………………………………………………….……………... 74
9.6 Covalent Group Transfer ………………………………………………………………….………... 76
9.7 The Serine Protease Catalytic Triad Mechanism ………………………………………..………...... 77
9.8 The Active Site of Tyrosyl-tRNA Synthetase ………………………………………..….………..... 80
Chapter 10. Coenzymes …………………………………………………………….…………….…...... 81

10.1 Classification ……………………………………………………………….……………….…...... 81
10.2 Survey of the Coenzymes …………………………………………………………………….….... 81
10.3 Metals ………………………………………………………………..…………………….…….... 88
10.4 Carbohydrates-Based Cofactors …………………………………………………………………... 90
10.5 Fat Soluble Vitamins ………………………………………………………………….…………... 91
Chapter 11. Carbohydrates and Glycoconjugates …………………………………..……………...... 93

11.1 Carbohydrates: Definition …………………………………………………..……..…………….... 93
11.2 Monosaccharides: Aldoses …………………………………………………………….………….. 93
11.3 Monosaccharides: Ketoses …………………………………………………..…………..………... 94
11.4 Structural Features ………………………………………………………………….……………... 94
11.5 Intramolecular Cyclization ……………………………………………………………….……….. 95
11.6 Conformations: Sugar Puckers ……………………………………………………………….….... 96
11.7 Sugar Derivatives ………………………………………………………………..………………... 96
11.8 Disaccharides ………………………………………………………………..……………….…..... 97
11.9 Polysaccharides ………………………………………………………………..………..…. ……... 98
11.10 Carbohydrate-Protein Conjugates ……………………………………………………....………... 99
11.11 Synthesis and Structural Characterization …………………………………………..…………... 101
Chapter 12. Lipids ………………………………………………………………..………….……….... 102

12.1 Structural Overview ……………………………………………………………….…………….... 102
12.2 Saturated and Unsaturated Fatty Acids ………………………………………………………….... 102
12.3 Functions ………………………………………………………………..………………….……... 104
12.4 Diacylglycerol Lipid Derivatives ……………………………………………………………….... 104
12.5 Structural Motifs ……………………………………………………………….………………..... 104
12.6 Assembly ……………………………………………………………………….……….………... 105
12.7 Structural and Dynamic Characterization ……………………………………………………….... 106
12.8 Eicosanoids ………………………………………………………………………..………….…... 107
12.9 Phospholipases ……………………………………………………..…………………………….. 108
12.10 Phosphoinositides ………………………………………………………..…………………….... 108
12.11 Steroids ………………………………………………………..………………………….……... 109
12.12 A Potpourrié of Lipids …………………………………………………………………….…...... 109
Chapter 13. Membranes ……………………………………………………..……………………….... 111

13.1 The Fluid Mosaic Model …………………………………………………………………….….... 111
Contents ix
13.2 Detergents ……………………………………………………..………………………….…….. 111

13.3 Distribution of Lipids in Biological Membranes ………………………………………….……. 112
13.4 The Hydropathicity Scale …………………………………………………………………….… 112
13.5 Lipid-Anchored Membrane Proteins …………………………………………………………… 114
13.6 The Erythrocyte Cytoskeleton ……………………………………………….….…………...…. 115
Chapter 14. Transport Through Membranes ……………………………………………………… 116

14.1 The Transmembrane Potential ……………………………………………………….…………. 116
14.2 Active Transport ………………………………………………………..………………………. 116
14.3 Ionophores …………………………………………………………………………….…….….. 117
14.4 The Acetylcholine Receptor Ion Channel ………………………………………………………. 118
14.5 Lactose Permease and Secondary Active Transport ………………………………………..…... 118
14.6 Mechanism of Transport by Na+, K+ ATPase …………………………………………...……… 119
14.7 Ion Channel Blockers ………………………………………………………..………………….. 119
Chapter 15. Signal Transduction ……………………………………………………………….…… 120

15.1 Signaling Pathways: Hormones, GTPases, Second Messengers and Intracellular Regulation …. 120
15.2 The Adenylate Cyclase Signaling Pathway ……………………………………………..………. 121
15.3 The Inositol-Phospholipid Signaling Pathway …………………………………………..……… 122
15.4 Phorbol Myristyl Acetate …………………………………………………………………….…. 123
15.5 The Insulin Receptor …………………………………………………………………….……… 123
15.6 Glucagon …………………………………………………………………..……………………. 124
15.7 G-Proteins ………………………………………………………………….…………….……… 124
Chapter 16. Nucleic Acids: DNA ………………………………………………………..……..……. 125

16.1 DNA and RNA …………………………………………………………..……………………… 125
16.2 Physical Properties ………………………………………………………………………….…... 127
16.3 Secondary Structure ……………………………………………………..……………………… 128
16.4 Backbone Structure ………………………………………………………………..……………. 129
16.5 Counterions …………………………………………………………………..…………….…… 130
16.6 Chemical Synthesis ………………………………………………………………….………….. 130
16.7 Watson-Crick Base Pairs …………………………………………………………….………….. 130
16.8 Structural Modifications ………………………………………………………….…………….. 131
16.9 Three-Dimensional Structures ……………………………………………………………….…. 132
16.10 Recognition of Sequences ……………………………………………………………………... 136
16.11 Genetic Mutations and Antisense Nucleic Acids ………………………………………..…….. 136
16.12 Unusual DNA …………………………………………………………………….…………… 137
16.13 Stabilization of Nucleic Acids ………………………………………………………..…..…… 138
16.14 Secondary Structure Predictions ………………………………………………………………. 139
16.15 Chromosomes …………………………………………………………………………..……... 141
16.16 Some Protein Nucleic Acid Binding Motifs ……………………………………………...…… 144
16.17 Recombination ………………………………………………………….……………………... 145
Chapter 17. RNA ……………………………………………………………………………..……… 146

17.1 Cells Contain a Variety of Types of RNA ……………………………………………………… 146
17.2 RNAs Have Stable Secondary Structure ……………………………………………………….. 146
17.3 Tertiary Structure: Transfer RNA ………………………………………………………………. 147
17.4 Messenger RNA (mRNA) ……………………………………………………………………… 148
17.5 Eukaryotic Messenger RNA ……………………………………………………………….…… 150
17.6 Alkaline Hydrolysis of RNA ……………………………………………………….…………… 152
17.7 Small Interfering RNA ……………………………………………………..…………………… 153
Chapter 18. Biotechnology …………………………………………………………….…………….. 154

18.1 Restriction Endonucleases ……………………………………………………….……………… 154
18.2 Cloning in a Nutshell ………………………………………………………………….………… 156
18.3 DNA Preparation: Phenol-Chloroform Extraction ………………………………..…………….. 157
x Contents
18.4 Polymerase Chain Reaction …………………………………………………………….………. 158

18.5 Probe DNA …………………………………………………………………..…………………. 161
Chapter 19. Metabolism ………………………………………………………………..……………. 163

19.1 Overview …………………………………………………………………..…………….……… 163
19.2 Metabolic Pathway Types ………………………………………………………………….…… 164
19.3 Energy Conservation …………………………………………………………………….……… 165
19.4 Key Pathways/Reactions …………………………………………………………………….…. 165
Chapter 20. Bioenergetics …………………………………………………………………….……… 167

20.1 Reaction Equilibria: Standard and Actual Free Energies ………………………………….…… 167
20.2 Metabolically Irreversible and Near Equilibrium Reactions …………………………….……... 168
20.3 Energies and Regulation of Glycolysis ………………………………………....………………. 169
Chapter 21. Bioelectrochemistry ……………………………………………………………….…… 171

21.1 Redox Reaction Principles ……………………………………………………….……………... 171
21.2 Redox Energetics: The Nernst Equation ……………………………………………...........…… 171
21.3 Electron Transport Chains ………………………………………………………..……..……… 173
Chapter 22. Glycolysis ………………………………………………………………………….……. 174

22.1 Reactions 1 Through 10 ………………………………………………………..……………….. 174
22.2 Regulation: Activation and Inhibition ………………………………………………………….. 176
22.3 Four Fates of Pyruvate ………………………………………………………..………………… 177
Chapter 23. The Krebs Cycle …………………………………………………..……………………. 180

23.1 Pathway ……………………………………………………………….……………………..….. 180
23.2 Reactions ……………………………………………………………….……………………..… 181
23.3 Yields ……………………………………………………………….……………….…….……. 181
23.4 Cellular Redox Potential …………………………………………………………..……………. 182
23.5 Regulation ………………………………………………………………………………..……... 183
Chapter 24. Gluconeogenesis ……………………………………………………..…………………. 184

24.1 Reactions ……………………………………………………………….……………………..… 184
24.2 Regulation ………………………………………………………………………….…….……... 185
24.3 Sources Used to Produce Glucose ……………………………………………………………… 185
Chapter 25. Electron Transport and Oxidative Phosphorylation …………………………….…… 186

25.1 Mitochondria in Red and White Muscle ………………………………………………………… 186
25.2 Overall Process ………………………………………………………..…………………… …... 186
25.3 Chemical and Potential Energies That Drive Proton Transport ………………………………… 187
25.4 Mitochondrial Electron Transport ………………………………………………………………. 188
25.5 Electron Transfer and Proton Flow in Complexes I through IV …………………….………….. 189
25.6 Oxidative Phosphorylation ……………………………………………………………………… 191
Chapter 26. The Malate-Aspartate Shuttle and Proteomics ………………………………….…… 193

26.1 Getting NADH into the Mitochondrion: Isozymes ………………………………………..……. 193
26.2 Isozymes and Proteomics ……………………………………………………………..………… 194
26.3 Characterization by Two-Dimensional Gel Electrophoresis ………………………….…… …... 194
26.4 Mass Spectrometry and Proteomics …………………………………………………………….. 195
Chapter 27. Degradation and Synthesis of Lipids …………………………………………..……… 196

27.1 Beta Oxidation of Saturated Fatty Acids ……………………………………………………….. 196
27.2 Biosynthesis of Fatty Acids …………………………………………………………………….. 197
27.3 Length Determination of Fatty Acids ……………………………………………………… …... 199
27.4 Synthesis of Acidic Phospholipids ……………………………………………………………… 200
27.5 Cholesterol Biosynthesis ………………………………………………………………….…….. 200
Contents xi
27.6 Regulating Cholesterol Levels …………………………………………………………………. 203
Chapter 28. Photosynthesis ………………………………………………………………………..... 204

28.1 Light and Dark Reactions ………………………………………………………………….…... 204
28.2 Photo-Gathering Pigments ……………………………………………………………………... 204
28.3 Photosynthetic Electron Transport Pathway (Z scheme) ………………………………….…… 205
Chapter 29. Carbon Fixation: The Calvin Cycle and C4/CAM Pathways ………….......………. 208
29.1 The Dark Reactions: Carbon Fixation …………………………………………………………. 208
29.2 Biosynthesis of Ribose-5-phosphate …………………………………………………………… 208
29.3 RuBisCO Mechanism …………………………………………………………………….……. 209
29.4 The C4 and CAM Pathways ………………………………………………..………………..…. 210
Chapter 30. The Urea Cycle ………………………………………………………………….…….. 211

30.1 Purpose and Reactions …………………………………………………………………….…… 211
30.2 Regulation ………………………………………………………………………………..……. 212
30.3 Comparative Nitrogen Excretion ………………………………………………..…………..…. 213
30.4 Protein Degradation and Programmed Cell Death ………………………………..…………… 213
STUDY GUIDE ……………………………………………………………………….……….…….. 215

Review Session for Chapters 1–3 ……………………………..……………….……………….….. 215
Organelle Functions …………………………………………………………………………….. 215
Example First Quiz Questions …………………………………………………………………. 215
Organic Chemical Functional Groups and Their pKas ……………………………….……….… 215
Urea, Guanidinium (Guanidine); Pyrophosphate ………………………………….…….……. 215
Ionization at pH 7, pKas …………………………………………………………….…………. 215
Chemical Principles ……………………………………………………………………….……. 216
Oxidation, Reduction ………………………………………………………………….………. 216
Reaction Equilibrium and Thermodynamics …………………..…………………..………..… 216
Physical Chemistry Concepts ……………………………………....…………………………. 216
Water-Solute Interactions …………………………………………………….……………….. 216
pH and pKa ………………………………………………………………………..……………. 216
Review Session for Chapter 4 ……………………………………………………………….…….. 217
Quiz 2A: Example Quiz Format …………………………………………….…….………..….. 217
Structure of Amino Acids at pH 7 ……………………………………………………………... 217
Functional Groups ……………………………………………………………….…………….. 217
Quizzes 2B Through 2F (Same Subject) ………………………………………………….…..… 217
Review Session for Chapters 5 and 6 ……….……………………………………………………... 220
Van der Waals Interactions; Hydrogen Bonds ……………………………………………..……. 220
Edman Degradation ……………………………………………………………….………….…. 220
Disulfide Bonds ……………………………………………………………………….....……… 220
α-Helix, Hydrogen Bonding Pattern ………………………………………………………….…. 220
β-Sheet, Hydrogen Bonding Pattern …………………………………………………………….. 220
The Hydrophobic Effect ………………………………………………………….……………... 220
Ramachandran Plots …………………………………………………………….………………. 220
Quiz 3A …………………………………………………………………………………………... 221
Van der Waals Interaction, Interatomic Distances and Free Energies …………………………... 221
Protein Primary Structure, Quaternary Structure ……………………………………………...… 221
Quiz 3B …………………………………………………………………………………………... 221
Hydrogen Bond Interaction, Interatomic Distances and Free Energies …………………………. 221
Protein β-Sheet; Hydrogen Bonds ……………………………………………………………….. 221
Quiz 3C …………………………………………………………………………………………... 222
Protein Secondary Structure ……………………………………………………………….…….. 222
Protein Tertiary Structure …………………………………………………………………….….. 222
Edman Degradation; Key Reagent ………………………………………………………………. 222
Quiz 3D ………………………………………………………………………...………………… 222
xii Contents
Chaotropic Agent ………………………………………………………………………….……. 222

Protein α-Helix; Hydrogen Bonds ……………………………………………………………… 222
Quiz 3E ………………………………………………………………………………………….. 223
Disulfide bond ………………………………………………………………….…………….…. 223
Purpose of a Ramachandran Plot. What are φ and ψ? ………………………………………..… 223
Quiz 3F ……………………………………………………………………...…….……………. 223
Zwitterion: Generic Amino Acid ………………………………………………….……………. 223
Hydrophobic Effect ………………………………………………………………….....………. 223
Review Session for Chapters 7–8.6 ……………………………………………….….……..…….. 224
Initial Rate ……………………………………………………………………………….……... 224
Catalysis; Enzymatic Reaction …………………………………………………………….…... 224
Maximum Velocity ………………………………………………………………….……….…. 224
Michaelis-Menten Equation ……………………………………………………………………. 224
Definition of KM ………………………………………………………………….……….……. 224
Michaelis Complex ………………………………………………………………….……….…. 224
Definition of kcat ………………………………………………………………….……….……. 224
Linked Chemical Equilibrium Reactions; Michaelis-Menten Catalysis ………………………... 224
Competitive and Uncompetitive Inhibition of Enzyme Catalysis …………………………….… 224
Requirements for Enzyme Catalysis ……………………………………………………………. 224
Initial Rate and Steady State Postulates ……………………………………………………….... 224
Review Sessions for Chapters 8.7–8.8 …………………………………………..……….…….….. 225
Enzyme Catalysis; External Limiting Factors ………………………………………....…..…… 225
Draw Acetylcholine …………………………………………………………………….………. 225
Amino Acid in the Esterase Site of Acetylcholine Esterase ………………………..…………... 225
Show How the Electrons of the Key Enzyme Atom Attack the Substrate ……………………... 225
Nerve Gas Antidote Pyridine Aldoximine Methiodide Reactivates Acetylcholine Esterase …... 225
Bisubstrate-Enzyme “Ping-Pong” Reaction ………………………………………………..…… 225
Enzyme Cascade …………………………………………………………………….…….……. 225
Blood Clotting ………………………………………………………………………..…………. 225
Zymogens; Digesting Food ……………………………………………………….…………….. 225
Feedback Inhibition ……………………………………………………………….….…………. 225
Logical Way to Regulate a Metabolic Pathway ……………………………………….…….….. 225
Allosterism …………………………………………………………………………...…………. 225
Kinase and Phosphatase Reactions: Regulation of Glycolysis; Krebs Cycle …..……………... 225
Cyclin Kinase ………………………………………………………………………..………….. 225
Review Session for Chapter 9 ………………………………………………….………………….. 226
Reversible Factors that Control the Catalytic Capability of an Enzyme ……………………….. 226
Irreversible Factors that Control the Catalytic Capability of an Enzyme ………………………. 226
Chemical Modes of Catalysis …………………………………………………….……………… 226
Binding Modes of Catalysis ……………………………………………………….……………. 226
Uncatalyzed and Catalyzed Reaction Coordinate Versus Energy Trends ………………………. 226
Nucleophilic Substitution (SN2) Reaction; Nucleophile ………………………….………… ….. 226
Electrophile ……………………………………………………………………..………….……. 226
Why is Histidine Used So Often? ……………………………………………….………………. 226
Why Do Enzymes Typically Have an Optimal pH? ……………………………….……….…… 226
Draw the “Catalytic Triad” in Serine Proteases ……………………………….…………….….. 226
Arrhenius Equation ………………………………………………………….……………….….. 226
Thermodynamic Equation or a Kinetic Equation? ………………………………...……………. 226
Structure of ATP; Role of Mg2+ …………………………………………………….…………… 226
Review Session for Chapters 10–11.4 ………………………………………….………………..…. 227
(Quizzes Contain Notes With Answers From Here On)
Definition of a Coenzyme …………………………………………………………………….…. 227
Heavy Metal …………………………………………………………………………..........……. 227
ATP: Biochemical Applications; Use of Different Parts of the Molecule ……………………… 227
NAD+ (NADH): Oxidized and Reduced Forms; Use ……………………………….……….…. 227
FAD (FADH2); Oxidized and Reduced Forms ……………………………………………..…… 227
Contents xiii
Pyridoxal Phosphate; “Schiff’s Base” With … Lysine ……………………………….………... 227

Coenzyme A; Acetyl Group Attachment …………………………………………….……..…... 227
Crucial Link in Intermediary Metabolism ……………………………………………..……..… 227
Biotin-Avidin Noncovalent Binding/Capture …………………………………..………….…... 227
Role of N5, N10 Tetrahydrofolate in the Production of DNA …………………………….....….. 227
Strategy for Anti-Cancer Chemotherapy ……………………………………………..……..….. 227
Ketose; Aldose ………………………………………………………………………………...... 227
Pyranose; Furanose …………………………………………………………………………..… 227
UDP-Galactose; Used to Make Lactose ……………………………………………….......…... 227
Cis-Retinal; Transducing the Signal of a Photon of Light into a Chemical Form …….…….…. 227
Review Session for Chapters 10–11.4: Key ……………………………………………...……….. 228
Review Session for Chapters 11.5–12.5 ………………………………………………..………..… 231
Chair, Boat Conformations of the β-D-Glucopyranose ……………………….….………… ….. 231
Most Radical Change in Chain Direction ……………………………………………...........….. 231
Hexaatomic Ring of Inositol; Stability vs. Galactose ……………………………..……………. 231
Inositol vs. Glucose or Galactose; Capabilities in Serving as Nucleophilic Centers ………..…. 231
Nomenclature: NAG-α (1→6)-NAM-α (1→4)-glc-β (1→4)-gal ……………………….……… 231
Meaning of α and β ……………………………………………………………………..………. 231
Glycogen; Amylopectin in Starch ………………………………………………………………. 231
Function of Glycogen …………………………………………………………………….……... 231
Penicillin …………………………………………………………………………………........... 231
Cell Surface Carbohydrates; Osmotic Pressure ……………………………………….……..…. 231
Export and Clearance …………………………………………………………………….……... 231
Phospholipase C; Two Different Second Messengers; Signal Transduction Pathway ……..…... 231
Function of Chondroitin Sulfate; Cartilage and Skeletal Joints ………………………….…….. 231
Saturated or Unsaturated Fatty Acids; Melting Temperature ……………………..……..……... 231
Lipid Bilayers and Micelles …………………………………………………………………….. 231
Phosphatidyl Choline ………………………………………………………………….………... 231
Review Session for Chapters 11.5–12.5: Key ……………………………..……………..…..…… 232
Review Session for Chapters 13–14.7 ……………………………..…….…………………….….. 235
Fluid Mosaic Membrane Model ……………………………………………………….……….. 235
Four Nucleic Acid Bases in DNA; Structure ……………………………………….........……... 235
RNA Trinucleotide; Structure ……………………………………………………….……...…... 235
Watson-Crick Base Pairs ………………………………………………………….……………. 235
Glycosidic Bond ………………………………………………………………….…………….. 235
Eukaryotic mRNA Structure ……………………………………………………….…………… 235
5’-Terminal m7G “Cap” Structure …………………………………………….………………… 235
Monocistronic ………………………………………………………………………..…………. 235
Introns, Exons ………………………………………………………………………..……….… 235
Poly (A) Tail ………………………………………………………………………..……….….. 235
Review Session for Chapters 13–14.7: Key ……………………………..…….……………….…. 236
Review Session for Chapters 16.2–17.6 …………………………………….…………..……….… 238
Absorbance at 260 nm; Formation/Melting of a Double Helix ………………………………… 238
Base Stacking ………………………………………………………………………………..….. 238
Predominant Forces ………………………………………………………………………….…. 238
Counterions ……………………………………………………………………………..………. 238
Histones; Function …………………………………………………………………………..…... 238
G•C Base Pairs, A•T (or A•U) Base Pairs; Relative Stabilities ………………………….……... 238
Differences Between A and B Forms of DNA ……………………………………….…….….... 238
Alkaline Hydrolysis of RNA ………………………………………………………….………… 238
Antisense Oligonucleotide; Inactivation of mRNA ………………………………….…….…… 238
Four Classes of RNA ……………………………………………………………………..…….. 238
Eukaryotic mRNA ……………………………………………………………………….……… 238
DNA Probe ……………………………………………………………………….…….……….. 238
Restriction Endonuclease ……………………………………………………………….………. 238
Produce, Manipulate and Clone Specific Pieces of DNA ……………………………….……… 238
xiv Contents
Two Functional Ends of Transfer RNA ……………………………………………………….. 238

Review Session for Chapters 16.2–17.6: Key ……………………………………..………….….. 239
Review Session for Chapters 19–20 …………………………………………………….………... 242
Purpose of the Three Central Catabolic Pathways of Intermediary Metabolism …………..….. 242
Two Ways Energy is Captured in a Chemically Usable Form ………………………………… 242
Reaction; Standard Gibbs Free Energy Change (∆G°') …………………………..……………. 242
Actual Gibbs Free Energy (∆G) of the Reaction; at Equilibrium ……………………………… 242
Equilibrium Poise of the Reaction; Mass Action Ratio (Q) …………………………………… 242
Three Steps in Glycolysis Control Most of the Flux Through the Pathway …………………… 242
Metabolically Irreversible; Near Equilibrium ………………………………………...………... 242
Enzyme Reaction Control When KM is Equal to the Actual [Reactant] ……..……………..….. 242
Pyruvate to Acetyl CoA Coupled to FAD Formation; NAD+ Formation …………………….... 242
At 1 mM Pyruvate vs. at 1 M ……………………………………………………………….… 242
Triose Phosphate Isomerase ……………………………………………………….…………… 243
Citrate; Negative Regulation of the Phosphofructokinase-1 Reaction ………………………… 243
Fructose-1, 6-Bisphosphate; Stimulation of Pyruvate Kinase …………………………………. 243
Review Session for Chapters 19–20: Key ………………………………………………………... 244
Review Session for Chapters 21–25 ……………………………………………….....…………... 247
Three Branching Catabolic Fates of Pyruvate ……………………………………………..…... 247
Draw the Three Reaction Sequences, Including Cofactors and Enzymes ……………………... 247
Absence of Alcohol Dehydrogenase Produces Scurrilous Behavior ……………………….….. 247
Role of Pyridoxal Phosphate ……………………………………………………………….….. 247
Carbonation Accompanying Ethanol Production ………………………………………............ 247
Bread Production ………………………………………………………………………….….... 247
Dihydrolipoamide Acetyl Transferase; Coenzyme; Fueling the Krebs cycle …………………. 247
Symport; Import of Pyruvate ……………………………………………………………….…. 247
pH of the Cytoplasm …………………………………………………………………………... 247
“Oxidative Decarboxylation” Reactions of the Krebs Cycle …………………………….….... 247
Reactions, Coenzyme(s), Cofactor(s) and Enzymes ………………………………….…….… 247
“Substrate-Level Phosphorylation” Reaction of the Krebs Cycle …………………………….. 247
“Energy Conserving” Compounds Formed by the Krebs Cycle …………………………….… 247
Number of ATP Equivalents ……………………………………………………….………… 247
Fumarase and Malate Dehydrogenase …………………………………………………………. 247
“Fixing” a Carbonyl Group on Succinate; Production of Oxaloacetate ………………………. 247
Crucial 2 Carbon Compound ………………………………………………………………..... 247
Principle Substrates for Gluconeogenesis …………………………………………..……….… 247
Amino Acid, Product of Pyruvate Metabolism …………………………………….……….… 247
“Protonmotive Force”; Enzyme Complex ……………………………….………..…………... 247
Electron Transport Drives Production of the Protonmotive Force …………………....…….... 248
Review Session for Chapters 21–25: Key …………………………………………....…………... 249
Review Session for Chapter 27 ………………………………..……………………………..….... 253
β-Oxidation of a Fatty Acid; Products; ATP Equivalents ………………………………….….. 253
Coupled Cofactor Cycles Coupled to the First Oxidative Step; Coenzyme Q ………......…….. 253
Three Steps of the Kreb’s Cycle Resemble Steps of Fatty Acid β-Oxidation ……………..…... 253
Analogous (but backward) Reactions of Fatty Acid Synthesis ………………………………... 253
Condensation Substep; Acyl-Carrier Protein-Mediated Fatty Acid Synthesis ……..…...…..…. 253
Review Session for Chapter 27: Key ………………………………..………………………..…... 254
Practice Exam 1, Key ………………………………………………………………………..….…. 256
Practice Exam 2, Key ……………………………………………………………………….……... 260
Practice Exam 3, Key ……………………………………………………………….................…... 267
Practice Exam 4, Key ………………………………………………………………….................... 276
Final Test Review …………………………………………………………….………………….… 281
Final Test Review (with answers) ……………………………………………………………….… 291
Index ………………………………………………………………………………………………..... 317
Chapter 1
Biochemistry: Subject Overview
Definition: Study of the chemical reactions and linked metabolic processes that sustain a reproducing line
of viable organisms.
1.1 Central Themes

Energy. Organisms break down food (substrates) to produce the common molecular currencies that drive
energy-requiring transformations in cells. Important examples include adenosine triphosphate (ATP),
reduced nicotinamide adenine dinucleotide (NADH), reduced coenzyme Q and phosphoenolpyruvate.
Thermodynamics. Ground state differences in the energy of the reactants versus that of products. The sign
and magnitude of the Gibbs free energy tells one about the degree of spontaneity and poise (extent of
reaction) of a metabolic step. The focus is on the effect of mass action on the reaction equilibrium.
Kinetics. Rates and resulting excited-state energies required for a reaction to proceed, which depends on
details of the mechanism and any drive imposed by the reverse reaction. The focus is on how the reaction
depends on time and how to use this dependence to uncover details of the mechanism.
Enzymes. Typically, but not always, a protein that catalyzes a reaction by lowering the transition-state
energy of the key energy-requiring step. The catalyst is regenerated in its original form after catalysis is
completed. Several metals, RNAs, carbohydrate complexes and lipid aggregates have also been shown to
support catalysis.
Genetics. Characterizes the transfer of information from one generation to the next. Stable storage occurs
with DNA. RNA participates in transient use to mobilize new proteins when required and then to get rid of
the message. The genetic material in a “pluripotent” cell contains all of the information necessary to
recreate the new organism. An overview of the processes involved is captured by an overarching concept
called the central dogma of molecular biology.
1.2 Central Dogma of Molecular Biology
transcription translation
DNA RNA protein
reverse transcription
replication Post-transcriptional Post-translational

modifications modifications
This scheme summarizes the respective transfers of genetic information that are required to support the life
cycles and process of reproducing life forms. Replication involves reproducing the two daughter
chromosomes from the maternal copy of double helical DNA. Transcription involves making RNA, based
on the template information residing in the replicated DNA sequence. These processes are orchestrated by
the DNA and RNA polymerase complexes, respectively. Translation of messenger RNAs to form proteins
is catalyzed by the ribosome, a huge complex composed of 2/3 RNA and 1/3 protein.
Chapter 2
Cell Biology Review
2.1 The Animal Cell. (Adapted from McKee and McKee, Biochemistry The Molecular Basis of Life [5th ed.],
2012, p. 43; Fig. 2.11.)
While eukaryotic cells have a wide range of morphologies, with unique and distinguishing properties, most
cells have comparable structural features.
Nucleus Nucleolus Nuclear
Ribosomes Envelope
Rough
Endoplasmic
Reticulum Centriole
Nuclear
Pores
Mitochondrion
Vesicles
Lysosome
Smooth
Endoplasmic
Reticulum
Microtubules
Golgi
Cytoplasm Apparatus
Plasma Membrane
(complicate external
detail not shown)
One specific example, fibroblasts, synthesizes the extracellular matrix (ECM) and collagen. The
ECM is composed of structural proteins and complex carbohydrates that are secreted from fibroblasts to
form a viscous surface that aids in cell-cell adhesion. It also serves functions such as providing support and
anchorage for the cell, protection and regulation of intercellular communication. Membrane receptors
control various chemical and mechanical signaling processes.
Rat liver cells have been used to characterize many biochemical systems. The following table shows
the percentage of membrane in several cellular compartments and substructures.
Table 1. Membranes of the Rat Liver Cell
Membrane Type % of total Membrane Type % of total
membrane in membrane
cell in cell
Plasma membrane 5 Lysosomes, peroxisomes 6
and other compartments
Rough endoplasmic reticulum 30 Mitochondria
Smooth endoplasmic reticulum 15 Outer membrane 7
Nuclear membrane 1 Inner membrane 30
Golgi apparatus 6
(Adapted from Moran et al., Biochemistry [2nd ed.], 1994, p. 20-10; Table 2.2.)
Chapter 2 Cell Biology Review 3
2.2 The Plant Cell

Two crucial biochemical processes that distinguish plant cells from animal cells are photosynthesis and
carbon fixation, which both occur in plants.
Plasma Membrane
Microfilaments
Mitochondrion
Cell wall
Rough
Endoplasmic
Chloroplast Reticulum
Ribosomes
Vacuole
Nucleus
Golgi apparatus
Nuclear Envelope
Cytoplasm Nucleolus
Smooth
Endoplasmic
Reticulum
Vesicle
(Adapted from McKee and McKee, Biochemistry The Molecular Basis of Life [5th ed.], 2012, p. 43; Fig. 2.12.)
2.3 Selected Organelles

Endoplasmic Reticulum
Rough and smooth variants exist. The rough ER is rough due to the presence of ribosomes, the
ribonucleoprotein machines that synthesize proteins (see the Protein Maturation section). The smooth ER
does not. A variety of post-translational modifications occur there, for example, disulfide bond formation,
proteolysis, addition of carbohydrate and lipid molecules, acetylation, and so on.
Rough
Endoplasmic
reticulum
Smooth
Endoplasmic
reticulum
Lumen
(Adapted from Moran et al., Biochemistry [2nd ed.], 1994, p. 2.16; Fig. 2.13.)
4 Chapter 2 Cell Biology Review
Golgi Apparatus
This organelle is the location of lipid and steroid biosynthesis and specific post-translational modifications.
Transfer vesicles transport modified proteins to the extracellular interface.
Trans
Golgi sacs
Cis
Transfer
Vesicles
Lumen
(Adapted from Moran et al., Principles of Biochemistry [5th ed.], 2012, p. 21.)
Mitochondria
This organelle is used to produce cellular adenosine triphosphate (ATP). This process, called oxidative
phosphorylation is driven by a proton gradient, which is generated as a result of the electron transport
process. Reducing equivalent carriers, such as nicotinamide adenine dinucleotide (NADH) and reduced
coenzyme Q (CoQH2) provide the electrons (reducing power, reducing equivalents) that drive electron
transport.
Outer membrane
Inner membrane
Matrix
Intermembrane Crista
space
(Adapted from McKee and McKee, Biochemistry The Molecular Basis of Life [5th ed.], 2012, p. 54; Fig. 2.23.)
Evolutionary Origins. Both mitochondria and chloroplast were at one point independent blue-green
bacterial cells with their own chromosome. They were captured by other cells and subjugated. Much of the
deoxyribonucleic acids (DNA) was eliminated but they still function genetically like prokaryotic cells, with
their own polymerases, ribosomes and transfer RNAs. Mitochondrial DNA is inherited from one’s mother,
thus providing a way to reconstruct maternal lineages.
Chloroplast
This organelle is involved with light-driven ATP production, which is coupled to electron transport. Carbon
fixation occurs in the Calvin Cycle pathway.
Stroma
Inner membrane
Granum
(stacks of
thylakoids)
Thylakoid
Outer membrane
Functional aspects of the key features, the grana, thylakoid membrane, and stroma, are described in
the Photosynthesis section. (Adapted from Moran et al., Biochemistry The Molecular Basis of Life [5th ed.], 2012, p.
50; Fig. 2.25.)
The Cytoskeleton
The cytoskeleton is a protein scaffold network that consists of actin filaments, intermediate filaments, and
microtubules. The nature of incorporated biomolecules is very different in prokaryotes and eukaryotes. The
components support movement and traction. They are very dynamic, building and disassembling at the two
ends, in a controlled manner.
Membranes
These ubiquitous structures separate a variety of biochemical functions from each other by forming
separate compartments. They can both limit and direct the trafficking of biomolecules, and are crucial to
the function of signal transduction, active transport, cell division, nerve function, and many other processes.
2.4 The Cell Cycle: Mitotic Cell Division

The purpose of the cell cycle is to replicate the DNA and segregate it into two genetically identical
daughter cells. The S phase (for synthesis) requires ten to twelve hours and requires about half of the
typical mammalian cell cycle. M phase involves chromosome condensation, nuclear breakdown,
attachment of the mitotic spindle to microtubules, in order to align the chromosomes, then segregation to
form the two daughter cells. Cytokinesis (division) completes the process.
mitosis (nuclear division)
cytokinesis
(cytoplasmic division)
M
G2
G1
S
Specific stages in the mitosis process are shown in detail below:

Mitosis
prophase prometaphase metaphase anaphase
Cytokinesis
telophase
(Adapted from Alberts et al. Molecular Biology of the Cell [4th ed.], 2002, p. 985; Fig. 17-3.)
The regulation of mitosis in eukaryotic cells is described in the Phosphorylation and

Dephosphorylation section. This cyclin-dependent kinase mechanism involves a complex set of threonine-
and tyrosine-specific reactions. This system regulates progression through a series of checkpoints in the cell
cycle.
2.5 Viruses
The general structure of a virus with a membrane envelope is shown below. A detailed example is provided
by the Human Immunodeficiency Virus (HIV), which is described in the next section.
Membrane
envelope
Membrane-embedded
proteins
Capsid protein
assembly
(Adapted from Moran et al., Biochemistry [2nd ed.], 1994, p. 2.30; Fig. 2.27.)
(1) The following structure shows a T-even bacteriophage, a virus that infects bacterial cells, such as
Esheria coli, the grand old work horse of biochemists and molecular biologist alike.
Genomic
DNA Capsid
Tail spikes
Injection
apparatus
Structural elements include the viral capsid (the head), which contains the DNA, the tail spikes and the
injection apparatus at the bottom center of the virus.
(2) Viruses are symbiotic with their hosts. They require their host’s functions to complement their typically
very limited capability to grow and reproduce on their own.
(3) In some cases, a payload of extra DNA can be included in the viral DNA, piggy-backing with the
normal genomic DNA. Following injection into the host bacteria, the (enlarged) DNA sequence is
replicated by (e.g.) E. coli DNA polymerase. The DNA is cloned in the viral genome and propagated in the
bacterium. Plasmids can carry much more payload DNA than these less efficient viral systems, so they are
generally preferred in cloning strategies.
Human Immunodeficiency Virus Structure

The HIV virus is surrounded by the viral envelope which is composed of two layers of phospholipids.
Embedded within the viral envelope are proteins from the host cell known as HLA (human leukocyte
antigens) that protect the particle from the immune system and copies of a complex HIV protein such as
gp120 and gp41. Within the envelope is a matrix composed of the viral protein p17 that enclose the capsid
and guarantee the viral particle’s integrity. The capsid contains two copies of single stranded RNA coding
for the virus’s nine genes and the viral proteins p6 and p24. The RNA is bound to p7 and p9. The enzymes
involved in the replication process are reverse transcriptase (RT), integrase, and protease.
HLA class II DR
gp120 HLA class I
gp41
p6
p17
Protease
p24
Lipid capsid
Integrase
Reverse
Transcriptase
RNA
and
p7/p9
(Adapted from McKee and McKee, Biochemistry the Molecular Basis of Life [5th ed.], 2012, p.642; Fig. 17K.)
Reproductive Cycle of the Retrovirus HIV

The viral particle binds to the surface receptors of the host cell (1) and the viral envelope fuses with the cell
membrane (2), releasing the HIV capsid into the cell (3). The contents of the capsid, RNA and many viral
enzymes, also enter the cytoplasm (4). An enzyme called reverse transcriptase frees the single-stranded
RNA from the viral proteins and makes a copy into complementary DNA. DNA polymerase then uses this
DNA strand to make a complementary DNA sense strand from this antisense strand (5). This double-
stranded viral DNA travels to the nucleus where (6) another viral enzyme, integrase, incorporates it into the
host cell chromosome. This newly formed provirus gets replicated with each new DNA synthesis of the
cell.
As this viral DNA undergoes transcription (7), it forms two types of RNA transcript: (8) RNA
molecules that are used in the viral genome and (9) molecules making up the components that are used to
remake the viral protein such as reverse transcriptase, capsid proteins, envelope proteins, and viral
integrase. (10) The protein molecules combine with the RNA genome, creating a new virus which (11)
makes budding on the surface of the host cell and (12) goes on to infect other cells.
Infected cell 2
HIV Particle
12.
1. 3.
10.
2.
11.
Nascent
HIV HIV particle
RNA 8.
CO-Receptor 4.
5.
CD4 HIV 9.
DNA Copy proteins
of HIV RNA
Infected HIV RNA
cell 1
7.
6. HIV
Integrase Provirus
Cellular
DNA
(Adapted from McKee and McKee, Biochemistry the Molecular Basis of Life [5th ed.], 2012, p. 642; Fig. 17L.)
Chapter 3
Chemistry Review
This review begins with the functional groups in organic chemistry and the relation between structure and
properties in different atomic contexts, which is the foundation of biochemistry. It then reviews physical
chemistry ideas one typically learns in basic chemistry course, applied to biochemical molecules and
principles.
3.1 Organic Compounds

The following functional groups play central roles in biochemistry. You will review them in the Review
Sessions and in the context of drawing structures throughout the course.
3.1.1 Functional Groups
O O O O
R OH R1 C R2 R1 C O R2 O OH
R P R R O P
O
Hydroxyl Carbonyl Acyl Phosphoryl
Phosphate Phenol
(pKa ~ 2, 6 and 12) (pKa ~ 11)
O O
R SH R S
H
Thiolate R C O-H C O
Thiol R C R
(Sulfhydryl) Carboxylic acid Carboxylate
(pKa ~ 8) H
(pKa ~ 5)
Phenyl Benzyl
3.1.2 Classes
R2 R4 O O O O
R2
R OH R1 C O C R6 R C H R1 C R2 R1 C O C R4 R3 C N R1
R3 R5 R3 R2
Alcohol Ether Aldehyde Ketone Ester Amide

(pKa ~ 15)
R2 R2 Amines can also be protonated:
R NH2 R NH3 R1 N H R1 N R3 R1 R1
Primary amine Ammonium Secondary amine Tertiary amine R NH3 R NH2 R NH R2

(pKa ~ 9)
3.1.3 Linkages
O O O O
R1 R2 R4
O P O C R3 R1 C O P O C R5 R1 O P O P O R2
O R2 R3 R6
O O O
Phosphate ester Phosphodiester Phosphoanhydride

10 Chapter 3 Chemistry Review
3.2 Chirality
Specification of Molecular Configuration: The Designations R and S
(Adapted from Morrison and Boyd Organic Chemistry [3rd ed.], 1973, Allyn & Bacon, NY.)
Chirality has had a profound effect on the structural and functional properties of biomolecules. Molecular
configurations can be determined in a more simple way than drawing out the structure. A generally useful
way is the use of the prefixes R and S.
Step 1. Following a set of sequence rules (detailed in the following section), we assign a sequence of
priority to the four atoms or groups of atoms attached to the chiral center.
In the case of C, H, Cl, Br and I, for example, the four atoms attached to the chiral center are all
different and priority depends simply on atomic number, the atom of higher number having higher priority:
I > Br > Cl > H.
Br Br
I I .
Cl Cl
Bromochloroiodomethane
H H
I II
Step 2. We visualize the molecule oriented so that the group of lowest priority is directed away from
us, and observe the arrangement of the remaining groups. If, in proceeding from the group of highest
priority to the group of second priority and thence to the third, our eye travels in a clockwise direction, the
configuration is specified R (Latin: rectus, right); if counterclockwise, the configuration is specified S
(Latin: sinister, left).
Thus, configuration I and II are viewed as follows:
Br Br
H H
Cl Cl I
I
R S
and are specified R and S, respectively.

A complete name for an optically active compound reveals—if they are known—both configuration
and direction of rotation, as, for example, (S)-(+)-sec-butyl chloride. A racemic mixture can be specified by
the prefix RS, as, for example, (RS)- sec-butyl chloride.
Sequence Rules
For ease of reference and for convenience in reviewing, we shall set down here those sequence rules we shall
have need of. The student should study Rules 1 and 2 now, and Rule 3 later when the need for it arises.
Sequence Rule 1. If the four atoms attached to the chiral center are all different, priority depends on
atomic number, with the atom of higher atomic number getting higher priority. If two atoms are isotopes of
the same element, the atom of higher mass number has the higher priority.
For example, in chloroiodomethanesulfonic acid the sequence is I, Cl, S, H; in α-deuterioethyl
bromide it is Br, C, D, H.
Cl H
H C SO3H H3C C Br
I D
Chloroiodomethanesulfonic acid α-Deuterioethyl bromide
Chapter 3 Chemistry Review 11
Sequence Rule 2. If the relative priority of two groups cannot be decided by Rule 1, it shall be
determined by a similar comparison of the next atoms in the groups (and so on, if necessary, working
outward from the chiral center). That is to say, if two atoms attached to the chiral center are the same, we
compare the atoms attached to each of these first atoms.
For example, take sec-butyl chloride, in which two of the atoms attached to the chiral center are
themselves carbon. In CH3, the second atoms are H, H and H.
H
H3C CH2 C CH3
Cl
sec-Butyl Chloride
In C2H5 they are C, H and H. Since carbon has a higher atomic number than hydrogen, C2H5 has the higher
priority. A complete sequence of priority for sec-butyl chloride is therefore Cl, C2H5, CH3, H.
In 3-chloro-2-methylpentane the C, C, H of isopropyl takes priority over the C, H, H of ethyl, and the
complete sequence of priority is Cl > isopropyl > ethyl > H.
CH3 H CH3 H
H3C CH C CH2 CH3 H 3C CH C CH2Cl
Cl Cl
3-Chloro-2-methylpentane 1,2-Dichloro-3-methylbutane
In 1, 2-dichloro-3-methylbutane the Cl, H, H of CH2Cl takes priority over the C, C, H of isopropyl.

Chlorine has a higher atomic number than carbon, and the fact that there are two C’s and only one Cl does
not matter. (One higher number is worth more than two - or three - of a lower number.)
Sequence Rule 3.
Where there is a double or triple bond, both atoms are considered to be duplicated or triplicated. Thus,
A C
C A equals C A and C A equals C A
A A C
For example, in glyceraldehydes the OH group has the highest priority of all,
H
C O H H
H C OH C O equals C O
CH2OH O C
Glyceraldehyde
The O, O, H of –CHO takes priority over the O, H, H of –CH2OH. The complete sequence is then –OH, –
CHO, –CH2OH, and –H.
3.3. Chemical Reactions

Acid-Base Chemistry
The key idea with acid-base reactions is that H+ is gained by one species and lost by another. The following
scheme shows an example of a protonation-deprotonation reaction:
O O O OH
C C
+ H
H C H H C H
H H
Acetate Acetic Acid
Assume that the pKa that governs this equilibrium is 5. Draw a plot that shows the number of H+ atoms
removed as the y axis and pH as the x axis. Recall that:
- pH (1)
pH = -log [H+] and [H+] = 10
The Henderson-Hasselbalch equation is expressed in terms of the conjugate base (A) and conjugate acid
(HA) as follows:
[A-]
pH = pKa + log (2)
[HA]
In the case of the equilibrium shown above, this equation is:
[acetate-]
pH = pKa + log
[acetic acid]
This equation allows one to calculate the following relations between protonated and deprotonated species:
(1) When the pH is equal to the pKa, 50% of the population is protonated and 50% is deprotonated.
(2) When the pH is 1 unit less than the pKa, 90% of the population is protonated and 10% is deprotonated
(3) When the pH is 2 units less than the pKa, 99% of the population is protonated and 1% is deprotonated
(4) When the pH is 1 unit more than the pKa, 90% of the population is deprotonated and 10% is protonated
(5) When the pH is 2 units more than the pKa, 99% of the population is deprotonated and 1% is protonated.
1.0
0.9
acetate
Number of
H+ Removed 0.5
acetic acid
pKa = 5
0.1
0
0 2 4 6 8 10 12 14
pH
The logarithmic plot has a sigmoidal shape and gives equal consideration to each concentration range,
that is, molar (M), millimolar (mM), micromolar (µM), nanomolar (nM), and picomolar (pM) each
correspond to a pH unit. In contrast, the nonlogarithmic curve is hyperbolic and emphasizes the high
concentration. Lower concentrations are “crunched” into the left portion of the curve.
Oxidation Reduction Reactions

The following scheme illustrates the relation between the different classes of two-carbon organic
compounds.
Oxidation
OH H O HO H
O
C C H C H methane
H C H
H C H H C H H
H C H
H +
H H
O C O CO2
Ethanol Acetaldehyde Acetic Acid
Reduction
The key idea is that electrons are gained by one compound and lost by another. This is captured by the
mnemonic acronym OIL RIG, which abbreviates the phrase: Oxidation Is the Loss of electrons; Reduction
Is their Gain. The electrons are sometimes attached to a hydrogen nucleus, forming a species called a
hydride.
Applying the redox series shown above to the 3-carbon compound glycerol:
H O O
H O O O
C O
H C OH C C
H C O P O
H C OH H C OH H C OH
H C OH H C OH H C OH H C H O
H H H O
O P O
Glycerol Glyceraldehyde Glycerate O
2,3-Bisphosphoglycerate
The compound shown to the right, 2, 3-bisphosphoglycerate (BPG), will be discussed as an allosteric
regulator of oxygen binding to hemoglobin in the Ligand Binding and Functional Control section. (Note
that the prefix is bis not bi.)
3.4 Physical Chemistry Concepts

Water: Structure and Properties
Water is composed of a tetrahedrally linked lattice of hydrogen-bonded molecules. The connections
resemble the methane molecule, but each oxygen is connected to 2 hydrogens by covalent bonds and 2
hydrogens by H-bonds.
H H H
H O O
O H
105o H H
O H H H H
H .
The two electron lone pairs O O O O
hydrogen bond with other H H H H
waters.
H H
O O
H H
.
Lone pair electrons donate and bond to H acceptors (δ+ sinks). The lone pair of one H2O’s oxygen
hydrogen bonds with another’s hydrogen. The gradient of electronegativity (electron retention) produces an
overall molecular dipole, which is indicated by the symbol to the left of the water molecule below.
membrane
H2 O bilayer H2O
δ+ -
H
O δ- -
+
δ+ H +
dipole -
direction H 2O
-
+
+
The polarity of water produces a property called hydrophilicity, in which polar biomolecule solutes
are soluble in the solvent. The high electron density on oxygen, nitrogen, and metals make them
polarizable, which, in turn, leads to effective ionic and polar bonding with water and other fully or partially
charged compounds.
nonpolar
H H
O O O
H C H
N H C N H H C
O N H
O H
H
polar amide imide
Acid-Base “Ionization” of water occurs, with a pKa of 14. This is the basis of the pH scale, which
ranges over 14 orders of magnitude in H+ concentration—from 1 M to 10-14 M. As a result, at pH 7, both
H+ and OH- are present at 10-7 M. This becomes important in biochemical reactions because it means that a
reasonably large concentration (in biochemical terms) of each is always around to aid in the catalytic
reaction mechanisms.
Three types of reactions can lead to the release either hydroxylate OH-, a proton H+ or both.
2 H2O H3O+ +
-
OH H2O disproportionation
-
R-H H+ + R H+ and R - anion formation
-
R- + H2O R-H + OH R - uptake and OH - formation
Water is a dynamic moldable liquid, so it is well adapted to filling grooves and gaps formed on the
exterior surface of biomolecules. Moreover, the large enthalpy associated with hydrogen bonding between
water molecules provides the energetic drive that leads to the folding of most native biomolecular
structures.
Hydrophobicity
Water binds other H2Os, which leads to the exclusion of nonpolar molecules; for example benzene, alkanes,
alkenes, including the aromatic and aliphatic side chains of biomolecules. Carbon-bound H’s are not very
polarizable, producing the important incompatibility of “oil” and “water” which is responsible for the
separation of different compartments—a key requirement for molecules to organize together into cells,
capsids, organelles, and so on.
The hydrophobicity of nonpolar molecules can drive assembly of a variety of biomolecular structures,
that is, folded proteins, assembled membranes and vesicles, and nucleic acid double helices.
External waters act like a cage, at temperatures below about 60ºC, enclosing the components of the
folded biomolecule within their collective volume. Formation of the maximal density of hydrogen bonds
among waters in the external solvent network leads to a large advantage in terms of gained enthalpic
energy (ΔH). This offsets the entropic contribution to the Gibbs free energy , that is, –TΔS, which typically
opposes folding. Recall that the Gibbs free energy (ΔG) is defined as:
ΔG = ΔH - TΔS (3)
These ideas will be described in detail in the context of protein folding in the Protein Structure section
(Sect. 6.8).
Van der Waals Forces

One must consider the effects of all atoms within 20 Å.
the repulsion energy increases with

(+) closer interatomic overlap at r -12 separation
ΔG vdW
0
(kcal/mol) Attractive energy also decreases
with larger r -6 separation
(-)
optimum interatomic distance
of the Van der Waals interaction
o
(~ 1.2 to 1.5 A)
o
0 rij (A)
The Van der Waals Interaction Free Energy (ΔGvdW) is proportional to the interatomic distance. More negative values
indicate more stable interatomic distances.
i j
ΔG vdW rij -6 + rij -12 D

distance between
two atomic centers
means "is proportional to" i and j.
ΔG vdW depends on both the negative 6th and negative 12th powers of the interatomic distance.
attractive repulsive (if the atoms are too close)

(if rij is greater than the
Van der Waals radius) (due to the Pauli Exclusion Principle)
(1) Attraction: Coordinated alignment of the dipoles, like a synchronized dance.
(2) Repulsion: Atom-atom electronic overlap is opposed by the Pauli Exclusion Principle, that is, two
electrons with the same spin cannot coexist in the same space.
(3) The parameter D is the dielectric constant. H2O has a large D (~ 80) so molecular charges are well
shielded from each other in H2O. This is much less true in nonpolar solvents, which enhances the
effectiveness of charge-charge interactions.
Hydrogen Bonding
They form between polar atoms such as oxygen and hydrogen or nitrogen and hydrogen.
ΔG HB rij -10 + rij -12 / D
10th 12th very narrow range of

stability
power power
the repulsion energy increases with

(+) closer interatomic overlap at r -12 separation
ΔG HB
0
(kcal/mol) Attractive energy decreases
with larger r -10 separation. It
(-) falls to zero by 2 to 3 Å.
optimum interatomic distance

in the hydrogen bond
0 rij (Å)
i j the electrons are not equally shared

rij
center-to-center distance (ΔG ~ 1-2 kcal/mole)
between two atoms
Van der Waals

or R2
H-bond distance R1 N H O C
R3
unfavorable
electronic rij
overlap covalent bond H-bond
Note the much narrower range of stability of hydrogen bonds relative to Van der Waals interactions.
Some Typical Biochemical Hydrogen Bonds

Amide-carbonyl: R1 R3
N H O C
R2 R4 Hydroxyl-hydroxyl:
R1 O H O R2
Amide-hydroxyl: R1
H
N H O R3
R2 H Hydroxyl-carbonyl:
R2
R1 O H O C
Amide-imidazole nitrogen:
R3 R3
R1
N H N NH
R2
Low-Barrier Hydrogen Bonds
The Catalytic Triad mechanism of proteolysis by Serine Proteases is driven by formation of this type
of bond.
LBHBs are characterized by the very far downfield chemical shift values in the hybridized (simultaneous)
proton nuclear magnetic resonance spectra (> 18 ppm). Examples have been studied in covalent-hydrogen bonds
. molecule model sytems.
small
LBHB formation is essential to formation of the aspartate-histidine hydrogen bond
that plays a key role in the 'charge relay' mechanism used by serine protease. LBHB R3
R1 .
formation activates the strong nucleophilic capability of the histidine to initiate
catalysis. The histidine nitrogen is activated to remove the proton from the serine, N H C
which, as a hydroxylate, subsequently attacks the carbonyl oxygen of the peptide bond R4
that will be cleaved in the target protein. R2
high energy: ΔG > 20 kcal/mol e more equally shared
Salt Bridges (also called Ionic Bonds) form between two fully or partially charged atomic species. Two
-
examples are the Glutamate–COO • • • +H3N–Lysine interaction and the Mg2+ - ATP4- complex (see the
Coenzyme section).
R1 X Y R2
(b) Mg2+ (c)

(a)
O δ-
H
δ- O
O O O P δ+
R1 C R2 δ- δ+ δ+ R1 O Ca2+
N H O P O P O R1 O O R2
H O + H
Na O O
K+
Osmotic Pressure
This is not a charge-dependent phenomenon. It is driven by the tendency of the molecules to equalize the
number of configurations in the two compartments (entropy). When more and less concentrated molecules
are released on two sides of a permeable barrier, they will try to equalize concentrations.
more possible
configurations
exist
higher lower
concentration gradient
concentration gradient reaches equilibrium poise
Remember the Ideal Gas Law: PV=nRT (In doing concentration-pressure-volume

work.)
Rearranging produces: P (V/n) = R T
The molar volume (V/n) balances the pressure (P) at constant temperature. If the volume changes and
concentration remains constant, the density (n/V) changes. The pressure that drives densities to equilibrate
is the osmotic pressure.
Water goes into cells from blood in an attempt to dilute the cellular salts and other solutes, which are
more concentrated within the cell. This creates the “turgor pressure” that maintains cells in the inflated
state.
Amphipathicity
This occurs when a molecule is composed of subcomponents that prefer formation of two different
segregated phases, one hydrophobic and one hydrophilic. For example, some alpha helices in proteins have
one hydrophobic face and one hydrophilic face.
(1) One important example is a fatty acid:
O
Carboxylate
C
O Alkane chain
Polar Nonpolar
Interface
(2) Sodium Dodecyl Sulfate (SDS): This compound denatures proteins by disturbing the ‘hydrophobic
effect,’ so it is a common additive in Polyacrylamide Gel Electrophoresis (SDS PAGE) samples.
O
O S O
12 carbons
O
(3) Diacylglycerol phosphate
O P O CH2 O
O
HC O O
H2C O
3.5 Buffering of Blood: The Bicarbonate System

The blood is buffered by dissolved bicarbonate anion. The following scheme shows the three forms of
dissolved carbon dioxide:
CO2 (g) CO2 (d) + H2O H2CO3 carbonic acid
(lungs) (blood)
(dissolved) H+ H+ 3
H+
2
HCO3 CO3 2-
+
Na
stabilized bicarbonate H+ carbonate
The two pKa values for deprotonation of carbonic acid (1) to form bicarbonate (2) and then carbonate
(3) are shown in the following pH profile. At pH 7, bicarbonate predominates.
1 2 3
100
Percentage
of species 50 .
present
0 pH
0 2 4 6 8 10 12 14
pKa,1 = 6.4 pKa,2 = 10.2
Garrett and Grisham provide a careful quantitative analysis of blood buffering. (See Biochemistry, 3rd
ed., 2005, Brooks-Cole Publishing Company, p. 47.). Conversion of dissolved CO2 (d) and H2O to carbonic
acid is catalyzed by the enzyme Carbonic Anhydrase. The equilibrium constant for hydration of CO2 (Kh) is
-
0.003. This reaction is coupled to the H2CO3 ↔ HCO3 + H+ equilibrium (Ka = 2.69 x 10-4). The overall
equilibrium constant for deprotonation of carbonic acid in equilibrium with dissolved CO2 is:
[H+][HCO3-]
Koverall = Ka Kh = = 8.07 x 10-7 ; pKoverall = 6.1
Kh [CO2 (d)]
The resulting Henderson-Hasselbalch equation is:
[HCO3-]
pH = pKoverall + log
[CO2 (d)]
The total concentration of the “Carbonic Acid Pool,” CO2 (d) + H2CO3, in blood is ~ 1.2 mM.
Gaseous CO2 at a partial pressure of 40 mm Hg in lung alveoli drives CO2 (d) and H2CO3 formation, which
produces a ~ 24 mM HCO3- concentration. Gaseous CO2 stabilizes the pH of blood by contributing a large
buffering effect on the total “carbonic acid pool.”
Chapter 4
Amino Acids
4.1 Basic Structures

All of the amino acids typically found in proteins have the L configuration about the alpha carbon (Cα).
Some less common structures contain D amino acids, for example, the mold-generated antibiotic
valinomycin (see Ionophores).
pKa = 2.2
O O "aaRoot" is the root structure
Both charges are favored at pH 7, in C of all amino acids.
part due to self-attraction, H
i.e. salt bridge formation.
H N Cα H
α
pKa = 9.5 H
R
The "R group"
In the case of proline (containing Cβ, Cδ, etc.)
the R-group is connected
intramolecularly to the
amine nitrogen.
Zwitterion. The word “zwei” means “two” in German. Both charges are favored at pH 7, due to self-
attraction between the positive charge of the ammonium group and negative charge of the carboxylate.
(Incidently, “zwitter” means “hermaphrodite.”)
The convention for naming the isomers derives from L-glyceraldehyde.
O H
C
(levorotatory) L-glyceraldehyde
HO C H
(S form conform-
(sinistra) ational isomer) CH2
HO
4.2 Amino acid “R Groups”

Twenty standard R groups occur in most natural proteins. The Root and R group atoms are often modified
in real proteins for specific biochemical reasons. The standard set is like a biochemical equivalent to the
alphabet. These modifications constitute a language. Deencrypting how the language is stamped into the
structures is often the key to understanding the regulation of intermediary metabolism, genetic functions,
cellular and organism-wide developmental strategies, modes of adaptation, and so forth.
Alkyl Side Chains

The following amino acids are aliphatic. Alanine, valine, leucine, and isoleucine are hydrophobic.
Chapter 4 Amino Acids 21
Glycine (Gly, G) Alanine (Ala, A) Valine (Val, V) Leucine (Leu, L)

H H
aaRoot H aaRoot CH3 aaRoot C CH3 aaRoot CH2 C CH3
CH3 CH3
R groups
Isoleucine (Ile, I) Proline (Pro, P) O

H H C O
H
aaRoot C CH2 CH3 pKa = 10.6 N+ C H
CH3 H 2C CH2
C Cα
(hydrophobic)
H2
Proline is a cyclized amino acid. It reinforces U-turns in protein chain structures.

Aromatic Side Chains. The following R groups occur in the aromatic amino acids. They are all
hydrophobic.
Phenylalanine (Phe, F) Tyrosine (Tyr, Y) Tryptophan (Trp, W)
aaRoot aaRoot aaRoot
CH2 CH2 CH2

H
H C H H C H C C
C C C C H C C C H
C C C C C N
H C H H C H C C Indole
H H ring system
H O H
H
pKa = 10.5
All aromatic N lone pair electrons

are in the π system.
Ultraviolet Absorbance at 280 nm
The aromatic amino acids absorb ultraviolet light at a wavelength of ~280 nm.
Beer-Lambert Law:
Absorbance
A=ε bc
molar
concentration
0 molar
200 280 extinction pathlength of light
coefficient in sample cuvette.
Wavelength (nm) absorbance
22 Chapter 4 Amino Acids
The Beer-Lambert relation (“Beer’s Law”) is commonly used to determine protein and nucleic acid
concentrations. If one knows the value of ε at the wavelength of interest, one can measure the absorbance
and calculate the molar concentration from it.
Hydroxyl-containing Side Chains. The following amino acids contain alcohol side chains:
Serine (Ser, S) Threonine (Thr, T)
aaRoot aaRoot
CH2 H C OH [a secondary (2o) alcohol]
OH CH3
pKa ~ 15
Sulfur-containing Amino Acids

Cysteine (Cys, C) Methionine (Met, M)
aaRoot aaRoot
a thiol CH2 CH2
(sulfur "alcohol")
S CH2
H S a thioether
pKa = 8.4 linkage
CH3
Carboxylate-containing Side Chains
Aspartate (Asp, D) Glutamate (Glu, E)

aaRoot aaRoot
CH2 CH2
C CH2
O O
pKa = 3.9 C
O O pKa = 4.1
Amide-containing Amino Acids

Asparagine (Asn, N) Glutamine (Gln, Q)
aaRoot aaRoot
CH2 CH2
C CH2
O NH2 C
O NH2
Basic Side Chains
Lysine (Lys, K) Arginine (Arg, R) Histidine (His, H)

aaRoot aaRoot aaRoot
(CH2)4 (CH2)3 CH2
H N H N H H C C N H imidazole = deprotonated
H C imidazolium = protonated
H N N H N C
pKa = 10.5
H H H
H
pKa = 12.5 pKa = 6
4.3 Ionization Properties

The following chart shows a series of key pKa values placed on the pH scale. Reactions occurring at each
relevant pKa (letters below the scale) are listed. They are average values, which can be very different,
depending on their environment within a protein (see Section 4.5).
protein C-terminal carboxylate
pKa Scale : (5)
α COOH COO (8.4)

(12.5)
SH Cys S NH2
(3.9) NH
C NH
(9.5) Arg N 2
Asp COOH COO
α NH2 H
NH3
(4.1)
lone amino acid (2.2) (10.5)
Glu COOH COO
α COOH COO Tyr OH O
(6)
Lys NH4 NH2
His H
0 2 4 6 8 10 12 14
pH : acidic neutral alkaline/basic
Protonated form Deprotonated form pKa

-
α–COOH R–COO 5.0 (C-terminal amino acid)
-
α–COOH R–COO 2.2 (lone amino acid)
-
Asp COOH R–COO 3.9
-
Glu COOH R–COO 4.1
His–H+ R 6.0
-
Cys–SH R–S 8.4
α–NH3+ R–NH2 9.5
-
Tyr–OH R–O 10.5
Lys–NH3+ R–NH2 10.5
Arg (guanidinium–H+) guanidine 12.5
4.4 Drawing Peptide Titration Plots

The isoelectric point (pI) is the pH at which the net charge of a biomolecule is equal to 0. Calculating the
charge of a protein at a given pH involves calculating all of their charges, taking into account the
percentage of protonation and deprotonation of each species. The following examples demonstrate how to
calculate isoelectric points in various situations.
(1) For an amino acid, average the pKa values. For example, with alanine only the following equilibria are
involved:
α-COO- ↔ COOH, α-NH2 ↔ α-NH3+
We find that the pI is midway between the 2 pKa values:
pI = [(pKa(R - COOH) + pKa(R—NH3+)] / 2 = (2.2 + 9.5)/2 = 5.85
(2) When three functional groups contribute to the molecular charge in amounts whose charges vary with
pH, you must consider the three pKa values, whether protonation or deprotonation occurs to get to the pH
of interest, and whether charge is produced or decreased when the protonation or deprotonation occurs. The
following points will help you visualize the calculation by helping to keep the whole set of contributing
reaction equilibria in mind and considering whether your answer is consistent with them.
24 Chapter 4 Amino Acids
(i) Write the structure with all the sites protonated.

(ii) Determine the net charge. If it is 0 or -, there is no pI.
(iii) Rank the pKas numerically from low to high.
(iv) N = the charge, pI = (pKa, N + pKa, N+1)/2
(3) If the curves overlap, which they will if two pKas are within two units of each other, one sketches in the
curves for each deprotonation, as if in isolation, then draws the best possible compromise between the
isolated curves. Visually, the curves appear to be “merged” into a single transition. The following curve
shows them as if the two transitions are clearly resolved. In reality, they will not be.
Case 1: Tyr—Asp—Ala
pKa = 10.5
OH
pKa = 9.5 pKa = 5 pI = (5 + 9.5)/2

CH3 CH3 = 7.25
+ aa2
NH3 aa1 aa3 COO
3
Equivalents Average of the two
curves one would
of H+ removed 2 pI draw if each reaction
occured in isolation
1 (the dashed patterns).
0 10 12 14
0 2 4 6 8
(1) (1)
pH
(1) (1)
(1) Protonate everything … then determine the net charge (= +1 in this case).
(2) What is the pI? In other words, at what pH will the charge = 0? Where one negative charge is acquired
(where half of the –COOH groups are deprotonated).
pI = average of the two of –COOH pKas = 4.45
Case 2: Tyr—Ala—Ala
pKa = 10.5
OH
pKa = 9.5 pKa = 5 pI = (5 + 9.5)/2

CH3 CH3 = 7.25
+ aa2
NH3 aa1 aa3 COO
3
Equivalents 2 pI
of H+ removed
1
0 10 12 14
0 2 4 6 8
pH
(1) (1) (1)
(1) The charge of fully protonated peptide = +1.

(2) At what pH will the charge be 0?
When all of the –COOH is deprotonated and none of the –NH3+ is deprotonated.
pI = (5 + 9.5)/2 = 7.25
Case 3: Tyr—Glu—Arg
pKa = 10.5 H2N + NH2 pKa = 12.5

OH C
pKa = 4.1 N H pI = 7.25
pKa = 9.5 COO (CH2)3
+ aa2
NH3 aa1 aa3 COO pKa = 5
3
Equivalents
of H+ removed 2
1 pI
0
0 2 4 6 8 10 12 14
pH
(1) (1) (1) (1) (1)
The charge of fully protonated peptide = +2.
pI = [(5 + 9.5] / 2 = 7.25
4.5 Factors That Influence the pKa of Protonatable/Deprotonatable Groups

in Proteins.
The standard pKa values can shift, sometimes dramatically, when influenced by solvent factors, as shown in
the following examples.
(1) Dehydration (Born Effect): The pKa changes due to the Born effect when a functional group is buried
within the interior of the protein because the dielectric constant is lower than that of water. Lower dielectric
constant conditions favor the neutral form. The pKas of asp, glu, cys, and tyr increase. Those of his, lys, and
arg decrease.
-COOH ↔ -COO- + H+ ↑ nonpolar environment ↑ pKa
-NH3+ ↔ -NH2 + H+ ↑ nonpolar environment ↓ pKa
When valine 66 in the protein staphylococcal nuclease is replaced by asp, the pKa of the carboxylate is 8.9,
5 units higher than that of asp in H2O. When val-66 is replaced by lys, the ammonium group has a pKa of
5.5, 4.9 units lower than the pKa in H2O.
(2) Charge-Charge Interactions (Coulombic):

-COOH ↔ -COO- + H+ ↑ positive charge ↓ pKa
-NH3+ ↔ -NH2 + H+ ↑ positive charge ↓ pKa
(3) Charge-Dipole Interactions (Hydrogen Bonding):
-COOH ↔ -COO- + H+ ↑ hydrogen bonding to protonated form ↑ pKa
-NH3+ ↔ -NH2 + H+ ↑ hydrogen bonding to protonated form ↑ pKa
(Adapted from: Pace, C., N., Grimsley, G. R., and Schultz, J. M. (2009) Protein Ionizable Groups: pKa Values and
Their Contribution to Protein Stability and Solubility, J. Biol. Chem., 284, 13285–13289.)
Chapter 5
Proteins
5.1 Peptide Bonds

Definition: Proteins are polymers composed of α-amino acids linked by sequential peptide bonds. The most
basic is the dipeptide:
1 2
R1 O R2 O
H 3N N C O a dipeptide
Cα C Cα
H H H
peptide bond
The peptide bond is the fundamental linkage that results in the creation of a protein chain.
In a long polypeptide, the pKa of the C-terminus increases from 2.2 to 5.
Nomenclature:
The ‘ine’ suffix is usually replaced by ‘yl’.
e.g.: glycine → glycyl
Note that the amino acids are listed in the chain from N-terminus to C terminus, the accepted standard
nomenclature.
For example, the substance Aspartame, which is used as an artificial sweetener:
O O C-terminus
N-terminus C
CH2 O CH2 O
H3 N Cα C N Cα C O CH3 modified
H H H
aspartyl-phenylalanyl-methyl ester
Exceptions: asparagine → asparaginyl

glutamine glutaminyl
cysteine → cysteinyl
tryptophan → tryptophanyl
The ‘in’ is retained, while only the terminal ‘e’ is replaced by ‘yl’
Chapter 5 Proteins 27
5.2 Purification and Characterization of Proteins

Solid-Phase Chemical Synthesis
The key to the synthetic technique is the solid phase synthesis approach. This technique involves binding
reactants to a ligand, which is bound to an insoluble bead material held inside a pyrex column by a set of
“frits,” which allow flow of reagents in and out, yet prevent loss of the matrix and bound synthetic
intermediate biomolecule. Reactions are carried out by passing reactants into the column, allowing them to
react with the column-bound reactant, then flushing them out with suitable solvents. The column is rinsed
with the new reaction solvent and new reactant is introduced. The cycle is repeated until the desired length
is made. The product is release from the column with a decoupling reagent.
X
H
aa1 X R1 R1 aa1 NH2
N X
deprotection
aa2 X
Bead in the condensation
column matrix + dehydrating
agent
H2O
More
cycles
H R2
R1 aa1 N C C N X
O H H
added aa2
This approach employs similar logic to that used in the chemical synthesis of DNA and RNA.
A key result is that synthetic peptides and protein are as functionally active as “biological isolates.”
This proved that synthetic and natural are functionally the same. This was the first clear indication that
“living” is a process that is completely the result of interactions between a complex set of coupled chemical
reactions.
Isolation from Cells

A typical purification approach involves the following steps:
(1) Grow the cells under conditions in which the protein is present in significant amounts, (2) disrupt the
cellular membranes, (3) Remove the large insoluble “debris” by centrifuging the cell homogenate. Larger
particles will sediment to the bottom of the tube. One pours off the solution to separate this debris from the
dissolved crude sample solution. (4) It is important to include additives to preserve catalytic/functional
activity: This involves keeping samples cold (“on ice”) and using protease inhibitors. Buffers are used to
maintain a protective pH. (5) Add ammonium sulfate to “salt out” either the desired protein or
contaminants. In the former case one collects the precipitate, resuspends it in a planned buffer solution then
dialyzes it to remove the salt. In the latter, the desired protein is in the supernatant.
Purification Using Gel Chromatography

This is typically performed using chromatography, the structural purity is assessed using gel electrophoresis.
The functional activity is determined using a biochemical assay.
Separation is based on the intrinsic molecular differences in affinity of a solute (sample plus
impurities) for a moving material (flowing buffer) through a column packed with a “stationary” gel.
Binding/unbinding cycles result in differential separation of different materials. This behavior is called
partitioning and, in gas chromatography, plating.
28 Chapter 5 Proteins
Gel Filtration (Gel Permeation) Chromatography. Smaller molecules are retained longer, while larger
molecules elute earlier. Larger molecules are less included within and therefore less well retained by the gel
network.
Load
column
A B
eluted eluted
(mostly)
A B
Chromatograms, activity activity

"elution profiles" A A B
fraction # fraction #
Ion Exchange Chromatography. The column matrix is modified with either cation or anion ligands. For
example, negatively charged DNA binds to the cationic diethylaminoethyl (DEAE+) ligand. More negative
(and more tightly bound) molecules require higher salt to be dislodged relative to less negative molecules.
Separation is based on different mixtures of exposed charges on the biomolecules one separates.
Ligand-dependent Affinity Chromatography. An example involves purifying ATP-binding enzymes by

using their tendency to bind to a column containing bound ATP. Free ATP in buffer is used to displace the
enzyme from the column matrix, purified from the pre-eluted impurities.
Polyacrylamide Gel Electrophoresis

SDS-Induced Protein Denaturation. The following reaction occurs when SDS binds to a protein:
n-
protein (+ or -) + n SDS- → [protein—(SDS-)n]
native denatured
(structurally intact) (little stable internal structure)
Electrophoresis.
In the electrophoresis process, (1) the current (I) carries the charged proteins, and (2) the voltage (V)
provides the energy that drives their movement. Ohm’s Law (V = I R) captures the relation between the
two. The resistance (R) is provided by the gel as the proteins migrate from the upper negatively charge well
to the lower positively charged buffer reservoir.
The negative charges of the n SDS molecules overwhelm the native charge(s) of the protein.
Therefore, the proteins adopt worm-like polyanionic SDS-coated structures, which differ in length.
Separation by electrophoresis is predominantly protein size-dependent, and not charge-dependent. Different
molecules have different hydrodynamic “sizes,” which leads to different mobilities.
upper reservior
smaller/ faster
unknown
mobility
~ 100 V
larger/
slower
~ mA's an
electrophoretic
"band"
lower reservior
log Mr
Determining Molecular Weights. One can determine the molecular weight (MW) of "unknown" proteins
using a mobility versus log MW plot. The method involves running suitable calibration proteins along with
the sample/unknown protein of interest. Note that the abbreviations MW and Mr are used to indicate
molecular weight. The latter actually refers to an experimentally determined “hydrodynamic” value, which
also depends on the rotational properties of the molecule.
Assessing Purification. The following figure illustrates an idealized electrophoresis pattern at four
different stages during purification of the glycolytic enzyme Lactate Dehydrogenase. Contaminant proteins
are somewhat randomly distributed prior to the ammonium sulfate stage and become less so after
ammonium sulfate fractionation, which selects a specific subclass of proteins. The (NH4)2SO4- step
eliminated proteins based on insolubility. Dialysis removes proteins with molecular weights below the
“cutoff” value of the dialysis tubing—typically 12 kDa.
1 2 3 4
(1) initial lysate
(2) ammonium sulfate fractionate
(3) post-dialysis
(4) post-affinity chromatography
The Differences in the Electrophoresis and Gel Filtration Mechanisms

Note that separation by Size Produces Opposite Migration Rate Trends in Electrophoresis and Gel
Filtration Chromatography.
(1) Electrophoresis: Larger molecules move less rapidly in the gel, ending up toward the top of the gel.
Larger molecules are held up by the tortuous network of the gel. Smaller molecules move most rapidly,
ending up at the bottom of the gel.
(2) Gel Filtration Chromatography: Larger molecules are not impeded as much as smaller molecules as
they flow through (and by) the column matrix material. Smaller molecules keep entering the beads, holding
them up. Larger molecules emerge most rapidly; smaller molecules emerge later.
Protein Quantification
Absorbance Spectroscopy.
A280—depends on aromatic amino acid content, which varies with the amino acid content and
pH of the protein.
A205—every peptide bond absorbs in this region. Problem: Interferences due to the buffer and
other solution components that typically accompany a semipurified protein.
Chemical Detection Methods.
(1) “Biuret” reaction:
Cu 2+
(alkalinep H)
peptide bond pale blue complex read

absorbance
Cu1+
(2) Lowry reaction: dye-coupled Biuret method; depends on comparison with standard protein samples
(usually bovine serum albumin, BSA).
(3) BCA assay: (Pierce kit) This is an improved version of the dye-coupled Biuret technique. It has fewer
interferences. This is generally considered the preferred technique. The Cu2+ reduction reaction is sensitive
to cysteine, cystine, tyrosine and tryptophan residues, in addition to the peptide bonds.
(4) Bradford assay: involves binding Coomassie blue dye to protein. The dye binds primarily to arginine, as
well as weaker interactions with his, lys, trp, tyr and phe. The dye is also typically used to visualize
proteins on electrophoretic gels.
Amino Acid Content and Sequence Determination.

Edman Degradation.
This technique involves making PITC-derivatives of amino acids. Because all of the amino acids are
labeled with the same reagent, one can compare their contents on a quantitative basis.
Technique 1. Hydrolysis, 6 M HCl,

110 oC, 1-3 days H H C OO
H COO
Protein H N C H N C N C H
R S R
N C S amino acids
δ- δ+ (all of them at the
phenylisothiocyanate same time)
(PITC)
trifluoroacetic acid
O (TFA)
H C protein
H n-1 F3CCOOH
N C N C H aa1-PITC
S R1
Technique 2. Reaction with the N-terminus of O
intact protein (Edman degradation) H
C protein n-2
H N C H
another R2
cycle
Methods 1 and 2 both involve the use of high pressure liquid chromatography (HPLC) to identify and
quantify the amino acid. While this approach has been superseded by the use of mass spectrometry, the
“microsequencing” version of it played an especially important role in the 1980s as a way to determine the
N-terminal amino acids, thereby allowing one to make a probe DNA suitable to search for the gene in a
suitable “DNA library” preparation.
Sanger’s Reagent
This technique was developed by Fred Sanger, double Nobel laureate. He won his first Nobel prize
for protein primary structure determination.
O
F F-
H C (protein)
N C H
O2N NO2 O 2N NO2 H
R
2,4-dinitrofluorobenzene HF
(DNFB)
DNP aa1 HPLC to identify the aa
DNA-Based Sequence Analysis.
(1) Dideoxy Sequence Determination. Fred Sanger won his second Nobel prize for inventing a
technique that allows one to determine the sequence of DNA. The method uses DNA Polymerase, primer
DNA and special “terminator” nucleotide triphosphates called dideoxy NTPs (ddNTPs). In modern
applications, each of the four nucleotides is labeled with a different colored conjugate molecule, allowing
one to determine the nucleotide at the terminated end. This is the basis of most modern machine-based
determinations.
(2) Reverse Translation of cDNA. One can determine the DNA sequence that encodes the protein to
indirectly obtain the amino acid sequence of the protein. The sequence of the transcribed mRNA and the
‘genetic code’ are used to work backwards to deduce the amino acid sequence encoded by the DNA. This
procedure is called “reverse translation.” It is done using reverse-transcribed mRNA sequences called copy
DNAs (cDNAs). The advantage to this approach versus sequencing the genomic DNA is that the introns
have been removed.
Proteins sequence information is also used to get a foot in the door. The sequences of the ends are
determined, then the information is used to determine the DNA sequence, which is used to make a probe
DNA. This probe is used to isolate the genomic DNA, whose sequence is then determined. This information
is also used to make primer DNAs, which allow one to carry out the Polymerase Chain Reaction, as a means
to obtain sufficient DNA to clone and manipulate it, if desired.
Sequence-Specific Enzymes. Three examples of sequence-specific digestive enzymes, along with their
specificities, are:
(1) Trypsin: specific cleavage for Lys and Arg
(2) Chymotrypsin: specific for Phe, Tyr, Trp. It also has a “loose specificity” for bulky side groups.
(3) Thermolysin: specific for Ile, Leu and Val
By carrying out cleavage reactions with different enzymes, then reconstructing the overlapping
patterns, researchers were able to reconstruct the sequence of the entire protein fragment. A similar
approach, using DNA fragments, is the typical way to reconstructing the sequence of genomic DNAs.
Computers are used to align the fragments and piece together the full sequence. For example, the protease
Carboxypeptidase B cuts the protein chain, leaving either arg or lys as the C-terminus. Note that the real C-
terminus, whatever it is, also remains, allowing one to determine its identity.
CB CB CB
H3 N Met Arg Leu Lys Ala Ala Phe Arg Tyr COO
Carboxypeptidase B
. H3 N Met Arg COO
H3N Leu Lys COO
H3N Ala Ala Phe Arg COO This is the C-terminus

because cleavage only
occurs at lys or arg.
H3 N Tyr COO
Chemical Modification by Cyanogen Bromide. Cyanogen bromide modifies proteins at methionine

residues. This provides a way to obtain protein subfragments. Each can then be sequenced using the Edman
technique or otherwise manipulated or studied.
Bioconjugates. Many other residue-specific protein modification methods exist. They form the basis of the
vast literature on uses of different bioconjugate molecules, in which either another type of biomolecule or
some artificial molecule is hooked to the initial biomolecule. Two examples include: (1)
immunoconjugates, in which an antibody is connected to an enzyme, and (2) biotinylated DNA, a
coenzyme connected to a nucleic acid. Each is described in detail in the Coenzyme and Biotechnology
sections, respectively.
Disulfide Bond Maintenance: β-Mercaptoethanol and Dithiothreitol.
Disulfide bonds: crosslinks between two Cys residues.
H H O H H O
R1 N C C R3 H2 R1 N C C R3
CH2 CH2
S oxidation S
Intervening portion H H
of the protein chain
reduction Intervening portion S
S of the protein chain
CH2
R2 H2
R2
2 sulfhydryls
Reversible disulfide
reaction bond
β-mercaptoethanol (BME).
This reagent traps reduced sulfhydryl groups. BME and DTT (see below) are used to protect proteins.
Most solutions used to prepare proteins contain one of these reagents. At lower concentrations (e.g., 1
mM), both BME and DTT will prevent proteins from forming unintended disulfide bonds. At higher BME
or DTT concentrations, the reagent will break disulfide bonds, forming two sulfhydryl groups.
The cell is generally held under reducing conditions, so relatively few cysteines in most cytosolic
proteins are involved in disulfide bonds.
R1 H2 R1
S S CH2 R1 S H
CH2 OH S H + R2
S
+ 2 HS CH2 CH2 OH +
S S CH2 CH2 OH
R2 (BME) S S CH2 CH2 OH +
R2 S CH2 CH2 OH
Dithiothreitol (DTT): This reagent is a more efficient reducing agent than BME.
OH
R1 S S
2 H2
R1 OH HS OH
S S
+ 2
S +
S OH
R2 OH
R2 S S
Dithiothreitol
(DTT) HS OH
Iodoacetic acid undergoes irreversible reaction with cys –SH groups. It acetylates and protects the cysteine
from reforming disulfide bonds. Note that the sulfur is a good nucleophile and iodine is a good leaving
group.
+
R CH2 S + I CH2 COO R CH2 S CH2 COO + H + I
H
Chapter 6
Protein Structure
6.1 Conformation
A generic dipeptide is shown to illustrate the structure of the peptide bond, which is formed as a result of a
dehydration reaction.
H H H H H H H H
H N Cα C OH + H N Cα C OH H N Cα C N Cα C OH + H2O
R1 O R2 O R1 O R2 O
amino acid 1 amino acid 2 water
dipeptide
The arrangement of atoms in three dimensions depends on the patterns of connectivity between the
atoms and the intrinsic rotational capabilities of the bonds, which is characterized in terms of torsion
angles.
H R2 H
O
Φ Ψ
N-terminus C Cα N C-terminus
Cα N C C
H
R1 H O H R3
Trans is preferred
in proteins
The peptide bond has double bond character due to enamine-ketamine tautomerism, which is
analogous to the more familiar enol-keto tautomerism. The atoms shown in the boxes are constrained to
being coplanar. The bonds, which are called φ and Ψ, can rotate, although the rotation is restricted when the
chain adopts a particular structure.
H H
R1 C N R2 R1 C N R2 R1 C N R2
+
OH O O-
enamine ketoamine hydroxylate-quaternary amide cation
H H H
R1 C C R2 R1 C C R2 R1 C N R2
δ+
OH O H Oδ -
enol keto net hybrid structure
Two types of bonding structures occur among the affected atoms. The first is the ketamine form shown
above. The second structure has two changes. (1) The carbonyl oxygen has a negative charge and is connected
by a single bond to what is normally the carbonyl carbon (as a hydroxylate). (2) The peptide bond is a double
bond and the tetravalent amide nitrogen has a positive charge. The net hybridized structure due to the two
contributing structure has delocalized electrons spanning the bonds between the carbonyl carbon the carbonyl
oxygen and the amide nitrogen, with a partially negative charge on the carbonyl oxygen and partially positive
charge on the amide. As a result, the peptide bond has a net bond order of 1.5 and does not rotate easily.
34 Chapter 6 Protein Structure
6.2 Classification of Substructure

The first level of classification involves the degree of compatibility with water.
(1) Water Soluble: many enzymes fall into this category. They are typically “globular” in shape.
(2) Water Insoluble: structural materials, such as Keratin, a primary component in hair and fingernails.
X-ray Diffraction Analysis. The wavelength used is similar to interatomic distances. Light is
diffracted by the electron density of an array of proteins embedded within a crystal. This produces a
specific pattern of spots on an imaging apparatus that surrounds the mounted crystal. Using computer
programs and careful human analysis, one can work backward to calculate the structure of protein.
In an early, highly instructive set of studies, John Kendrew and colleagues solved the structure of
myoglobin, which carries molecular oxygen in muscle tissues. The structure revealed many general features
of protein architecture. (The structure is shown in the Myoglobin section.)
Primary Structure. This is the sequence of covalently linked amino acids.

Secondary Structure. These are the regular folded structures found within most proteins, for example,
α-helix, antiparallel and parallel β-sheets. Others are the U-turn and extended chain, which are described in
detail below.
Tertiary Structure. This refers to the packing and stabilization of regular interconnected
substructures from a single chain into the overall globular structure; side chains accommodated. An
example of the ATP-binding domain of hexokinase, which is composed of an α/β-domain, is shown below.
1
2
4 3
C-terminus
4
N-terminus
3
2
1
(Adapted from McKee and McKee, Biochemistry The Molecular Basis of Life (5th ed.). 2012, p. 149; Fig. 5.21.)
Quaternary Structure. This involves the packing of subunits (different chains) into multisubunit
complex. An example is provided by hemoglobin, which forms a tetrameric quaternary structure:
2 α + 2 β → α2 β2
Non-covalent quaternary structure formation often modulates regulatory behavior. Multisubunit proteins
exist for three main reasons.
(1) Having several subunits is likely to be more efficient than adding more sequence to the length of one
polypeptide chain.
(2) Replacing depleted or damaged portions in multisubunit complexes, such as collagen fibers, can be
more efficiently managed.
(3) Interactions within multiple subunit complexes commonly aid in regulating the biological function of a
protein monomer.
Chapter 6 Protein Structure 35
6.3 Alpha (α) Helices

The α-helix was first described by Linus Pauling. Most α-helices in proteins are right-handed due to steric
interference between the carbonyl oxygens and amino acid side chains. The helix conformation is reinforced by
hydrogen bonding between each carbonyl oxygen and the amide hydrogen of the fourth amino acid toward the C-
terminus.
.
R2 H H O R4 H H O R6 H H O
R1 N C C N C C N C C N C C N C C N C C R8
H H O R3 H H O R5 H H O R7
Hydrogen Bonding
(13 inclusive atoms)
The hydrogen bonds that form between the N, O, and H atoms are approximately parallel to the long axis of the
helix. The distance between equivalent positions on the α helix is called the pitch and recurs every 0.54 nm. The rise of
the helix is referred to as how far each amino acid advances the α helix and is 0.15 nm. Per turn of the helical structure
there are 3.6 amino acid residues.
back
side
Pitch
(advance
front per turn)
side
. 0.54 nm
Rise per
amino acid
residue
α helix
axis
An α-helix is a secondary structure of polypeptides and could be right-handed or left-handed. In the

ideal α helix, the distance each amino acid advances the helix, or the rise, is 0.15 nm. For one complete turn
of the α helix, 3.6 amino acid residues are required, which consists of approximately one carbonyl group,
three N-Cα-C units, and one nitrogen. The pitch of the helix is the distance of equivalent positions on the
helix and is usually occurs every 0.54 nm.
The helical structure contains stabilizing hydrogen bonds between each carbonyl oxygen and the α-
amino nitrogen of the fourth residue toward the C-terminus. The atoms circumscribed by the hydrogen
bonds form a ring structure containing the 13 atoms, carbonyl oxygen, 11 backbone atoms, and amine
hydrogen.
A variant of the α-helix called a 3-10 helix has a slightly different hydrogen bonding pattern.
6.4 Beta Sheets

Beta sheets form when two or more polypeptide chain segments either fold back upon themselves
(antiparallel), or line up side by side in the same direction (parallel), to form an interconnected sheet. These
sheets are stabilized by hydrogen bonds between the polypeptide backbone amide hydrogen and carbonyl
groups of adjacent chains. The hydrogen bonds are nearly perpendicular to the extended polypeptide chains.
(1) In parallel β sheets, the polypeptide chains are arranged side-by-side in the same N- to C- terminal
direction. The hydrogen bonds are evenly spaced but slanted, as shown in (A).
(2) Antiparallel chains run in opposite directions with respect to N- to C-terminal directions. The hydrogen
bonds are perpendicular to the strands and the space between the bonds alternates between wide and
narrow, as shown in (B). Antiparallel β-sheets are more stable than parallel β-sheets and there can be
occasional mixing between the two to form pleated sheets.
A B
O R H H O R
R H O R H H O R H H
R H
C C N C C R C C N C C
C N C C N C N C C N C C N
R
H H H
R H H O R H H O R R H O R
O H H O R
R H H R O R O
R H H H H
R R
C C N C C N C C N C C N C C N C C N C C N C C
R
R H H O R H H O R H H R O H H R
R H O R H H O R H H
O R H H O R
C C N C R R H
C N C C C N C N C C
C C N C C N C N C
R H H O R H H O R H
R
R H O R H H
β-Barrels. β-Sheet structures are often skewed from planarity. Human retinol-binding protein is
characterized by its β-barrel domain. Retinol is a light antennae pigment that is used for vision and bone growth.
N-terminus
1 6
8 5
2 7
3
C-terminus 4
CH3
1 5
7
HO
4 3
bound retinol
(Adapted from McKee and McKee, Biochemistry The Molecular Basis of Life (5th ed.). 2012, p. 149; Fig. 5.21.)
6.5 U-Turns
The structure of proline is nearly locked in, but exchanges slowly between cis and trans structures. The cis
form supports formation of U-turns, resulting in a reversal of the direction of the protein chain.
O
C-terminus N-terminus H2
O H C
N-terminus C
C N CH2
C C versus
N α R
C CH2 H Cα C
R H H2
H2C C
H
H2
trans peptidyl proline cis peptidyl proline C-terminus
6.6 Ramachandran Plot

The Ramachandran Plot shows the coordinates corresponding to a variety of protein conformations. Solid lines
indicate the range of commonly observed φ (phi) and ψ (psi) values (as defined on the first page of this chapter).
Large dots correspond to values of φ and ψ that produce recognizable conformations such as the α-helix and the
β-sheet. The unenclosed portions of the plot correspond to values of φ and ψ that rarely or never occur.
Each (Φ, ψ) coordinate pair gives the coordinates for one amino acid. The plot consists of all of the
data pairs for a given protein. It is constructed after the structure has been determined (by crystallography
or NMR analysis). The plot is a fingerprint for the protein structural topography present in the protein
under analysis. It is not used to determine structure. It is a postdetermination characterization tool. One
could, for example, compare the plots of two proteins to get a rapid assessment of the types of secondary
structures in each and their relative populations.
1800
Collagen helix
Antiparallel β sheet
Type II
turn
(residue 2)
α helix
(left-handed)
Parallel
β sheet
ψ 00 Type II turn
310 helix (residue 3)
α helix
(right-handed)
Fully extended chain
-1800
-1800 00 1800
φ
Secondary structures are specific repeating patterns. They occur when consecutive amino acid
residues have similar φ and Ψ values. These patterns occur when all the φ bond angles (rotation angle about
N–Cα) in a polypeptide segment are equal and all the Ψ bond angles (rotation angle about Cα–carbonyl C)
are equal. Because peptide bonds are rigid, the α-carbons are swivel points for the polypeptide chain. If
these angle values are distorted in any way, the secondary structure can be disrupted. The vacant areas in
the Ramachandran plot represent conformations that are rare because atoms would be too close together.
Many amino acid residues fall within the permitted areas on the plot, which are based on alanine as a
typical amino acid. Since there are limits on the values of the φ and Ψ angles, certain amino acids are not able
to form a secondary structure. Proline is restricted to a φ value of about -60º to -77º because it contains a rigid
ring that prevents the N–Cα bond from rotating. By contrast, glycine residues are exempt from many steric
restrictions because they lack β-carbons. Thus, they are very flexible and have φ and Ψ values that often fall
outside the shaded regions of the plot.
Table 1. Ideal φ and ψ values for some recognizable conformations
Conformation φ Ψ
α Helix (right-handed) -57˚ -47˚
α Helix (left-handed) +57˚ +47˚
310 Helix (right-handed) -49˚ -26˚
Antiparallel β sheet -139˚ +135˚
Parallel β sheet -119˚ +113˚
Collagen helix -51˚ +153˚
Type II turn (second residue) -60˚ +120˚
Type II turn (third residue) +90˚ 0˚
Fully extended chain -180˚ -180˚
6.7 Stabilizing Factors

(1) The “Hydrophobic Effect.” This is the key driving force. It involves the balance between two huge
energies. The first is due to the intrinsic entropy of the chain, which always favors unfolding because that state
presents more possible configurations to occupy. The second huge energy is the enthalpy accrued by hydrogen
bonding of water molecules surrounding the macromolecule. A third contribution involves “freeing” the water
molecules that are “peeled away” from the surfaces that interact when two molecules bind each other and
returned to the bulk solvent pool.
(2) Hydrogen Bonding: This is ubiquitous in biomolecules and provides the major contribution to the
enthalpy that drives the “Hydrophobic Effect.”
(3) Disulfide Bonds. They form in an oxidative reaction between the sulfhydryls of two cysteines. The
bonded pair is called cystine. They can scramble and thereby inhibit folding. They were a key focus of the
proof by Christian Anfinsen that structure begets function in enzymes.
(4) Van der Waals Forces. Occur between every atom and every other atom; they are significant up to 20
Å. All pair-wise interactions must be calculated to simulate them.
(5) Dipole-dipole Interactions. Occur between partial charges. In the transient version, the electron density
in the two lobes on the barbell-shaped π orbital can switch back and forth in either correlated or anti-
correlated synchrony, depending on the attractive or repulsive nature of the interaction.
(6) Ionic Bonds. Also called a salt bridge. This is the interaction between two full charged species, for
example, an aspartyl carboxylate group with a lysyl ε-ammonium group. A more relaxed definition
includes charge-dipole atom interactions, such as a Na+ ion with a carbonyl oxygen.
6.8 Thermodynamics of Protein Folding: The Hydrophobic Effect

The primary driving force for protein folding is described as the “hydrophobic effect.” Consider the Gibbs
free energy for protein unfolding, the measurement of the degree of spontaneity (– sign) or lack thereof (+
sign).
ΔGu = ΔHu - TΔSu

where ΔSu is the entropy, which is affected by the number of accessible configurations, ΔHu is the enthalpy,
which is gained by bond formation, and T is the temperature in Kelvin. The gross numbers under the
energy terms are intended to show how the net energies accrue. A more specific plot is described below.
ΔHu - TΔSu → ΔGu
(+2 ) (-1 ) (+1) Folding is spontaneous. The
protein is predominantly folded.
ΔHu - TΔSu → ΔGu
(+1.5) (-2 ) (-0.5)
Unfolding is spontaneous.
Temperature-Dependant Denaturation. Unfolding is more spontaneous due to the increase in temperature,
which affects the contribution due to entropy.
(1) At low T (20˚C) the protein is stable.
(2) At higher T (> 70˚C) the protein denatures (unfolds).
Why? Proteins either fold or unfold depending upon what happens to the surrounding water.
(1) At low temperature, hydrogen bonds among surrounding water molecules are intact. They are an
example of an enthalpic contribution to the ΔG for folding. Note that the contribution is a positive ΔG (+)
for unfolding, since it would be disfavored. The protein always wants to unfold because it becomes more
disordered (entropy), but hydrogen bonds overcome that tendency. The encaged protein folds to a small
volume to maximize hydrogen bonding.
(2) At high temperature, hydrogen bonds are driven apart by more vibrational energy, and so on. The
T is always multiplied by ΔSu and the ever-present tendency to unfold is enhanced. Also, the many released
water molecules gain a lot of entropic energy releasing the folded protein from its cage, allowing it to
unfold. The enthalpy is overcome.
Note that the ∆G values are subscripted “f,” which means they corresponding to folding, not the
subscripted “u” used in the beginning of the section.
(1) folded state (2) at the Tm (3) unfolded state
200
- T∆S f = + 200 + 210 = - T∆S f + 220 = - T∆S f

100
∆G f
0
(kcal mol-1) ∆G f = 0
∆G f = -10 ∆G f = +10
-100
∆H f = - 210 ∆H f = - 210 ∆H f = - 210
-200
Two huge energies that almost Two huge energies Two huge energies produce a
offset each other, leaving a that exactly offset small destabilization energy.
small stabilization energy. each other, leaving Note that when ∆G f = +10 kcal
∆G f of zero. mol-1, ∆G u = -10 kcal mol-1.
6.9 Chaotropes and the Hofmeister Series

Salts such as sodium perchlorate favor protein denaturation. As a result, they are not used to precipitate
proteins, where it is very desirable to maintain the native form. Salts such as NaClO4 and guanidinium
chloride are called “chaotropes” because they disturb the hydration cage around the protein. As a result, the
protein can unfold to the entropically favored denatured form. The ammonium cation and sulfate dianion
actually foster protein folding, so (NH4)2SO4 is called an “anti-chaotropic” salt. The chaotropic salt
guanidinium chloride (GuHCl) is commonly used to disturb the structure of a protein and determine its
relative stability in a quantitative manner. The effect produced by increased [GuHCl] matches the behavior
found in spectroscopic thermal denaturation studies.
The relative propensities of salts to induce denaturation or foster folding were first classified by
Hofmeister in the early 1900s. The experiments involved placing defined protein and nucleic acid
preparations in a broad array of solutions whose cations and anions were varied systematically. Tendencies
of salts to induce denaturation were sorted out and placed on a pair of scales, one corresponding to the
respective tendencies of a series of anions, and one to those of an array of cations. Some cations, such as
sodium and chloride, are only moderately denaturing, Ammonium and sulfate foster folding, so they’re the
preferred choice in precipitation protocols. (Details are described on pp. 198–200 in Hardin et al., Cloning,
Gene Expression and Protein Purification, Oxford University Press, 2002.)
6.10 Sodium Dodecyl Sulfate (SDS): Chaotrope Action

Sodium Dodecyl Sulfate is an amphipathic compound that has a polar head group and a nonpolar alkane
chain. When SDS is present with a protein in solution the polar head groups encapsulate the protein with
the hydrophobic tail positioned to the interior against the folded protein. Because SDS is covering the
protein, the hydrophobic effect is disrupted and the protein is then able to hydrogen bond with the
surrounding solution. This causes the protein to unfold. Note that not all proteins fully denature with SDS.
This reagent is used to convert the proteins to denatured wormlike shapes for analysis of molecular
weights using Polyacrylamide Gel Electrophoresis, as described in that section.
O
+ O S O
O
folded protein SDS-bound

unfolded protein
6.11 Visualizing the Energy Landscape

Polypeptides undergo folding to return to their original condition. Some do so by way of forming
alternatively folded intermediates while others become trapped in an incorrectly folded manner.
The term “molten globule” is used to refer to a partially folded protein intermediate state found during
denaturation within many molecules or their domains. They are collapsed and often have some native,
secondary structure features, but have an interior tertiary structure which contains interactions between
amino acid side chains that are not yet stabilized.
(1) Often with small proteins, folding is cooperative, not involving intermediates.
(2) With large proteins, a molten globule is often formed first followed by the return to its native
formation.
(3) Large proteins with multiple domains often fold separately in each domain, ending with the whole
molecule returning to its native conformation.
other folding C
A pathways
pathway pathway
1 2
Collapse into Alternative

molten Cooperative Molten Globule
formation of all folding pathway
globule Intermediate native interactions
energy wells
native misfolded
protein protein Rearrangement of
partially folded structure
pathway 1 pathway 2 (flipped 180o)

B
larger -
∆G folding
Unfolded
folding alternative folding
intermediates folded intermediates
(Adapted from McKee and McKee, Biochemistry the Molecular Basis of Life (5th ed.), 2012, pp.158–159; Fig. 5.29–
30.)
6.12 Protein Maturation

Making Proteins for Export: The Signal Recognition Process in Translation
The first post-translational processing event occurs while the protein is still being synthesized. Proteins that
are targeted for processing in the endoplasmic reticulum (ER) contain a hydrophobic set of amino acids in
the region located N-terminal to the first amino acid in the mature protein called the signal peptide, pro-
peptide or leader sequence. The following figure shows a schematic of the translation process catalyzed by
the ribosome and using transfer RNAs, the messenger RNA and the signal recognition protein (SRP). The
emerging nascent signal peptide binds SRP, which directs the translating complex to the signal recognition
receptor complex, which removes the signal peptide by proteolysis once the “homing” process has been
accomplished. The remaining protein is extruded into the lumen of the ER.
70S ribosome
aminoacyl
and
peptidyl mRNA
tRNAs
Signal
recognition
protein
ER lumen
SRP receptor
Signal peptidase
Signal peptide Ribophorin
(Adapted from Moran et al., Principles of Biochemistry (5th ed.), 2012, p. 693; Fig. 22.32.)
The Molecular Chaperones

Molecular chaperones assist with the folding and assembly of proteins in living cells. Most are classified as
heat shock proteins (hsp), which are present in plants, animals, and bacteria. They are also found to be
active in eukaryotic organelles such as mitochondria, chloroplasts, and the endoplasmic reticulum.
Molecular Chaperones briefly bind to nascent proteins and unfolded proteins that have undergone
stressful conditions that have led to denaturation. The chaperone hsp70 can aid in stabilizing nascent
proteins (i.e., newly made, yet not fully folded) and reactivating unfolded proteins. Chaperones in the
hsp60 family are beneficial in helping proteins reach their developed conformation. If a protein is incapable
of folding correctly, the chaperones destroy it.
Note that arginine residues line the inside surfaces of the barrel-like multisubunit chaperonin
complex. Recall that the R group of arg is terminated by a guanidinium group. Both guanidinium and urea
disrupt the structure of water. Inside the chaperonin complex, they are thought to “tickle” the water around
the folding protein so that it unfolds and refolds correctly. This solves an essential problem in protein
folding, recovering misfolded proteins and sending them down the correct pathway.
Model of the E. coli Chaperonin Complex GroES-GroEL

GroES (a co-chaperonin, or hsp10) is a seven-subunit ring chaperonin that often works in conjunction with
GroEL (a chaperonin, or hsp60). GroES sits on top of GroEL which is made up of two seven-subunit rings
that are stacked to form a hollow space where protein folding occurs with the aid of ATP.
Unfolded protein
(with bound
hsp70 proteins)
bound protein
GroEL complex (lower ring expands)
(2 stacked rings
containing 7 proteins
each)
GroES cap
n ADP
+ Pi n ATP
hsp60 protein complex

Released GroES-EL Released folded protein (GroES-EL)
(Adapted from McKee and McKee, Biochemistry the Molecular Basis of Life (5th ed.), 2012, p. 161; Fig. 5.32.)
Disordered Protein Binding

Some proteins occur as disordered coil forms, which fold into regular secondary structures upon binding a
suitable “template” binding partner.
For example, the transcription regulatory protein, CREB, binds to DNA and increases or decreases
the transcription of downstream genes. The CREB protein has a phosphorylated disordered domain called
pKID that binds the KIX domain on the transcription co-activator protein CBP. A pair of helices is formed
as the two domains bind and undergo folding.
The following figure shows the process (panel A) and the ordered KIX domain alone in the tube and
ribbon formats, and the complex between the peptide and the pKID domain, in ribbon structure format.
disordered pKID domain KIX domain of CBP ordered pKID

of pCREB bound to the KIX domain
B C
Folded pKID, tube and ribbon structures [pKID-KIX] complex, ribbon structure
(Adapted from McKee and McKee., Biochemistry the Molecular Basis of Life (5th ed.). 2012, p. 155; Fig. 5.27.)
Measuring Reversible Folding and Linking it to Catalytic Activity
Christian Anfinsen demonstrated that the activity of the enzyme Ribonuclease A (RNase A) depends on the
integrity of the protein structure. RNase A can be denatured using 8 M urea and a thiol, a sulfur-hydrogen
bond (R-SH) which reduces disulfides to sulfhydryl groups. The enzyme can be renatured (returned to the
native structure) by simply removing the urea and R-SH group and using air to oxidize the reduced
disulfides.
SH
native active denatured
HS
Ribonuclease SS Add 8 M urea Ribonuclease
HS
HS
and R-SH
SS
SS
Remove urea HS
and R-SH
HS
SH
S
S HS
Other Types of Protein Crosslinks. Desmosine is present in elastin, where it allows for elasticity and
flexibility. Allysine involves cross-linkage between two lysine residues via Schiff base formation. It is
present in both elastin and collagen, a triple-helical protein structure that provides the structural rigidity in
connective tissue.
H H O
A B
R8 N C C R7
R1 R6 R1 R3
H C H
O C H H 3 H C O O C H H C O
H
H C C C C C C H H C C C N C C H
H H + H H N H H N H H H N H
H N 3 4
N
R2 R5 R2 R4
H C H
4
Desmosine Allysine
R3 C C N R4
O H H
Insulin Maturation. The physiological processing of insulin begins in specialized Islet of Langerhans cells
in the pancreas then proceeds through several steps as outlined in the following figure. Blockage of proper
folding is predicted to lead to neonatal-onset Diabetes Mellitus.
pre Preproinsulin C domain

B C A A domain
S
S S
1. Signal Recognition Particle (SRP), S
SRP-Receptor bediate translocation
2. Endoplasmic Reticulum-mediated
pro peptide oxidative folding N terminus
B domain SS
C terminus
A Nascent Insulin
B C
3. Post-Golgi granules: Zinc-stabilized C domain

C domain hexamer assembly
A domain
S
B A Mature Insulin S S
S
4. Release to portal circulation
5. Hexamer dissociation to form N terminus

bioactive monomers
B domain SS
C terminus
(Adapted from Weiss, M. A. (2009) Proinsulin and the Genetics of Diabetes Mellitus, J. Biol. Chem. 19159–19163,
Fig. 1.)
The Signal Recognition mechanism leads to removal of a hydrophobic signal peptide, which directs
the protein from the ribosome into the lumen of the endoplasmic reticulum, where further processing
occurs. Eventually the protein is packaged into a hexameric zinc-containing complex for transport through
the circulatory system. See Weiss, 2009 for details.
Amino Acid Biosynthesis, Catabolic Degradation and Protein Breakdown.

Most biochemistry textbooks contain sections that describe the biosynthesis and breakdown pathways for
the common amino acids. While this information is important, as well as relevant to a number of genetic
diseases, they are beyond the scope of this one semester treatment. Their relation to the Urea Cycle and
protein degradation is described in Section 30.4.
Chapter 7
Ligand Binding and Functional Control
7.1 Oxygen Transport in Blood

Myoglobin and Hemoglobin
Hemoglobin binds molecular oxygen (O2) and transports molecular oxygen from lungs to tissues within red
blood cells. It has a tetrameric quaternary structure with four O2-binding pockets containing one heme
molecule each. Myoglobin binds O2 in muscle tissues.
These proteins provided the basis for our first clear understanding of the relation between protein
structure and how their functions are modulated by solution conditions. This “workbench” was used to
develop and test many fundamental ideas involving how cooperative ligand binding by multi-subunit
ligand-binding proteins controls their functional activity. It is a great example of how physiological and
biochemical levels of function can intercommunicate through allosteric interactions.
Heme. Hemoglobin and myoglobin contain the bioinorganic cofactor called heme (iron-
protoporphyrin IX). Heme consists of a porphyrin macro-ring composed of four pyrrole rings. Reduced
iron (Fe2+) is held by chelation in the center of the macro-ring.
CH2
CH3 CH
HC CH
H3C N CH3
2+
N Fe N
H2C CH
N
HC CH
H2C CH2
CH2 CH3
OOC
CH2
COO
(Adapted from Moran et al., Biochemistry (2nd ed.), 1994, p. 5.28; Fig. 5.34.)
Although the figure shows iron covalently bound to only two nitrogen atoms, iron binds equally to all four
nitrogen atoms. Both myoglobin and hemoglobin bind to molecular oxygen via this chelated iron.
Cofactor Binding. Globular proteins contain cavities or clefts, whose structural components are
complementary to the structure of the specific ligand. These clefts typically contain ionizable residues that
perform crucial functions by participating in the chemical mechanisms of ligand binding. When required
cofactors are bound within a protein it is called a holoprotein. When the cofactors are absent, it is call an
apoprotein.
4 globins + 4 hemes → hemoglobin
apoprotein holoprotein
Myoglobin
Myoglobin is composed of eight helices. The heme group alternately binds iron and oxygen reversibly.
Only the α-carbon atoms of the globin polypeptide and the two histidine residues on the side chains are
illustrated. In order to make the visual clear, one of the heme acid side chains is moved.
The protein is found at high concentrations in skeletal and cardiac muscle and gives these tissues their
characteristic red color. This red coloration resides in the iron atoms of the heme prosthetic groups, where
molecular oxygen molecules bind. Heme consists of a tetrapyrrole ring system called protoporphyrin IX
with an ionic iron bound in the center. The four pyrrole rings of this system are linked by methylene (-
CH=) bridges, so that the entire porphyrin structure is unsaturated, highly conjugated, and planar.
46 Chapter 7 Ligand Binding and Functional Control
50
44
heme
C-terminus
96
56
148
27
117
69
140
O2-binding 84
iron atom
131
78
121
N-terminus 12
Myoglobin
(Adapted from McKee and McKee, Biochemistry The Molecular Basis of Life (5th ed.), 2012, p. 165; Fig. 5.37.)
The protein component of myoglobin is a single polypeptide chain called globin, which contains eight
sections of α-helix. The folded chain forms a crevice that almost completely encloses the heme group. His-
64 forms a hydrogen bond with oxygen, and His-93 is bound covalently to the iron atom within the heme.
Hemoglobin
The protein contains four subunits. Each subunit contains a heme group that binds reversibly with oxygen.
Hemes 1 and 2
+
+
+ + BPG
+
+
+
+
+
+
+
Hemes 3 and 4
The central cavity of hemoglobin contains six positively charged side chains. The N-terminal α-amino
group of each β chain form a cationic binding site. (Adapted from Moran et al., Biochemistry (2nd ed.), 1994, p.
5.50; Fig. 5.62.)
Chapter 7 Ligand Binding and Functional Control 47
7.2 Hemoglobin Oxygen Binding: Cooperativity

Titration Curves Are Ligand-Lattice Binding Curves
So far, we have looked at binding in terms of proton removal from a conjugate base, such as the titratable
amino acid functional groups. The pKa is a benchmark that characterizes the capability of the molecule to
bind the proton. A lower pKa means that a higher H+ concentration is required to jam the proton onto the
molecule.
The standard format for plotting a binding curve places the ligand concentration on the x-axis and the
percentage of the sites on the bound (lattice) molecule that are occupied on the y-axis. This plot is typically
a hyperbolic shape, as shown for the binding of O2 to myoglobin below.
The pH titration plot and the hyperbolic ligand-binding curve contain the same information. Consider
the two-proton/conjugate base binding reaction A2- + 2 H+ ↔ H2A. The pH-titration plot shows the number
of protons removed from the lattice molecule in the two deprotonation steps, with pKas at 2 and 6. Panel B
shows a plot in which the [H+] corresponding to the pH values in panel A are replotted as [H+]. The number
of H+ bound to the conjugate base (the lattice) are plotted on the y-axis. This transformation converts the
pH titration curve into a conventional binding curve. Note that a log scale is used. If the scale is not
logarithmic, the plot would have the expected hyperbolic shape and all of the lower H+ concentration
information would be pushed into the left side of the plot. The semilog presentation retains the advantage
of showing each concentration range with equal emphasis.
A. Titration
Curve :
2
H+
(2 H+ released)
removed
from the
1
A2- lattice
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
pH
1M 10-14 M
H+ H+
Reverse the scale direction
B. Binding
Curve :
2
[2 H+ bound]
H+ bound
to A2- 1
[2 H+ released]
0
10 1 pM 1 nM 0.1 µ M 1 mM 1M
fM (pico) (nano) (pH 7) (milli) H+
(femto)
1 µM 10 mM
(micro)
H+ Plotted on a - log scale

Hemoglobin: Oxygen Transport and Binding Equilibria.

Hemoglobin transports molecular oxygen from lungs to tissues. It is found in red blood cells. The binding
curves for binding of oxygen to myoglobin and hemoglobin are shown below.
Tissues Lungs
1.0
Myoglobin
Hemoglobin
Y
.
(fractional saturation 0.5
of the lattice) P50 = 26
0
20 40 60 80 100
pO2 (torr)
partial pressure of O2
(not - log [O2])
The parameter P50 is the partial pressure of O2 required to achieve 50% occupancy of the binding sites
on the molecule. Of course, myoglobin only binds one O2, while hemoglobin binds four O2s to achieve
100% occupancy. Be clear that we are talking about populations of molecules, not necessarily single
species. The P50 value for myoglobin-O2 binding is 2.8 torr. In contrast, hemoglobin requires a P50 of 26
torr. This shows that hemoglobin binds O2 with a lower affinity than myoglobin.
Biologically, this means that hemoglobin is nearly saturated with oxygen in the lungs, where the
partial pressure of oxygen is high. In contrast, in the tissue capillary beds where exchange between
circulatory and cellular systems occurs, the partial pressure of oxygen is low enough that oxygen is released
from hemoglobin and transferred to myoglobin, which holds it in the cells. This transfer provides an
efficient fuel-supplying system for delivery of oxygen from the lungs to the muscles.
Cooperativity In O2 Binding
Hemoglobin displays a sigmoidal (S-shaped) curve, which usually indicates the presence of cooperativity
in the reaction equilibria. The hyperbolic curve for myoglobin does not. A common reason for multimer
formation is because it introduces the possibility of controlling the differential binding characteristics of
different sites, depending on how much ligand is present.
With hemoglobin, binding the first O2 makes subsequent molecules bind more easily. This is called
positive cooperativity. When O2 binds to the first site, the next binding site changes, making it more
susceptible to ligand binding. The third O2-binding site has approximately the same affinity as the second,
and the fourth has a lower affinity. If each subsequent association/binding constant increased relative to
that of the previously bound site, the system would show purely positive cooperativity. Hemoglobin shows
positive cooperativity in the site 1 to site 2 case, but negative cooperativity in the site 3 to site 4 case. At
low [O2], it picks up O2 fairly efficiently, at higher O2 pressures it tends to try to release the O2. This is
called mixed cooperativity—positive at low [O2], negative at high [O2].
Chapter 7 Ligand Binding and Functional Control 49
BPG Inhibits Binding of O2 to Hemoglobin. Allosteric Inhibition

Hemoglobin binds the compound 2, 3-bisphosphoglycerate (BPG), which is derived from the glycolysis
intermediate 3-phosphoglycerate.
O O O
C
P O
O
H C
.
O 2, 3-bisphosphoglycerate
H C
O O
H P
O O
BPG is an allosteric effector because it binds to the central cavity in the tetrameric complex, a site other
than the oxygen binding site, the active site of this binding protein complex.
(1) When hemoglobin is in the deoxy conformation, the positively charged groups bind to the five negative
charges of BPG. When the BPG concentration is low, hemoglobin has a very high affinity for oxygen. As
with H+ and CO2, binding BPG stabilizes the deoxygenated form of hemoglobin.
(2) When hemoglobin is oxygenated, the β chains are closer together so the allosteric binding site is too
small to bind BPG.
(3) In the absence of BPG, hemoglobin has a higher affinity for oxygen. When BPG is present, the affinity of
hemoglobin for oxygen decreases. BPG increases the proportion of deoxy (T) hemoglobin. The deoxygenated
conformation of hemoglobin is often referred to as the T (taut) state. Oxygenated hemoglobin is said to be in
the R (relaxed) state. Oxygen and BPG have opposite effect on the R ↔ T equilibrium.
(4) Hemoglobin transfer oxygen to myoglobin at the low partial pressures of oxygen in the tissues.
(5) At the high partial pressures of oxygen in the lung alveoli, and in the absence of BPG, hemoglobin
binds oxygen.
The important physiological consequence is that our breathing is connected to our metabolic status.
Oxygen is required as the terminal acceptor of electrons in the Electron Transport process. The allosteric
role of BPG provides a direct link between the status of glucose metabolism and the extent of oxygen
uptake. When more ATP is around, the glycolytic compound 3-phosphoglycerate is converted to BPG,
which limits the amount of O2 bound by hemoglobin. The BPG concentration is an indicator of the status
of the metabolic pathway.
Fetal Hemoglobin
Regulation of hemoglobin oxygenation by BPG plays a unique role in the delivery of oxygen to the human
fetus from the maternal placenta. Fetuses produce a specific hemoglobin called hemoglobin F (Hb F). Like
adult hemoglobin, Hb F is tetrameric and contains two α chains. Instead of the two β chains found in adult
hemoglobin, Hb F contains two γ globins. The γ globins contain serine instead of the His-143 found in β
globins. As a result, Hb F has two fewer positive charges in its central cavity, making fetal hemoglobin
have a lower affinity for BPG. In the absence of BPG, oxyhemoglobin forms more easily. Since fetal
hemoglobin binds BPG poorly, it has a greater affinity for oxygen than adult hemoglobin.
The Bohr Effect.

Christian Bohr, the father of the famous physicist Neils Bohr, won the Nobel prize for his work on the
effect of H+ on the binding of O2 to hemoglobin. He demonstrated that H+ is an allosteric effector, which
like BPG favors deoxygenated hemoglobin.
What Do Allosteric Effectors Change?

In the case of allosteric control of a ligand-protein binding equilibrium, the inhibitor or activator modifies
the binding protein, which changes the Ka of the binding equilibrium.
We will encounter another case, in which an enzyme is affected by an allosteric activator and an
allosteric inhibitor, in the Allosteric Feedback Inhibition: Aspartate Transcarbamoylase section. In that
case, the allosteric effectors change the Michaelis constant (KM) of the catalytic reaction.
Many other examples of allosteric effectors have been characterized. See the book Binding and
Linkage by Wyman, J. and Gill, S. J. (University Science Books, 1990) for a thorough description of this
subject and the mathematical approaches used to model the cooperativity.
7.3 Antibodies: Immunological Recognition

Immunology, Protection and “Self.” Antibodies circulate in the bloodstream and recognize non-self
molecules called antigens. The immune system is broadly divided into two general types called cell-
mediated and humoral. The latter depends on antibodies; the former involves a complex set of proteins
called “complement.” Antibody recognition of foreign antigen leads to proliferation of specialized cells that
make many more of the antibody of that specific type. Also, the bound antibody tags the antigen as being
non-self, which leads to elimination of the antigen by another class of circulating cells called macrophages.
Several different classes of antibodies exist. The most abundant in blood are of the immunoglobulin G
class. This specific antibody is Y-shaped and is composed of four polypeptide chains: two identical light
chains and two identical heavy chains. The heavy chains attach to carbohydrates and the light chains attach
to antigens. Disulfide bonds connect the chains so that the N-termini coincide and create the antigen
binding sites. These sites are important for the antibody to recognize specific antigens.
Structure of Immunoglobulin G. The IgG tetramer is composed of two heavy chains (H) and two light
chains (L) that are linked to each other and the opposite chain by disulfide bonds and noncovalent
interactions. This forms a tetramer with two identical halves that form a Y-like shape. Each chain contains
a constant (C) region and variable (V) region composed of β-barrel domains. Each constant domain gains
structure from its disulfide bridges. The two variable domains exist on the end of the fork and contain an
identical antigen binding site. A variety of foreign antigen proteins bind to the outer surface of the sites.
N-terminus 2 N-terminus 3
Antigen Antigen
binding VL VL binding
site 1 site 2
N-terminus 1 CL CL N-terminus 4
VH VH
C-termini C H1
CH1 2 and 3
C-terminus 4
C-terminus 1
C H2 C H2
C H3 C H3
C-terminus 1 C-terminus 4
(Adapted from McKee and McKee, Biochemistry the Molecular Basis of Life (5th ed.), 2012, pp. 152; Fig. 5.25.)
Antibody Repertoire and Diversity. Genes that encode antibodies undergo a complex rearrangement
process called hypervariable switching. The resulting antibodies formed after transcription and translation
have a wide variety of antigen-binding sites. This process leads to an adapted immune response by allowing
recognition and removal of foreign molecules that cover a wider variety of structures. They are a diversified
form of protection.
Chapter 8
Enzymes
8.1 Enzymes are Biological Catalysts

Purpose and Definitions
(1) Catalysts increase the rate of reaction by lowering the activation energy required to cross the transition
state barrier.
rate = velocity = activity
rate = k1 c (1)
The lower case ‘k1’ is the rate constant for a simple one-way (irreversible) reaction and c is the reactant
concentration in a simple irreversible reaction. It describes the relation between the rate and reactant
concentration. Larger c leads to a larger rate. Enzyme-catalyzed reactions involve a more complicated
scheme, which is captured in a generic reaction scheme called the Michaelis-Menten model.
(2) Reactants are called substrates in biochemistry. The overall goal of biochemical kinetics studies is to
characterize the rate of product formation at different substrate concentrations.
(3) Enzymes are very specific and produce high yields. One product is typically made, which is usually
stereochemically pure (a single chiral isomer).
Classification
The suffix used to designate an enzyme is -ase. The following classes have been defined:
(1) Oxidoreductase. These enzymes catalyze reactions involving redox changes. NAD+/NADH and
FAD/FADH2 are common cofactors.
(2) Transferase. A-X + B ↔ B-X + A The X group is commonly phosphate (Pi), which is often
transferred from ATP. Kinase reactions catalyze many crucial regulatory processes. Substrates range from
small molecules to proteins. GTP, UTP and CTP are also used as energy sources in the construction of
biomolecules, e.g., proteins, carbohydrates and lipids, respectively.
O
O P O
O
(3) Hydrolase. These reactions break a bond by adding water. Examples include protease, nucleases and
lipases.
(4) Lyase. This involves adding a molecule to a double bond, without breaking the bond.
The term synthase is used. (Note that ligases are synthetases, not synthases.)
(5) Isomerase One reactant → one product This involves moving a group to another site within a molecule.
They are also called racemases.
(6) Ligase This class typically require ATP to drive the reaction. They are called synthetases. (Lyases are
synthases.)
52 Chapter 8 Enzymes
Table 1. Enzyme Class and Subclass Definitions

Class Name Reaction Properties Enzyme names
1 Oxidoreductases A(ox) + B(red) ↔ Involving oxidation and Dehydrogenase
A(red) + B(ox) 1 reduction pairs; usually have Oxygenase
NAD/NADH or Oxidase
FAD/FADH2 Peroxidase
Reductase
2 Transferases AX + B ↔ A + BX Transfer of one group from Trans-
one molecule to another Transferase
Kinase
3 Hydrolases AB + H2O ↔ A + B Addition of water to cleave Many different names
a double or single bond
4 Lyases A (=) X + B ↔ A=B Addition to a double bond Synthase

or cleavage to yield a double
bond
5 Isomerases A↔B Interconversion of two Isomerase

isomers; always reversible; Racemase
not limited to stereoisomers
6 Ligases A + B + NTP ↔ AB + Joining two molecules Synthetase

NDP + Pi or AB + NMP together with input of
+ PPi energy from nucleotide
triphosphate
1
Note that the symbol ↔ is sometimes used in this book to indicate a reversible reaction. The more
commonly used symbol involves two half-headed arrows in opposing directions.
k1
. Aox + Bred Ared + Box (2)
k -1
A mnemonic can be used as an aid to remembering these classes:
Example used to remember the enzyme classes: O-T-H-L-I-L
Other Teachers Have Lied In Litigation.
8.2 Enzyme Function: Activity Assays and Enzyme Kinetics

Overview: Kinetics in Three Steps
The purpose of the Michaelis-Menten approach to kinetic analysis of enzyme-catalyzed reactions is to
determine how the velocity of the reaction depends on the concentration of added substrate [S]. The rate of
the reaction varies from zero to the value of the maximum velocity (Vmax). The midpoint of the trend is
characterized by the Michaelis constant (KM).
Curve 1: The Progress Curve. Dependence of product formation on time. One measures the rate as the
slope. The slope changes as the amount of reactant dissipates. The key misconception students have is
captured in the following question:
Q: Why does the measured rate/velocity (V) decrease to zero at lower [S]?
The naïve idea many reach is that [S] decreases to zero, so the rate becomes 0. This not true and highlights
a key point that is central to understanding the benchmark-like nature of KM.
Chapter 8 Enzymes 53
The correct answer is that [S] has decreased to less than ~1/20 the value of KM. This occurs because too
little substrate remains to drive the mass action requirement for catalysis to occur.
If KM is a large [S], the physiological concentration remaining when the enzyme turns off can be
significant, i.e. 50 µM for a KM of 1 mM.
V0 = the 'initial' slope
Tangental definition, approaching the maximal initial slope:
d[P]
equilibrium product V0 = "initial velocity" = limit (3)
concentration dt
t 0
[P]
Two-point approximation method, which must be used with a
(M) judicious choice of points to capture the real slope.
poorly chosen
"two-point slope"
time interval to V0, two point = [P]
fit the data t
0 time (sec) [P]2 - [P]1
=
t2 - t1
Curve 2: The logarithmic Michaelis-Menten Plot. Dependence of V0 on the logarithm of the Substrate
Concentration.
Note that the non-logarithmic version of this plot has a hyperbolic shape. Do not confuse the
sigmoidal nature of the logarithmic plot with the situation in which sigmoidicity appears and the x-axis is
[S] not log [S]. In that situation, sigmoidicity is typically an indication that the catalytic mechanism
involves significant cooperativity.
V0 = Vmax
V0
V0 = 0.5 Vmax
(M/s) [S] = KM
V0 = 0
log [S]
(M)
Note: each circle is drawn from the data of a different progress curve.
Vmax is the "maximum velocity," which is the fastest possible initial velocity under the defined conditions.
V0 reaches the limiting velocity Vmax.
Vmax [ S ]
. Michaelis-Menten
V0 = Equation (4)
KM + [ S ]
The equation reproduces the following intuitive behaviors.
(1) At low [S], the [S] is << KM. In this case, V0 ~ (Vmax/KM)[S]. Doubling [S] doubles V0. This is called a
first-order kinetic response.
(2) At high [S], the [S] is >> KM, and V0 ~ Vmax..

Comparing a Michaelis-Menten Plot to a Standard Titration Curve

It is instructive to point out the similarities in the pH-dependence plot and the logarithmic version of the
Michaelis-Menten plot. In each case, the x-axis is a function of a concentration and the y-axis is the
variable that is affected by the change in concentration. In the H+ titration plot, the number of protons
bound ranges from 0 at low [H+] to 1 at high [H+]. In the logarithmic version of the Michaelis-Menten plot,
one sees the same sigmoidal behavior, but in this case V0 varies from 0 to Vmax. If one divided V0 by Vmax,
the y-axis would also vary from 0 to 1. In each case the benchmark at the center of the transitional action is
an equilibrium constant. Note that plot B is an inverted pH titration curve, that is, an H+-binding curve (see
Section 7.2). It plots the number of protons bound, not the number removed, for three representative amino
acid R groups.
A. Michaelis Menten Plot
Vmax
V0 0.5 Vmax
(M/s)
[S] = KM
log [S]
B. Amino Acid R-groups: Representative Binding Curves (i.e. Inverted pH Titration Curves).
cys asp
Number of his
0.5 R-S-H R-COO-H
R-N-H
H+ bound pKa = 8.1 pKa = 3.9
pKa = 6.0
10 1 pM 1 nM 1 mM 1 mM 1M
fM
pH 7
log [H+]
Curve 3: The Lineweaver-Burk Plot (The Double-Reciprocal Plot):

(1) 1/V0 is plotted versus the corresponding 1/[S] values as data pairs : (1/V0, 1/[S]).
(2) This provides an analytical method to calculate the Vmax (i.e. 1 divided by the y-intercept) and the KM
(i.e., Vmax times the slope).
One determines the properties of the substrate in the enzymatic reaction by observing how changes in
[S] affect the rate of product formation. This is like feeling inside a black box to determine the properties of
what’s inside. Or, more precisely, like putting something into a tube and determining what’s inside based
on what comes out the other end.
Consider the following two-step reaction, the generic reaction corresponding to the situation governed
by the Michaelis-Menten treatment:
"Catalytic rate
constant "
k1
k cat
E + S ES E + P
k -1
Vmax
.
kcat = (units: seconds-1) (5)
[ E ] tot
The units of the quantity 1/kcat correspond to time. This time corresponds to the period required to complete
one catalytic event.
The Steady-State Kinetics Reaction Technique

The “steady-state approximation” was developed by Briggs and Haldane. It postulates that the rate of ES
formation is equal to the rate of ES consumption. This simulates the situation in which a constant supply of
substrate is supplied to the metabolic system. It also assumes that no intermediate compounds are
accumulating.
Note that the Briggs-Haldane “steady-state” approach is very different from the Michaelis-Menten
method of running the reaction, in which a specified amount of substrate is supplied only at the beginning,
then the reaction is allowed to occur until the rate decreases to zero. This occurs because [substrate] is well
below the value of KM. Note that although it is very low it is not zero.
If the [ S ] is low: V0 = k1 [ E ] [ S ]
By definition, Vmax = k cat [ E]tot, so:
k cat [ E ]tot [ S ]
. V0 = (6)
KM + [ S ]
The denominator approximates
KM at low [ S ].
kcat
V0 = [ E ]tot [ S ] (at low [ S] )
KM
The ratio kcat/KM is called the "specificity constant." It gives a quantitative way to make
the following decisions:
(1) Which of a set of different substrates does a given enzyme prefer?

(2) Which of a set of enzymes most rapidly produces product from a single target
substrate in cross-referenced tests?
Diffusion and Geometric Considerations of Mutual Approach

Catalysis is limited by the maximal rate of possible diffusion of E and S together. The rate constant for
diffusion-limited movement is around 108 to 109 M-1 s-1 for most biomolecular interactions. This is a
second-order rate constant, signifying the fact that two entities must meet to produce a productive
encounter.
"Active Site"
cleft
approaching/diffusing
substrate molecule
nonproductive
approach
Increasing target size
The Lineweaver-Burk Equation: The Double Reciprocal Plot
Vmax [ S ]
Michaelis-Menten
. V0 = Equation .
KM + [ S ]
Taking reciprocals:
1 KM + [ S ] KM 1 1 [S]
. = = + (7)
V0 Vmax [ S ] Vmax [S] Vmax [ S ]
=1
The corresponding
linear equation is: y = m x + b
(slope) (y-intercept)
Plot the (1/[S], 1/V0) data pairs as follows to determine KM and Vmax:
slope = KM Vmax
1
V0
Allows
(s M-1) measurement of
KM & Vmax
1
KM
1 Vmax
0
1 [S] (M-1)
0
(larger [S])
8.3 Requirements for Catalysis

(1) The catalyst must be regenerated in the same form following catalysis.
k1 kcat
E + S ES E + P
k -1
(participation in another
round of catalysis)
(2) It does not change the equilibrium free energy difference. It lowers the activation energy (Ea), the
energy required to form the activated complex and cross the transition state barrier.
(3) A product must form (E + S → ES → E + S is a “futile cycle”).
The Reaction Coordinate. This diagram characterizes the energies corresponding to each progressive state
as the reaction proceeds.
k1 kcat
E + S [E S] E + P
k -1 (product)
(reactant)
uncatalyzed
transition state
Ea
(forward
Energy direction) catalyzed
Ea
reaction (reverse
direction)
reactants
G products
Transition State Activation Energy. The activation energy in the forward direction controls the rate of the
forward reaction, which occurs while reactions are also going in the reverse direction. The ground state
Gibbs free energy (ΔG) characterizes the degree of spontaneity, and poise, of the reaction. As typically
defined, negative ΔG values correspond to spontaneous reactions in the direction of product(s) formation,
however one defines it. Positive ΔG values indicate reactions that favor flow in the reverse direction,
toward the reactant(s).
ΔG = -R T ln K,
where ΔG is the key thermodynamic parameter, R is the universal gas constant, the temperature is in
Kelvin, and K is the equilibrium constant.
Table 2. Enzyme Kinetics Plot Summary
Plot Number Plot Parameters Purpose
x y
1 Progress Curve Time [P] Experimental V0 determined
2 Michaelis-Menten Plot [S] or log [S] V0 KM & Vmax intuition
3 Lineweaver-Burk Plot 1/[S] 1/V0 a. Calculating KM & Vmax
b. Noncovalent inhibition patterns
8.4 Comparing Enzymes and Relative Efficiency of Use of Substrates

Uses of the Specificity Constant. If KM is a larger value, for example, in the millimolar range, a large
amount of substrate (in biological terms) is required to achieve the same rate.
Two types of comparisons are important to consider when, for example, trying to determine the
usefulness of a new pharmaceutical candidate:
(1) One can compare two enzymes that can use the same substrate.
E1
S
E2
(2) Alternatively, one can compare how well two variant substrates work with one type of enzyme.
.
E
S1 P1
same E
S2 P2
Efficacy is a central concept in the pharmaceutical industry. It is a measure of two opposing characteristics
of a prospective treatment method (type of molecule). One wants to maximize the effectiveness of action of
the drug/approach, yet many very strong drugs have one or more such negative side-effects that the drug is
not considered useful. A highly efficacious drug will give an effective result with minimal side effects and
downsides.
The comparisons described here encompass a large percentage of the types of problems researchers
want to understand when they quantify the relative efficiencies of two drugs with respect to each other.
Another scenario occurs when a given pharmaceutical interacts with two alternative enzymes. For example,
one might need to compare results obtained with two contrasting patients who have a genetic difference
that leads to vastly different amounts of a key enzyme, which is required to metabolize the drug properly.
8.5 Drug Design

Table 3. Design and Discovery of Ligands That May Be Strong Pharmaceutical Lead Compounds.1,2
Step Activity Purpose
1 Target selection and validation Informed by medicinal chemistry experience,
Protein (or nucleic acid) identified genome data, and comparative bioinformatics,
chemical validation, gene KO, or RNAi.
2 Target characterization Obtain recombinant source of target, purify, and
3D structure determined, drug ability develop appropriate assays for biochemical and
assessed kinetic analyses, crystallize, and determine the X-
ray or NMR structure. Orthologues and
construction of homology models are considered.
3 Ligand/inhibitor studies Exploit the structure in VS, identify ligands from
Binding and inhibition data (hit discovery) HTS or focused screens, fragment screening by X-
ray or NMR methods, and so on utilize de novo
design, seeking to discover appropriate chemical
scaffolds.
4 Target-ligand structure determination Derive accurate structures of target-ligand
Elucidate feature that determine affinity complexes by X-ray/NMR methods, alternately
and selectivity (Initiate hit-to-lead employ docking calculations
progression)
5 Design chemical modification to ligands Molecular modeling of scaffold modifications
Improve target affinity and develop SAR seeking to enhance affinity for the target or address
for compound series (expand to lead potential ADMET issues, bioavailability, and so on,
series) docking calculations can assist modeling
information and compounds obtained here can feed
back into an iteration of further characterization and
design.
6 Lead series developed, cell-based assay Testing against whole cells and elucidation of
and ADMET studies commence efficacy in a disease model. ADMET investigations
Investigate biological/pharmacological are carried out.
activity to select preclinical candidates
1
Adapted from Hunter, W. N. (2009) Structure-based ligand design and the promise held for antiprotozoan drug
discovery, J. Biol Chem. 284, 11749–11753.
2
Compounds acquired in Stage 5 go into Stages 3 and 4 in an iterative process. ADMET, adsorption, distribution,
metabolism, excretion and toxicity; HTS, high-throughput screening; KO, knock-out; NMR, nuclear magnetic
resonance; RNAi, RNA interference; SAR, structure-activity relationships.
8.6 Enzyme Inhibitors

Reversible Inhibition
Inhibitors are used to investigate the mechanism of an enzyme and decipher metabolic pathways. Natural
inhibitors can be used as regulators, and constitute the mechanism of many medicines, toxins and
pesticides.
Relative capabilities are characterized by the inhibition constant (KI). For the reaction:
E + I ↔ [E•I]
KI = ([E] [I]) / [EI] (8)
Types of Reversible Inhibition.
Competitive Inhibition. (The inhibitor (I) binds free E and competes with S for the same site.)
E is subjected to direct competition between reactions with true and false “substrates” S and I. KM
increases, Vmax remains the same. Increasing KM makes sense since more [S] is required to attain the same
catalytic rate.
more I
1
E + S [E S] E + P V0
+ (s M-1)
I no I added
KI Vmax doesn't change
[E I] 0
1 [S] (M-1)
0
1
KM
Note that this -1

x-intercept =
indicates that [I] )
KM increases. KM (1 +
KI
Modified Michaelis-Menten Equation:

Vmax [S]
V0 = (9)
[I] )
[S] + KM (1 +
KI
Uncompetitive Inhibition. In this case, I binds the ES complex. Binding of S to E changes E by creating a
site for I to bind. I traps S in the enzyme. Both KM and Vmax decrease. Note that this makes sense with
respect to Vmax, but the decrease in KM is anti-intuitive.
more I
1
E +S [E S] E + P
V0
+
[I] (s M-1)
I (1 + )
KI'
slope = no I
Vmax
] E S I]
Vmax decreases
1 [S] (M-1)
1 KM
-1
x-intercept =
[I]
KM + [S](1 + )
KI'
The modified Michaelis-Menten Equation is:
Vmax [S]
V0 = (10)
KM [I]
+ [S] (1 + )
KI'
KI'
where ES + I IES.
Noncompetitive Inhibition. Here, I binds both E and ES. Note that S binds EI to create a cyclic connected
set of equilibria. Two separate sites for I and S exist. Binding of I changes the conformation of E. The KM
does not change, but Vmax does. Note that the product can be an inhibitor if the catalytic step is reversible
(by mass action).
more I
E + S [E S] E+ P
+I +I 1
V0
(s M-1)
no I
[E I] +S ] E S I]
Vmax decreases
Note: S binds [EI] too.
1 [S] (M-1)
Vmax [S] 1
V0 = KM
[I] 1 + [I]
KM (1 + )+
KI' KI' + [S]
1 [I]
y-intercept = 1 +
Vmax KI'
Irreversible Inhibitors. These are generally the result of a displacement reaction, with covalent bond
formation.
k1 >> k -1
E + I X E I + X
leaving group
Acetylcholine Esterase. Inhibitor and Antidote Actions

Nerve conduction involves the following reaction, which is susceptible to inhibition by an organic
fluorophosphate compound:
O
CH3 C O (CH2) 2 N (CH3) 3 messenger

diffuses to receptors
Acetylcholine across the neural gap
junctions
H2O
acetylcholine
esterase
H
O
CH3 C O + HO (CH2) 2 N (CH3) 3 inactivated messenger
resting state
acetate choline
.
This reaction is involved in the relay of impulses between nerves, and nerves and muscle cells.
.
1. Normal Reaction δ-
O
(CH3)3 N CH2 CH2 O C CH3

δ+
acetylcholine
O H
enzyme
anionic esteratic 'active site'
site site
2. Nerve Gas - diisopropylfluorophosphate (DFP); a "suicide substrate" or "dead-end inhibitor"
i-propyl O i-propyl i-propyl O i-propyl

O O O
P O
δ+ P
F
(inhibited esterase)
H O O
3. Antidote - pyridine aldoximine methiodide (PAM)
H
PAM
OO i-pr
+N CH N O removed
(reactivated esterase)
I i-pr
CH3 δ+ P O H
O
O H+
AZT (3’-azido-2’, 3’-deoxythymidine). This drug inhibits reverse transcriptase, which synthesizes DNA
from viral RNA during infection by HIV (see Section 2.4.2). It was the first widely used treatment for
AIDS, which has been replaced by protease inhibition-based approaches.
O
HN CH3
H H O
This very reactive electrophile, N H
HO O
which replaces the hydroxyl H H
group, inhibits the normal active H H
site nucleophile of the HIV .
N H
reverse transcriptase. N
N
Methotrexate. This drug inhibits dihydrofolate reductase, which is required to make the DNA precursor
dTTP. This inhibition selectively kills rapidly dividing cells, such as cancer cells. This technique is used as
a successful cancer chemotherapy agent, for example, childhood leukemia and autoimmune diseases such
as rheumatoid arthritis and Crohn’s disease.
folate para-amino benzoic acid

derivative (PABA) glutamate
H2N N N O COO
N CH2 N C N CH
N
NH2 CH2 CH2 COO
CH3 H
Bisubstrate Enzyme Kinetics

The Michaelis-Menten method can be modified and used to study more complicated enzyme-catalyzed
reactions, for example, two- and three-substrate reactions. The following generic reactions show two ways
in which the reactants can bind to the enzyme. The horizontal line represents the enzyme surface; the state
of the enzyme is indicated.
Sequential Reactions
(1) Ordered: Reactants bind in a specific order; products dissociate from the enzyme in a specific order.
A B P Q
EAB EPQ
EA EQ
E E
(2) Random: Substrates can bind in either order; products can dissociate in a specific order.
B P
A EA EQ Q
E EAB EPQ E
B
EB EP P
A Q
One determines KM values with respect to the concentrations of each substrate. For a given substrate, the
other substrate is held at a constant concentration. The analysis involves obtaining data then plotting 1/V0
versus 1/[A], at constant [B], and 1/ V0 versus 1/[B], at constant [A].
The Ping-Pong Reaction

A general scheme is shown below:
A P B Q
E EA FP F FB EQ E
This is the modified form of enzyme (E), with the amine

group bond to the PLP coenzyme: F = E - X.
(1) The EA transformation to form FP occurs as follows. The X portion of A (= PX) is attached to E, which creates F
(i.e. E-X) and the P portion is released. The A species leaves X behind.
(2) Aspartate Transaminase creates aspartate from oxaloacetate. The enzyme form designated as F is bound to the
coenzyme pyridoxal phosphate (PLP), which binds and holds the amino functional group that is subsequently transferred
to the proto-Cα atom of oxaloacetate to form aspartate.
A-NH2 'P' = A-C=O 'B' = Q-C=O Q-NH2
Glutamate α-Ketoglutarate Oxaloacetate Aspartate
E EA FP F FB EQ E
E + A-NH2 F = E-PLP-NH2 F = E-PMP E + Q-NH2
+ P Enzyme-bound
Pyridoxamine phosphate
Enzyme-bound P=A B=Q Amino group transferred,

Pyridoxal phosphate without -NH2 without -NH2 E -PLP regenerated.
(E -PLP)
Variations of this mechanism are (1) used in two different compartments in the Malate-Aspartate
Shuttle, by two different enzyme varients, and (2) to produce pyruvate from alanine, which provides fuel
for the Krebs Cycle.
Kinetic Regulation: Principles
Zymogens and Proteolytic Activation

Proenzymes are inactive precursors that are activated by removal of a peptide, which is an irreversible
covalent modification. Four examples of pancreatic digestive enzyme are:
Chymotrypsinogen → Chymotrypsin
Trypsinogen → Trypsin
Proelastase → Elastase
Procarboxypeptidase → Carboxypeptidase
The proenzymes on the left are activated by proteolysis to produce the active enzyme on the right.
Enzymatic Cascades
This involves a catalysis-mediated increase in number of enzymes that occurs at progressive stages in a set
of proteolysis reactions. At each step, a new protease proteolyzes a new protease precursor. This produces
the activated protease with the next required specificity to catalyze the next step in the cascade. The net
effect is to produce a very large number of enzymes, greatly amplifying the effect of the initial single
proteolysis step. In stage 1, the initiator makes a large number of activated enzyme, each of which activate
a large number of enzymes in stage 2. Each stage produces a multiplicative number of enzymes, leading to
a huge response, initiated by the single initiator enzyme.
one
initiator Many activated enzymes
E1 (by proteolysis to form the active zymogen)
nA n A* Many, many activated enzymes

(proteolysis) (amplified)
stage 1
inactive
proteases n x m B n x m B*
stage 2
The 'n' active enzymes A*
catalyze activation of
(further
'm' enzymes B to form
amplification)
'n x m' enzymes B*.
Examples:
(1) Blood Clot Formation (Coagulation): The reaction involving an enzyme called
Factor IX is missing in hemopheliacs, so their blood will not clot properly.
several Factor
cascade
stages
Factor
prothrombin thrombin
proteolysis
Fibrinogen Fibrin
clot
(2) Blood clot dissolution (breakdown): Used to dissolve and prevent clots in stroke victims.
several
cascade
stages activators
plasminogen plasmin
Fibrin degraded Fibrin

Note that zymogens need not be enzymes. For example, fibrinogen is the precursor to the structural
protein that forms the “logjam” in the clot, fibrin. Cascades occur in many biochemical processes.
(3) A third example of a cascade regulates the cell-mediated immunity system. The key reactions are
catalyzed by a set of proteases called complement.
Feedback and Feed-forward Regulation

Feedback inhibition occurs when the product of a set of linked reactions binds to an enzyme, or other
functional protein, in an earlier step in the pathway, thereby inhibiting the downstream reaction it was
created by and intervening set of reactions. Regulation is often controlled by allosteric effector molecules.
The related concept feed-forward activation involves stimulating a later enzyme, or other functional
protein, in a pathway by formation of sufficient amounts of a particular activator molecule produced by an
earlier reaction in the pathway. This serves to increase the flow by activating the later step and thereby
drawing reactants through the linked pathway.
8.7 Allosterism
In this mechanism, a regulatory effector, which is usually a small biomolecule, changes the activity of an
enzyme or binding protein (or nucleic acid) by binding to a site other than the active site. The result is
either an increase, or decrease in the capability of the protein to do its job.
(1) The effector can either increase or decrease the rate of catalysis. This is done by changing either the KM
(or Vmax) of the affected enzyme. Recall that the KM is a measure of the concentration of substrate that must
build up and be present to get a reaction to proceed.
(2) In the case of the binding protein, the effector increases or decreases the binding constant of the protein.
This increases the constant itself, which normally does not change.
(3) Note that allosterism is a separate concept from feedback inhibition or feed-forward activation. An
allosteric effector might lead to these effects; however, it might act on an entirely different metabolic
pathway/scenario. In addition, feedback inhibition or feed-forward activation might be produced by a
nonallosteric route, such as direct binding of an inhibitor to the active site, or recruitment of a third player,
such as a kinase or phosphatase, and so on.
Cooperativity and Induced Fit

Approaches used in this field have been developed in two main directions, involving mathematical models
that handle either a serial or parallel consecutive buildup of the allosteric effect. A serial approach is
incorporated into the Monod-Wyman-Changeaux Model of allosterism. The rate or binding constants
increase, or decrease at each consecutive linear sub-reaction step in the overall process. The degree of
cooperativity is quantified as the percent buildup, or decrease, in activity. It is calculated as the ratio of
binding constants, or rate constants, for two consecutive steps. Positive allosterism means this ratio is
greater than 1; negative allosterism means that the ratio is less than 1. The parallel approach, which is
incorporated into the induced fit model differs in that the buildup is affected by all possible parallel, but
linked, reactions.
Both approaches “model” a fit to the data, revealing evidence for cooperativity among the consecutive
(and flow-linked) events. For example, controlled switches between the “tense” (T) and “relaxed” (R)
states occur when O2 binds to the four ligand binding sites in the tetrameric lattice protein hemoglobin. The
R state occurs has an intrinsically higher affinity for O2 than the T state. This allows for a large range of
binding timescales, producing usage rates that vary over a broad range of O2 requiring activities. The ligand
concentration controls its own binding affinity to the lattice.
(a) R ↔ T: fast (seconds) changes in activity; (b) Covalent changes: slow (minutes to hours).
Allosteric Feedback Inhibition: Aspartate Transcarbamoylase

This reaction regulates the de novo synthetic pathway of the pyrimidines, that is, the uridine-, thymidine-
and cytosine-containing nucleotides. This highly controlled enzyme consists of 12 subunits. Six are
catalytic and six are regulatory, with a number of binding sites for different allosteric effectors.
NH2 COO
COO
δ+ CH2 ATCase
CH2
O C H 2N
H
C CH
O C
NH2 COO O N COO
Pi
PO32- H
carbamoyl phosphate aspartate carbamoyl aspartate
KM
increased (-) KM
(inactivation) (+) decreased
(activation)
UMP
2 ATP
amination
2 ADP
CTP UTP
The enzyme catalyzes the first committed step in the pathway, a common site of regulation. A series of
enzyme-catalyzed reactions produces uridine monophosphate (UMP), uridine triphosphate (UTP) and
cytidine triphosphate. Since UTP can be used to make thymidine triphosphate, this pathway leads to all of
the pyrimidine nucleotides.
Allosteric inhibition occurs when sufficient cytidine triphosphate (CTP) is produced to allow binding
to the allosteric inhibitor binding site on ATCase. This regulates against overproduction of CTP. The
pyrimidine nucleoside triphosphate binds ATCase. In this case, the enzyme is allosterically activated.
Thus, if ATP is present, CTP synthesis is promoted. This offsetting pair of effectors serves the grand
metabolic purpose of balancing the production levels of pyrimidines and purines.
The general effect of allosteric regulators on the KM is shown for the following cases: (1) activator
present, (2) no effector added, and (3) inhibitor present. Note that in some enzyme systems Vmax changes
instead of KM.
Vmax
Activator S P
V0 KM (activated)
Catalytic 0
active site log [S] (M)
Allosteric
activator Vmax
S P
binding
site V0 KM (no effector)
0
Allosteric log [S] (M)
inhibitor
binding site
Vmax
S P
Inhibitor V0 KM (inhibited)
0
log [S] (M)
Experimental results demonstrate that aspartate transcarbamoylase by is activated by ATP and

inhibited by CTP.
Vmax
2 mM ATP
V0 0.5 Vmax
.
(M s-1)
no effector
0.5 mM CTP
0
0 5 10 15 20
[aspartate] (mM)
(1) The results show how the V0 of the ATCase-catalyzed reaction depends on aspartate concentration, one
of the two substrates. The other substrate, carbamoyl phosphate, was held at a saturating concentration in
each set of measurements. Conditions are labeled on the plot.
(2) Note that the effect is subtle. The KM values only change by a few mM, yet a fairly large change in activity
occurs. For example, compare the V0 values at 5 mM aspartate: (i) no effector present, 40% of Vmax, (ii) 0.5
mM CTP, 15%, and (iii) 2 mM ATP, 50%. Both activation by ATP and inhibition by CTP are verified.
8.8 Phosphorylation and Dephosphorylation

These reactions are catalyzed by kinase and phosphatase activities, repectively. Kinases are transferases,
phosphatases are hydrolases. This ubiquitous mechanism of metabolic regulation; is very commonly
employed as an on-off switching technique. The serine hydroxyl group is typically phosphorylated by
kinases; threonine and tyrosine are also common substrates. Phosphorylation often leads to activation of
enzyme activity. However, in some cases the opposite is found. One example is the enzyme pyruvate
dehydrogenase. To illustrate some aspects of phosphorylation-dephosphorylation, four different situations
that lead to regulation of some key aspects of metabolism are described below.
Pyruvate Dehydrogenase
This enzyme complex releases acetyl coenzyme A to fuel the Krebs Cycle. It is controlled by two
counteracting enzymes, a kinase and phosphatase (circles 1 and 2)
Glycolysis:
pyruvate Pyruvate dehydrogenase (PD) 1 Phosphorylated

(dephosphorylated) ADP PD
ATP PD kinase
CoA-SH O
E O E O P O-
H O-
H2 O
(active) 2 H 2O (Inactive)
Pi
PD phosphatase,
acetyl CoA Ca2+ cofactor
CO2
Kreb's cycle Note: This is not the typical logic. Phosphorylation more often
results
in activation.
ATP activates PD Kinase. This is a form of feedback inhibition.
See the Pyruvate Dehydrogenase entry in the Glycolysis section for detailed mechanistic features,
including the roles of five different coenzymes to accomplish the net reaction.
Glycogen Phosphorylase
This reaction controls the release of glucose-1-phosphate from glycogen stores in the liver.
conformational
change
GP ADP
ATP kinase 2- arg
arg
O Pi arg active
OH arg site
active
site GP
phosphatase
Glycogen Phosphorylase b Glycogen Phosphorylase a
(1) Inactive form (1) Activated form
(2) No salt bridges (2) Two salt bridges
Glycogen α-D-Glucose-1-phosphate
breakdown
(released)
Glycogen Synthase
Gluconeogenesis Glycolysis
Glycogen Synthase—catalyzes production of glycogen, which offsets the effect produced by GP kinase.
Isocitrate Dehydrogenase
This enzyme is a key step in the regulation of the Krebs Cycle. Catalysis is inhibited by phosphorylation
because the dianionic phosphate repels the trianionic isocitrate from the active site.
From the COO COO COO

Krebs Cycle: CH2 CH2 NADH, H + CO2 CH2
NAD
HO C COO H C COO CH2
CH2 HO C H Isocitrate C O
Aconitase
Dehydrogenase
COO COO COO
Citrate 2 Rearrangement Isocitrate 3 First oxidative α -Ketoglutarate
decarboxylation
COO O
COO
O H CH2 O P O
H COO O CH2
C
H C COO
HO C H
HO C H
COO
Charge
COO
repulsion
Unmodified Phosphorylated
Isocitrate Dehydrogenase Isocitrate Dehydrogenase
(See Regulation in the Krebs Cycle section for the full context and further details.)
Cyclin Kinase
This system controls the “checkpoint” for entry into mitosis (and exit from S Phase) in yeast as well as
most organisms. It is “very, very, very” complicated according to the cited reference. The following
scheme shows the interconnected reactions.
Catalytic Subunit
synthesis: Cyclin
(aa, mRNA,
ribosomes)
O
wee1, mik1
HO YT OH HO YT OH O P O YT OH
cdc25 O
Regulatory subunit: Inactive
Cyclin Kinase M-phase Promoting Factor
(cdc2) (MPF)
Note: Cyclin kinase does not INH CAK

actually phosphorylate cyclin.
It binds to it and forms a
complex whose activity is
regulated by phosphorylation,
which is catalyzed by CAK and O O O
cdc25. wee1, mik1
HO Y T O P O O P O YT O P O
cdc25
O O O
Active
MPF
Promotes Mitosis T = threonine

i.e. Cell Division
Y = tyrosine
S-phase M-phase
(synthesis) (mitosis)
This mechanism leads to the cyclic nature of the cell division process. This is illustrated by the time-
dependent progression in synchronized yeast cell cultures shown in the following figure, which shows the
biochemical changes that occur during the cell cycle in wild-type yeast cells.
The plot shows time-courses for total cyclin (monomer + dimers), active MPF [Cdc25-P], and inactive
MPF [phosphorylated Cdc25-P], relative to total Cdc2. The dotted lines indicate the cyclin level and MPF
activity that would be measured in a partially synchronous culture. By the author’s convention, mitosis is
defined as the time period when the tyrosine is more than 50% dephosphorylated.
1
cyclin
0.8
0.6 Pi Y T Pi
0.4
0.2 HO Y T Pi
0 40 80 120 160 200 240 280 320 360

Time (min)
(Adapted from Murray, W. and Kirschner, M., Cyclin Synthesis Drives the Early Embryonic Cell Cycle, ... Mitotic Control in Fission
Yeast, J. Theor. Biol., 1995, 173, 283–305.)
Chapter 9
Metabolic Enzyme Action
Catabolic process refer to breakdown of mass to obtain chemically useful energy, as occurs with
carbohydrates in glycolysis and the Krebs Cycle, and lipids in the β-oxidation pathway.
The following enzyme classes are common in metabolism:

(1) Kinases (a Transferase; class 2); it is not a ligase.
(2) Phosphatases (a Hydrolase; class 3); water is the phosphate acceptor.
(3) Pyruvate Dehydrogenases (an Oxidoreductase; class 1). Reduced CoA-SH is converted to oxidized
CoA-S-R.
A typical logic in regulation: (1) Phosphorylation leads to activation, and (2) dephosphorylation
causes inactivation. However, this is not true in general.
9.1 Enzyme Mechanisms

Factors that Contribute to the Control of Enzyme Activities. The following list collects a series of ideas,
which were described in previous sections, allowing one a chance to review how each works.
(1) The enzyme concentration. This is controlled in cells by the levels of protein synthesis, modification,
and degradation.
(2) The substrate concentration produces changes when [S] is less than about 20 times KM (i.e., when V <
Vmax ).
When V = Vmax, the reaction is said to be saturated with substrate. When [S] is less than 1/20 of KM, the
rate of catalysis is 0.
(3) Inhibitors inactivate E, so the [E] decreases (Eact → Einact). The two general classes are:
(i) Reversible, which are generally not covalently bound.
(ii) Irreversible, which form covalent protein-inhibitor complexes.
(4) Allosteric regulators, which can inhibit or activate. The effect is often cooperative in nature.
(5) Protein covalent modification. Examples include:

(i) Zymogens cause change in sequence (1° structure) as a result of protein chain cleavage. This is
almost never reversible.
(ii) Phosphorylation-dephosphorylation. This reversible on/off switching mechanism involves kinases
and phosphatases.
(iii) Protein modifications. Examples include glycosylation, myristoylation (lipid addition), ADP-
ribosylation (nucleotide addition), ubiquitinylation (attachment of a small protein that tags the bound target
protein for controlled destruction), and the many types of cofactors and “natural products” that participate
in unique biological niches.
(iv) pH (H+ on/off); H-bonds, salt bridges
(6) Chaotropes: (urea, guanidinium, SDS…)
(7) Temperature: At least two factors affect the kinetics (rate) of a given reaction: (1) increasing the
numbers of collisions at low to moderate temperatures, and (2) increasing denaturation as the temperature
is increased above 60 to 70°C. The first idea is captured in the form of the Arrhenius equation:
kT = k0 exp [−Ea / (kB T)]
x
where exp x = e , kT is the observed rate constant (k1, k-1, kcat…) and k0 is the intrinsic rate constant. Ea is
the activation energy required to form the transition state configuration. Two Ea values exist correspond to
Chapter 9 Metabolic Enzyme Action 71
(1) reactant crossing to products, and (2) the converse crossing. The Boltzmann constant (kB) is 1.361 x 10-23
J K-1and T is the temperature in Kelvin. The latter idea, protein denaturation, is governed by the hydrophobic
effect.
(8) Variation in the (i) ionic strength of salt solutions and (ii) dielectric constant of the solvent
environment. Increasing the value of D of a solvent leads to more shielding of interactions between
charges.
Recall that ΔGvdW is proportional to (- r -6 + r -12)/D and ΔGHB is proportional to (- r -10 + r -12)/D.
Computational Chemistry. Many of these factors can be simulated, in solution environments, using
computational methods called molecular mechanics and molecular dynamics analysis.
(1) Energy averages are determined by intensive, “all-atom force field” calculations. Vibrations, rotations,
translations are accounted for completely, with respect to all types of energies. The theory comes from the
field called Statistical Mechanics.
j
i i
i
j
Vibrations Interactions Relative motion
(2) Energies can be calculated and resolved into various contributions due to molecular aspects such as
hydrated surface coverage, charge topography, motional rates, conformational accessibility, and so forth.
(3) One can simulate reaction mechanism by combining quantum mechanical treatment of the problem,
when important, with the dynamics approach, when it is not.
9.2 Modes of Catalysis

Chemical
(1) Acid-Base Catalysis. This reaction class involves catalytic transfer of an H+ species. The electron-rich
proton acceptor species (B) is called the conjugate base. Electropositive species, such as H+ and
carbocations, often act as electrophiles in the role of a conjugate acid.
(2) Covalent Group Transfer. A functional group, obtained from a first substrate, is transferred to a
second substrate. Alternatively, it is transferred to an enzyme-held functional group (i.e. a coenzyme),
which then passes it to the second substrate. Functional groups are classified according to their group-
transfer potentials. Two typical examples are: (i) amine transfer catalyzed by the pyridoxal phosphate-
bearing aspartate transaminase, and (ii) phosphate transfer catalyzed by zinc-containing polynucleotide
kinase.
Binding
(1) The Proximity Effect. Bringing the reactants together in the active site decreases the effective volume,
which increases the effective concentration of the substrate. This drives the reaction by mass action. Details
are described below.
(2) Transition-State Stabilization. The reactant and enzyme favor the transition-state configuration
over that of the reactants. The products must be able to dissociate from the active site. Typically, a set of
functional groups distort the substrate physically and electronically.
An example occurs twice during the catalytic triad mechanism catalyzed by the serine proteases (e.g.,
chymotrypsin). At one point in the mechanism, a hydroxylate nucleophile attacks a carbonyl carbon. Since
this would make the carbonyl carbon pentavalent, one bond must go. To make it happen, the carbonyl
(C=O) double bond is stretched to form a hydroxylate (C-O–) single bond. This stretch is mediated by two
amide hydrogens located in the oxyanion hole of the active site. (See the Enzyme Mechanism section for
details.)
72 Chapter 9 Metabolic Enzyme Action
9.3 The Reaction Coordinate

This reaction coordinate plot follows the energies of the different reactants, intermediates and transition
states and products. It is an energetic map of course of the reaction. They can involve one or several steps,
depending on the complexity of the mechanism and amount known. Each step has an activation energy-
requiring process in each direction. Enzymes are reversible, but their energetics are not the same in each
direction. Many reactions form transiently stable intermediates, which exist until the species reacts in the
next step of the mechanism.
The following reaction coordinate diagrams show what happens when the two primary contributors to
catalysis are engaged.
(1) The proximity effect is shown in going from plot A to B.
(2) Transition-state stabilization is added in going from plot B to C.
A B
A B C Transition-state
A B
stabilization
Decreases the
2 excited state energy,
reverse Eact which decreases Eact.
Eact - proximity
uncatalyzed effect engaged fully
forward Eact A-B A-B A-B
catalyzed
A ΔG prox Eact A
ΔG ground state E E
B 1 1 B
Proximity effect.
A+B Raises the ground state
potential energy, which
decreases Eact.
The Principle of Mass Action. The poise of a reaction equilibrium is established by the concentrations of
reactant(s) and product(s) as a result of the constraints imposed by the equilibrium constant.
Under Standard State conditions, all component concentrations are 1 M. The superscripts in ΔGo’ indicate
that the parameter pertains to those conditions. Calculating values at other conditions is addressed in the
Bioenergetics and Bioelectrochemistry chapters.
Enzyme-Substrate Interactions. Weak binding of a substrate to an enzyme is preferred. Strong

binding would inhibit catalytic transformations by blocking the release of product(s). As an indie
record producer once said, “I do their record and I kick ’em out.” Some of the important forces that
mediate mechanisms are: (1) electrostatic interactions (including charge-charge, charge-dipole, and
dipole-dipole), (2) hydrogen bonds, (3) the hydrophobic effect and hydrophobic interactions, (4) van
der Waals forces, and (5) the entropy associated with liberating water molecules from the bound faces
of the molecules.
The Proximity Effect. The catalytic groups and substrate(s) are clamped together by the enzyme within
the active site. Since the effective volume has been constrained to the active site, it decreases dramatically
compared to that of the bulk solvent. Recall that the concentration (c) is the number of moles per unit
volume (c = m/V). Because the components are constrained to react within the active site, the local
effective concentration increases dramatically. One problem, however, is that trapping the species in one,
or a few, configuration(s) costs entropic energy. Nothing is free.
The close juxtaposition of the catalytic groups in the catalytic triad mechanism discussed below is a
clear example of the proximity effect. As we shall see, the histidine and serine species undergo a
complicated series of binding and unbinding reactions in the course of the process required to break the
peptide bond.
Reaction Intermediate Formation and Reaction Coordinate Diagrams. The two-step reaction
coordinate plot applies the ideas of the one-step reaction to a two-step scenario.
A 2
B 2
C + transition-state
stabilization
1 1
2, cat
Eact - 1, cat
proximity
uncatalyzed effect fully
Eact I I A-B catalyzed I A-B
A-B engaged
Eact
Species:
+ proximity effect
I intermediate
A+B transition-state
i
configuration 1 or 2
9.4 Induced Fit Revisited

When more than one contact point binds to the substrate, it becomes progressively more inclined to proceed
through the reaction. This inclination is called cooperativity and regulates a large number of biochemical
processes, ranging from binding of O2 to hemoglobin, membrane-induced enhancement of catalysis during
blood clotting, driving the binding of repressor proteins to genetic operator sites, and so on.
Transition State Analogs

The induced fit concept has been studied using modified substrates that mimic the substrate. Because they
are structurally similar, they fit. Because they contain non-natural functional atoms, they are often potent
enzyme inhibitors. This has led to their use as probes of functional groups that contribute to active-site
reactivity and in the design of a vast array of pharmaceuticals. Many such compounds are fluorinated.
Specific examples can be found in the abzyme literature.
Catalytic Antibodies: Abzymes

Consider using a transition-state analog as an antigen to produce an antibody. Since the antigen resembles the
transition-state geometry of a reacting substrate within an enzyme, it was surmised that they might convert
normal substrates to the transition-state, and then on to products. If so, the antibody would be catalytic. A
series of these abzymes have been produced and do catalyze the intended reaction. The success of this design-
driven approach provides strong experimental evidence for the induced fit idea. While abzymes are natural,
they are artificial catalysts. The specificity of antibody-antigen recognition is used to accomplish a particular
catalyzed reaction. The immunized animal is used to produce the proactively designed catalyst.
Hexokinase: Large-scale Domain Movements

The active site of this enzyme shifts from opened to a closed structures upon binding the substrate glucose
and ATP substrates. This closure involves large-scale movement of the surrounding protein domains, which
shields the active site from H2O, a potent competitive inhibitor of the kinase reaction. Incorporating active
site closure slows the enzyme rate 10,000-fold, but is required to protect the mechanism, which would
otherwise be poisoned by the encroaching solvent.
A B
active site closed active site
with glucose bound
(Adapted from McKee and McKee Biochemistry the Molecular Basis of Life (5th ed.), 2012, p. 187; Fig. 6.2.)
Enzyme Specificity. Enzymes can often use more than one substrate. Recall that the specificity constant
(kcat/KM) can be used to compare specificities two different substrates with one enzyme. A larger value
means that the specified substrate is used more efficiently. This demonstrates that the enzyme can
sometimes adapt to different structures, suggesting some degree of plasticity in the recognition process.
Two examples of varied specificities are: (1) alkaline phosphatase, which can remove the phosphate from a
variety of substrates, and (2) alcohol dehydrogenase, which can convert a number of alcohols to aldehydes.
9.5 Acid-Base Catalysis

Acid-base Catalyzed Peptide Bond Cleavage. The following mechanism shows how acid and base
species (H+ and OH-) each participate in the hydrolysis of an amide bond, such as catalyzed by a protease.
Step 3 O
O O R1 C R2
R1 R2 R2 +
C N R1 C N OH N
Step 2 R3
R3 R3
HO H
H O
H
H B R4 Step 4 B R4 B R4
Step 1
The steps are:

(1) H+ is abstracted from H2O by the R4 -B: species
(2) OH- attacks the amide C=O
(3) carbanion I forms
(4) The amide/peptide bond is cleaved and C=O is restored. R4-B releases H+ and the secondary amine
product forms.
C-H Bond Cleavage. Here, a conjugate base extracts a proton from a substrate, which creates a carbanion.
Conjugate Carbanion Conjugate

base acid
R1 R1
R2 C H B R4 R2 C + H B R4
R3 R3
Nucleophile stronger Nucleophile
Why Do Enzymes Often Work Best at Neutral pH?

Enzymes depend on pH because the functional groups must typically be in a certain state of
protonation/deprotonation to support each of the proton-passage steps required in the mechanism.
Determining how an enzyme is affected by pH is a standard first step in characterizing it. It is useful
because it provides information on which functional groups are required for catalysis.
The following pH profile shows a situation in which protonation of something destroys the catalytic
mechanism below pH 4, and deprotonating something inactivates catalysis at pHs above 9. Why?
The optimum pH is typically
in the 6 to 7.5 range.
Enzyme activity
(e.g., initial velocity)
pH
4 6 8
essential groups typical essential groups

are protonated range are deprotonated
The explanation assumes that the activity of an enzyme depends on the pKa values of two functional
groups in the active site. Let’s set one acidic pKa at 3.9 and one basic value: 10.5 (i.e., asp and lys). These
could represent the carboxylate of an aspartate or glutamate residue and the ε-amino group of a lysine,
respectively.
BH2+ ↔ BH ↔ B -
H+ H+
NH3+ NH3+ NH2
AS AS AS
COOH COO - COO -
H+ H+
Reaction Reaction
1 2
charge = 1+ charge = neutral charge = 1-
In principle, four types of curves are possible. What are they? What does the enzyme’s active site look like,
in a simplified sense, in each situation?
(1) What does a plot of pH versus V0 look like when only the BH form is active?
The plot should have a maximal V0 at a mid-range pH and progressively decreasing values at lower and
higher pH values. Where is the pH optimum? In the middle range, around pH 7, with reasonably high
activity between pH 6 and 8.
Maximal activity
pKa of reaction 1 range
~ 4.0 pKa of reaction 2
~ 10.5
Vmax
Activity decreases due

Vmax to lysine deprotonation
V0 Activity decreases due
2 to aspartate protonation
0
2 4 6 8 10 12
pKa1 pKa2
pH
(For mathematical details see Piszkiewicz, D., Kinetics of Chemical and Enzyme-Catalyzed Reactions, Oxford Univ.
Press, NY, 1977, pp. 61–65.)
(2) Which amino acids participate in catalysis?

(3) What does the plot look like when only the B- form is active?
(4) What about when only the BH2+ form is active?
(5) What does the plot look like when the pH titratable residues in the active site don’t really affect
catalysis?
This can occur when substrates are nonpolar (aromatic, aliphatic) and the attraction between active site and
substrate is predominated by van der Waals forces and hydrophobic interactions.
Triose Phosphate Isomerase. Moving a Hydroxyl.

This reaction is discussed in the Glycolysis chapter. It converts a compound that cannot be used in
glycolysis to one that can. When the six-carbon compound fructose-1, 6-bisphosphate splits into these two
3-carbon compounds, the cell must recover the one that can’t be used. The reaction is:
dihydroxyacetone phosphate → glyceraldehyde-3-phosphate
(DHAP) (G3P)
The mechanism is a concerted acid-base reaction.
unusual
his, his
glu H , glu O H H O
H C O C
H H
C O H C O
CH2 CH2
O O
O P O O P O
O O
Dihydroxyacetone phosphate Glyceraldehyde-3-phosphate

(Substrate) (Product)
The acid reaction involves histidine imidazole/imidazolium protonation-deprotonation. The base

changes are glutamic acid/glutamate transformations. The following subprocesses occur during the
mechanism:
(1) C=O is converted to C-O-
(2) C-O- is protonated to form C-OH
(3) a C-H is abstracted to form C=C
(4) a proton of R=C(H)O-H is abstracted producing R=C(H)O-
(5) R=C(H)O- is converted to R-C(H) =O.
The measured reaction coordinate for triose phosphate isomerase has five resolved steps:
E + S → [E-S] → [E-I] → [E-P] → E + P

(1) (2) (3, 4) (5)
9.6 Covalent Group Transfer

Group Transfer and Leaving Groups
Many biochemical group transfer reactions occur. The good leaving potential of phosphate from ATP is
why it is a key carrier of biochemical form of energy. In the first step of a typical mechanism, part or all of
S is transferred to E or a bound coenzyme. In a second step, this piece is attached to a second substrate. The
product leaves the enzyme with the transferred piece from the initial S.
Group X is transferred from A to B via E. Two steps occur, then E is regenerated.
A X + E A + X E (step 1)
X E + B B X + E (step 2)
Leaving groups (X) have different group transfer potential levels, their propensities to leave.
Acetoacetate Decarboxylase. The Schiff Base. The next reaction illustrates the use of Schiff base. In the
acetoacetate decarboxylate mechanism the Schiff base facilitates elimination of a CO2 from the molecule.
Reversal of the Schiff base releases acetone.
In a contrasting case, the benzyl α-carbonyl group of pyridoxal phosphate forms a Schiff base with
the ε-amino group of a lysine of aspartate transaminase. The adduct serves to protect the aldehyde group
of bound PLP when it is not engaged in catalysis. Details are described in the Pyridoxal Phosphate section.
lys (E) ketamine Schiff base

acid-base
catalysis lys (E) lys (E) O
enamine
NH2 + C
O H+ H2O H N O N H O
δ+
CH3 C CH2 C O CH3 C CH2 C O CH3 C CH2
O (E S) (X)
(1) charge migrates (2) iminium formation
toward (tautomeric shift)
(E + S X) (E S X)
CO2 is eliminated H 2O
C C forms (3) reversal of acid-base
catalysis lys (E)
E is regenerated + H+
P forms NH2
(E)
acetone O
CH3 C CH3 ( P )
The steps involved in this mechanism are: (1) 1 H+ is used as an acid catalyst and O= leaves, (2) the
negative charge migrates toward -CO2, which is eliminated as the –C = C bond forms, (3) iminium
formation occurs by a tautomeric shift, (4) acid-base catalysis is reversed: E is regenerated and P forms.
9.7 The Serine Protease Catalytic Triad Mechanism

The Catalytic Triad. The goal of the enzyme is to use the catalytic triad to create a serine hydroxylate
nucleophile, which attacks the partially positive carbonyl carbon in the peptide. Two protein amide
hydrogens stretch the carbonyl carbon-oxygen double bond of the peptide substrate, driving it to form a
hydroxylate single bond. This type of deformation of a reactant to get the reaction to proceed is called
transition-state stabilization. This portion of the enzyme that stretches the bond is called the oxyanion hole.
His 57 Ser 195 Bound protein substrate with
E-S
N-terminus R1 and C-terminus R2
3
N N O O
H "Charge relay"
O H 2 C R2
O 1 +
Asp N H his, ser his H , ser O
C
102 OH
R1
Creates the ser O

nucleophile that attacks
the peptide δ+C O.
"oxyanion hole."
E - T1 His 57 Ser 195
(S)
+ N O O
N H
O H C R
Asp O N 2
(Protein fragment 2)
C R1 H (5 steps)
102 O
a bifurcated C R2
H-bond
O
R1 NH2
(Protein fragment 1)
9.7.2 The Charge Relay System. This mechanism creates the nucleophile that attacks the protein
substrate, in the first step of the reaction. Subsequently, the first product, the C-terminal fragment of the
cleaved protein chain (with the newly created amino terminus), is released. The reaction concludes as the
N-terminal fragment (with the newly created carboxylate) is released and the enzyme ‘active site’
components are returned to their pre-reaction states, in readiness for the next round of catalysis.
The mechanism can be broken into seven steps, as shown below. (Adapted from Moran et al., Principles
of Biochemistry (5th ed.), 2012, pp. 188-189; Fig. 6.28.)
Ser-195
The noncovalent enzyme-substrate complex His-57
is formed, orienting the substrate for N
reaction. Interactions holding the substrate E+S Cα H
CH2
in place include binding the R1 group in the CH2 Gly- 193
specificity pocket. N
N O H
N
The binding interactions position the O H H
carbonyl carbons of the scissile peptide O
bond (susceptible to cleavage) next to the C O
specificity
oxygen of Ser-195. Asp-102 R2 N C R1
CH2 pocket
(1)
Ser-195
His-57
Binding of the substrate compresses Asp-102 E-S N
Cα H oxyanion
and His-57. The strain is relieved by CH2 hole
formation of a low barrier hydrogen bond. CH2 Gly- 193
The raised pKa of His-57 enables the N
imidazole ring to remove a proton from the N N O O H
hydroxyl group of Ser-195. The nucleophilic H H
O C
oxygen of Ser-195 attacks the carbonyl O R1
carbon of the peptide bond to form a carbonyl C N
R2
intermediate E-TI1. This species is believed to Asp-102 CH2 H
resemble the transition state.
(2)
Ser-195
His-57
N
When the tetrahedral intermediate is formed E - T I1 Cα H oxyanion
CH2 hole
the substrate C - O bond changes from a CH2 Gly- 193
double bond to a longer single bond. This N
allows the negatively charged oxygen, the N + N O O H
"oxyanion," of the tetrahedral intermediate H H C
to remove to a previously vacant position, O
the "oxyanion hole." It hydrogen bonds to O N R1
C R2
the peptide chain N - H groups of Gly-193 H
and Ser-195. Asp-102 CH2
(3)
Ser-195
The imidazolium ring of His-57 acts as an acid Acyl E His-57
catalyst, donating a proton to the nitrogen of the + N
scissile peptide bond - facilitating its cleavage. PI CH2 Cα
H Gly- 193
CH2
N
N N O O H
The carbonyl group from the peptide forms a H C
covalent bond with the enzyme, producing an O
acyl-enzyme intermediate. The initial C- O R1
C H
terminal peptide product (P1), with its new
amino terminus is produced. This fragment Asp-102 CH2 R2 N
leaves the active site. H
Carboxy-terminal
product (PI)
(4)
Acyl E Ser-195
+ His-57
H 2O N
Cα H Hydrolysis (deacylation) of the acyl-enzyme
CH2 intermediate starts when Asp-102 and His-57
CH2 Gly- 193
N again form a low-barrier hydrogen bond and
N N O O H His-57 removes a proton from the H2O
H H C molecule to provide an OH- , which attacks
O O O R1 the carbonyl group of the ester.
C
H
Asp-102 CH2
(5)
Ser-195
His-57
A second tetrahedral intermediate (E-TI2) is
E - TI2 N formed and stabilized by the oxyanion hole
Cα H oxyanion
CH2 hole
CH2 Gly- 193
N
N + N O O H
H H C His-57, once again an imidazolium ion,
O donates a proton, leading to collapse of the
O O R
C 1 second tetrahedral intermediate.
H
Asp-102
CH2
(6)
Ser-195
His-57
E - P2 N
Cα H
CH2 The second product (P2) forms - the amino-
CH2 Gly- 193
N terminus of the original protein with a new
N N O O H carboxy terminus.
H H C
O O R
O 1
C H
Asp-102
CH2
(7)
Ser-195
His-57
E + P2 N
Cα H
CH2
CH2 Gly- 193
N
N N O H P2 is released from the active site,
H H regenerating free chymotrypsin.
O
O
C
Asp-102 O
CH2
C R
+ 1
H O
Amino-terminal product (P2)

9.8 The Active Site of Tyrosyl-tRNA Synthetase

The following figure shows a reconstruction by site-directed mutagenesis of the transition state for
activation of tyrosine by tyrosyl-tRNA synthetase. A mobile loop envelops the transition state in an
induced-fit mechanism.
Thr-
40 CH3
CH
O
H His-
45
Arg-
86 N N
NH H
Substrate 2: ATP
C
H2N NH H
O H N Lys-
H H 230
Lys-
82 O P O
Gln-
173 H
N O
NH2 H
C H H Lys-
O P O H N 233
O Substrate 1: Tyrosine H
O O
O O NH2
H
P
C O H N CH C O N
O O N
Asp- H
CH2 N N CH3
78 CH2
O O CH
O H H
H
Thr-
H H H 51
N N
O OH
O H His-
H 48
Tyr- H
169 O O
O
C
H
Asp- S
176
Cys-
Tyr- 35
34
Active Site Protein-Ligand Binding Interactions

This figure shows the large number of hydrogen bonds (at least seven) formed between tyrosyl tRNA
synthetase and the two bound substrates, tyrosine (left) and ATP (right). Lysine 82, Arg-85, Lys-230, and
Lys-233 all bind to the portion of ATP that becomes pyrophosphate. (Adapted from Fersht, A. R., et al. (1988)
Biochemistry 27, 1581–1587.)
Site-directed Mutagenesis
This technique involves changing the gene sequence in an expression plasmid vector. One plans which
sequence to replace in order to insert a desired amino acid in a particular position in the protein chain. By
making such replacements, one can proactively determine the degree to which a given amino acid
contributes to catalysis, binding and other aspects of a mechanism. This approach can be used, along with a
technique that determines the energetics of the reactions (e.g., calorimetry, spectroscopy), to determine how
much each site contributes energetically to binding, structural transformations, and catalysis.
Chapter 10
Coenzymes
10.1 Classification
Cofactors are divided into two subgroups, essential ions and coenzymes.
Essential Ions
(1) Activator Ions are usually loosely bound. Examples are:
(i) Potassium (K+ ) is crucial in nerve conduction.
(ii) Calcium (Ca2+) is carried by calmodulin and regulates a huge number of biological processes.
(iii) Magnesium (Mg2+) is present in chlorophyll and activates phosphate transfer from nucleotide
triphosphates.
(2) Metal Ions are usually tightly bound in metalloenzymes. Examples are:
(i) Iron (Fe2+) is the redox site in iron-sulfur clusters, which are common in electron transfer redox
complexes.
(ii) Zinc (Zn2+) is typically bound to sulfur and histidine ligands, for example, as in carbonic
anhydrase and zinc fingers.
Coenzymes
(1) Cosubstrates are loosely bound; for example, ATP and UDP-sugars are used to accomplish a reaction,
but are not tightly bound and dissociate after the reaction is complete.
(2) Prosthetic Groups are tightly bound; vitamins; for example, pyridoxal phosphate, a covalently bound
coenzyme within aspartate transaminase.
10.2 Survey of the Coenzymes

Adenosine Triphosphate (ATP)
Obtaining full activity with enzymes usually requires Mg2+.
Mg2+ H
H
N
O O O
H H N
C C C
O P O P O P O H N
C N C
O C
O O O N
C H H C H
H C C H
High energy
O O
bond
H H
Two important examples of uses of ATP are:
ATP + Glucose → ADP + glucose-6-phosphate Pi2- transferred (1)
The first reaction is the beginning of glycolysis.
ATP + ribose-5-phosphate → AMP + 5-phosphoribosyl-1-pyrophosphate PPi4- transferred (2)

(PRPP)
The product of reaction 2, PRPP, is attached to nucleic acid bases to make the nucleotides. The
pyrophosphate group leaves during nucleoside formation, driving the reaction to completion.
82 Chapter 10 Coenzymes
Redox Coenzymes (NAD+ and FAD)

Recall the mnemonic “Oil Rig”: Oxidation is the Loss of electrons; Reduction is the Gain of electrons.
These two reactions always occur in pairs, leading to the general name redox reactions. The generic
reaction is illustrated by the alcohol dehydrogenase reaction below. The generic reaction is:
Ared + Box Aox + Bred
where species A passes an electron to B.
Nicotinamide Adenine Dinucleotide (NAD+, NADP+) (Vitamin precursor: Niacin, that is, nicotinamide)
Oxidized form Reduced form

H H
H O H H O
C C C C
C C
C NH2 C NH2
H C H C
CH2 N C C
R CH2 N
O H O H
H H H H
H H H
H
O OH OH OH
OH
O P O
O
O P O
O CH2 Adenine
O
H H
H NADP+ (which is really NADP3- )
H
2-
OH O ( H or Pi )
(1) The oxidized form NAD+ is used to oxidize a substrate. The reduced compound NADH is used to
reduce a substrate.
(2) The coenzyme carries reducing equivalents for use in electron transport, which fuels oxidative
phosphorylation by creating a proton gradient across the inner mitochondrial membrane.
(3) It is typically used in ordered dehydrogenase reaction mechanisms:
Example. Alcohol Dehydrogenase. A typical redox enzyme.
H O H H O
H H
C NH C NH
2 2
OH H
H + H O
CH2 N H N H
Alcohol C
CH3 R Dehydrogenase R
CH3
ethanol + NAD+ acetaldehyde + NADH + H+
Ared Box Aox Bred
(1) NAD+ receives an electron from ethanol and forms NADH. The electron is transferred attached to
hydrogen, as a hydride.
(2) The kinetics can be measured in either direction, by putting in either the left or right components to
drive the intended reaction. The equilibrium and kinetics are reversible.
Chapter 10 Coenzymes 83
Flavin Adenine Dinucleotide (FAD, FADH2): Made from Vitamin B2
proton
hydride
O O
H H, H H
H3C C C H 3C H
N H C N C H
riboflavin C C C N C C C N
vitamin B2 C C
C C N C C C C
H3C C N O N N
H3C C H O
H CH2 H R
the redox
(CH OH)3 center
CH2
5' ADP
(1) Solutions of the oxidized form FAD (which is actually FAD2-) are intensely yellow. The reduced form
FADH2 is colorless. This change in absorbance provides an especially good way to follow the kinetics of
catalysis and other interactions of flavins in biology.
(2) The coenzyme carries reducing equivalents. It functions metabolically like NAD+ (and NADP+) but has
a different redox potential, so it can be used to oxidize more difficult functional groups.
(i) NAD+: used to oxidize R-OH, C = O, COOH (easier)
(ii) FAD is used to oxidize C = C (more difficult) and accept electrons from other carriers: for example,
NADH, ubiquinone.
Pyridoxal Phosphate: Contains Vitamin B6

Schiff Base. The covalent species [E-lys–PLP] is linked by an internal aldimine formed by covalent
bonding of the coenzyme to the enzyme active site.
E lys (CH2)4 NH3+ E

H2O
H H N H
O O
O C C
H2 H2
O P O C C O O P O C C O
C C C C salt bridge stabilizes
O O C C
the negative charge
C C
H N CH3 H N CH3
H H
PLP (really PLP2-) E lys PLP
The following general linkage is called a Schiff base.
E
E - lys - PLP
H N H Schiff base
C
R
Schiff base formation requires several individual reaction steps.

The Ping-Pong Mechanism

An example of this mechanism is catalyzed by Aspartate Transaminase. A number of other transaminases
(aminotransferases) exist, for example, (1) the isoenzymes involved in the malate-aspartate shuttle, and (2)
the enzyme that produces alanine from lactate, which fuels gluconeogenesis.
(1) The reactants are the amino acid glutamate and Krebs Cycle intermediate oxaloacetate. The products
are aspartate and another Krebs Cycle intermediate α-ketoglutarate.
(A) reactant 1 (P) product 1 (B) reactant 2 (Q) product 2
COO COO
H2 N CH C O COO COO
CH2 CH2 C O H2N CH
CH2 CH2 CH2 CH2

COO COO COO COO
Glutamate α-Ketoglutarate Oxaloacetate Aspartate

(glu) (α-KG) (OAA) (asp)
(E) E-LMP (F) E-PMP (E) E-LMP

"F" receives, retains and
transfers the amino group
(2) PLP and Schiff Base Formation. PLP has a positive charge and serves as a sink for electrons. The steps
in the mechanism are:
E
E
NH2 NH
NH
CH2 CH
CH
2- 2- 2-
O3 POH2C O O3 POH2C O O3 POH2C O
H CH3 H CH3 H N CH3

N N
H H H
Pyridoxal phosphate-lysine Pyridoxamine phosphate Schiff base adduct

Schiff base adduct (PMP) regenerated
(i) The ε-amino group of a lysine of the enzyme is normally bound to the PLP aldehyde site as a Schiff
base. This is like a sword, the aldehyde, being held inert and protected within the scabbard, the Schiff base
adduct.
(ii) In the first step of catalysis, this Schiff base-bound lysine group is freed from PLP, exposing it for use
(i.e., the sword is removed from the scabbard).
(iii) in the second, the aldehyde site binds the amino group, which is removed from glutamate. It holds on
to it as a-ketoglutarate is ejected and the second substrate oxaloacetate (OAA) binds.
(iv) In this step, the amine group is transferred to OAA, producing aspartate. Voila—but not done yet.
(v) To complete the mechanism, the exposed aldehyde, regenerated after the amine transfer, is reconnected
to the lysine as the protective Schiff base adduct. The sword has returned to the scabbard.
Another example of a Schiff base-mediated reaction is described in the Acetoacetate Decarboxylase

section.
Coenzyme A
The free form of coenzyme A (CoA-SH) is used to carry the acetyl group, which binds the circled
sulfhydryl sulfur, forming a thioacetate linkage.
The vitamin is Pantothenic Acid; the coenzyme includes ADP.
4'-Phosphopantotheine group
NH2
O O CH3 O O N N
5' H
HS CH2 CH2 N C CH2 CH2 N C CH C CH2 O P O P O CH2 N N H
H H O
OH CH3 O O
4' H H 1'
β-alanine H
3' 2' H
2-
O3PO OH
2-Mercaptoethylamine Pantothenic acid ADP with 3'-phosphate group
Activated Acetyl. CoA-SH is used in the Bridge Reaction, which links glycolysis to the Krebs Cycle. The
activated acetyl is transferred to oxaloacetate to produce citrate. Pyruvate is decarboxylated by Pyruvate
Dehydrogenase to yield CO2 and acetyl-CoA:
O O
CoA-SH CO2 CoA
C
S
C O
C O
CH3
NAD+ NADH, H+ CH3
pyruvate acetyl CoA
CoA-SH is used a second time in the Krebs Cycle to drive the substrate-level phosphorylation reaction
catalyzed by Succinyl CoA Synthetase.
Acyl Carrier Protein (ACP). This structure is very closely related to CoA. It is required to synthesize fatty
acids. Instead of being linked to ADP, the 4’-phosphopantotheine is linked directly to the protein via a
serine.
R1
O O CH3 O C O
HS CH2 CH2 N C CH2 CH2 N C CH C CH2 O P O CH2 CH

H H
OH CH3 O NH
R2
4'-Phosphopantotheine prosthetic group Protein
Fatty acids are synthesized two carbon units per addition, contributed by ACP-bound acetate molecules.
Biotin (Vitamin H)
Pyruvate Carboxylase.
This enzymatic reaction begins with ATP-dependent carboxylation of the biotin cofactor embedded within
the enzyme. Biotin and bicarbonate react to form carboxybiotin. The thioacetate bond is important in
metabolism due to the high group-transfer potential of the acetyl group.
COO
Phosphoenolpyruvate (PEP)
C O Pi2-
(from glycolysis)
CH2 Oxaloacetate (OAA)
Enolpyruvate
COO COO
C O C O
CH2 1. Enol-keto CH2

ATP ADP + Pi tautomerization
O 2. CO2 addition COO
O O O
O
C O + HN1 NH C
O N1 NH HN1 NH
HO
S Enz
S Enz Enz
S
Bicarbonate Biotin
Carboxybiotin Biotin
In the second step, the carbanion from enolpyruvate attacks the carbon of carboxybiotin and transfers a
carboxyl group. This then yields oxaloacetate and regenerates biotin.
Biotin-Avidin Affinity Chromatography.

The protein avidin (from egg white) binds biotin with Ka = 1014 M-1 (very strong). An important example of
the use of this phenomenon in biotechnology involves affinity capture and tagging systems.
Noncovalent Binding: Gel bead “matrix” packed in a chromatographic column.

Biotin
(The capturing ligand.)
Agarose bead Goal of the Method:
(One of many packed in a Captured Transcription Factor
chromatographic column.) (Commonly binds as a dimeric
complex.)
Chemical
linker
Avidin protein Synthetic DNA duplex
(Has a high affinity (Contains a sought
for biotin.) transcription factor
binding site.)
This technique involves the following steps:
(1) synthesize a specific biotinylated DNA,
(2) make a nuclear protein preparation,
(3) mix the DNA with the protein prep,
(4) place the DNA and captured protein(s) through an avidin-agarose affinity chromatography column,
(5) elute contaminants, and
(6) elute the biotinylated DNA-protein(s) complex with 2-aminobiotin. One recovers proteins that bind the
biotinylated DNA sequence.
Folate
Folate is composed of three parts: pterin, p-amino benzoate (PABA) and glutamate.
N N H2N N N
H2N 1 8 7 7
2 N N 2
8
O COO
HN 4
5 6 HN 5 9 10
N N N 6 CH2 N C N CH
N H H
4 O O CH2
Pteridine Pterin Folate PABA CH2
(2-Amino-4-oxopteridine)
COO
Reduction by transfer of hydride from NADPH occurs in two steps.
NADPH, H NADPH, H
H H
NADP H NADP H
R1 N H R1 N R1 N
8 7 8 7
H 8 7 H
6
5 6
Slow 5 6
Rapid 5 H
R2 R3 R2 R2 N
N N R3 R3
H
Folate 7,8-Dihydrofolate 5,6,7,8-
Tetrahydrofolate
Example: Dihydrofolate Reductase

Dihydrofolate reductase converts folic acid to its biologically active form, tetrahydrofolic acid (THF). The
carbon units carried by THF are bound to N5 and/or N10 of the pteridine ring forming N5, N10-
methylenetetrahydrofolate (N5, 10-THF). The enzyme Thymidylate Synthase uses N5, 10-THF to attach the
methyl group to deoxyuridine monophosphate (dUMP), and thereby produce deoxythymidine
monophosphate (dTMP). As the methyl group is transferred to form dTMP, the folate coenzyme is oxidized
to form dihydrofolate. N5, 10-THF is regenerated from dihydrofolate by dihydrofolate reductase and
NADPH.
H H
N N H m5,10-THF THF
H2N
HN H
dUMP dTMP
N5 CH2
10
O N
C Occurs by addition of -CH3 at
H2 PABA - Glu C5 of the pyrimidine ring.
Dihydrofolate reductase and thymidylate synthase have been studied as targets for cancer and Crohn’s
disease chemotherapy. See Methotrexate in the Enzyme Inhibitor section. Cells that grow, and replicate,
more rapidly, the hallmark of cancerous cells, are more susceptible to growth inhibition by the
chemotherapeutic agent than those that are not actively replicating, i.e. most normal cells.
Thiamine (Vitamin B1)

Thiamine is used by the “Bridge Reaction” enzyme complex Pyruvate Dehydrogenase to make
hydroxyethylthiamine pyrophosphate (HETPP). The circled site is used.
O O
H3C CH2 CH2 O P O P O
Thiamine NH 2 + O O
pyrophosphate CH2 N S
(TPP) N
C
hydroxyethyl group
H3C N H attachment site
TPP is also used by the enzyme Pyruvate Decarboxylase, which is required by yeast to synthesize
ethanol, and the transketolases, which interchange C2 units between sugars to make other sugars, for
example, in several Calvin Cycle reactions.
10. 3 Metals
Molybdenum.
The structure of the molybdenum cofactor used by xanthine oxidase is:
H
H
H2N N N H O
H
HN CH2 CH2 O P O
N C C
O H O
S S
Mo
O O
The cofactor contains molybdenum, FAD, and two different Fe-S centers. The enzyme oxidizes
hypoxanthine to xanthine, a metabolic intermediate in the biosynthesis of guanine.
Vitamin B12 (Cobalamin)

Methylated cobalamin transfers a methyl group to homocysteine to produce methionine. The methyl group
is initially transferred to cobalamin from the coenzyme 5-methyltetrahydrofolate.
NH2
H2N C O
transferred
O C CH2 group
H2N R
H2C CH2
O C H 3 C CH3
A N N
H3C O N Co N
H2C N R CH3
O CH2 C NH2
D N Co N B
N C CH2 CH2 N CH3
H2C CH2
H
H3C N O
CH C CH CH2 C NH2 N CH3
H3C H3C 3
O O
CH2 CH3 O
P N O
O O OH CH2 C NH2 HOCH2
N
H CH3 O
H H
CH3
HOCH2 O H
The corrin ring system contains four pyrrole rings. The nitrogen of each pyrrole is linked to a central cobalt atom.
Another group, 5, 6-dimethylbenzimidazole ribonucleotide, is also attached to cobalt. The structure of corrin is similar
to the porphyrin heme group, but lacks one methylene bridge between pyrrole rings A and D.
In another important application, the fatty acid precursor methylmalonyl coenzyme A is rearranged by
the the enzyme Methylmalonyl CoA Mutase. This reaction produces the Krebs Cycle intermediate succinyl-
CoA. The coenzyme is in the adenosylcobalamin form.
H O H
- Methylmalonyl-CoA -
OOC C C S CoA mutase OOC C H
H C H Adenosylcobalamin H C C S CoA
H H O
Methylmalonyl CoA Succinyl CoA
Iron-Sulfur Proteins
Iron-sulfur proteins form the core of several redox carrier proteins in mitochondrial electron transport. Free
sulfurs of the disulfides (–S2-) bind the iron atoms in a number of different ligand geometries and
stoichiometries. Two examples are:
protein
cys
protein S
cys cys cys
S Fe
S S S S
protein Fe S
Fe Fe
protein Fe S
S S S protein
cys cys protein
S Fe
S
cys S
cys
.
These centers are used in redox reactions in a number of oxidoreductases. In another example, the
protein thioredoxin and the small tripeptide compound glutathione are involved in maintenance of disulfide
bonds in proteins. Glutathione has access to active sites. Reactions in the Krebs Cycle and photosynthesis
depend on thioredoxin.
Transferrin and Ferritin. These two protein mediate the transport of the iron atoms, which are
intrinsically toxic, through the circulatory system and into cells.
Zinc
Carbonic Anhydrase: Zinc as a Lewis Acid.

This protein catalyzes the interconversion between carbon dioxide and bicarbonate, which leads to the
buffering of our blood (See that section for details.)
H his
N
his H
N H
O
O
N O
H N Zn2+ O C O C Bicarbonate
H H O
O
N
H2O is made acidic by Zn2+
his N binding; it is a "superacid."
H OH
H2O + H+
Zn2+
Zinc Fingers
Another important role of zinc is its use in transcription factor (TF) proteins. Zinc forms a nucleus for four
cysteine and/or histidine residues, stabilizing formation of a zinc finger (ZF) domain. Different amino acid
ligands form unique ligand spheres in different fingers, depending on the particular DNA-binding protein.
ZF domains usually occur in several copies in the DNA-binding domain of TF proteins. The fingers
fit into the DNA grooves in a sequence-specific manner. Successful recognition leads to transcriptional
activation of the bound gene sequence. (See the Nucleic Acids section for details)
10.4. Carbohydrates-Based Cofactors

3-Phosphoadenosine-5-Phosphosulfate
Sulfate is transferred to a recipient carbohydrate (e.g., heparin) from the activated coenzyme 3-
phosphoadenosine-5-phosphosulfate.
O O
Adenine 3', 5'ADP
O S O P O O
O O H H
O
O O
H H O S O
Pi 2- O 2-
O OSO3
NH2
Carbohydrate or or N H
R Carbohydrate
R
Note that phospholipid biosynthesis is analogous in that CDP leaves diacylglycerol when serine or inositol
diphosphate are attached to it. (See the Synthesis of Acidic Phospholipids section for details.)
Uridylyl Carbohydrates
UDP is a good leaving group. It has a high “group-transfer potential” and is used in the synthesis of lactose.
Lactose
HO O H
H HO H
CH2 H
H N
HO O glucose H H
H OH
OH H HO H H
OH
H O Pi Pi N O OH
HO O
O H
O
H OH H H OH
H H OH H
H
H OH glucose
HO OH UDP
UDP-galactose
galactose
Vitamin C
Vitamin C (ascorbic acid) is used as a reducing agent in the hydroxylation of collagen.
OH OH
H lactone 2H , 2e H
HO CH2 O HO CH2 O
O O
H H
HO OH O O
diol (reduced) 2H , 2e dione (oxidized)
Upon oxidation, the diol functional group, which is composed of two adjacent hydroxyl groups, is
converted to a dione, which has two adjacent ketones. This molecular system is an efficient free radical
scavenger. Note the resemblance between ascorbate, ubiquinone and vitamin E (α-tocophorol) and their
redox partners. Each supports the dione-to-diol transformation. They all function as both electron carriers
and free radical scavengers in a number of biological scenarios.
Ascorbate is used as a reducing agent in a lot of laboratory techniques, often to quench or prevent free
radical-mediated processes. It prevents oxidation of carbohydrates, proteins and lipids, yet is nontoxic, so it
is a common food additive.
10.5 Fat Soluble Vitamins

Vitamin E
Vitamin E (α-tocophorol) is a free radical scavenger. (For details, see the A Potpourrie of Lipids section.)
Vitamin A: Cis-Retinal and the Visual Response

The molecule β-carotene undergoes an oxidation reaction, cleaving a double bond and forming two
molecules of vitamin A. The alcohol group of vitamin A is then enzymatically oxidized to form cis-retinal.
Rhodopsin is the pigment of the retina that is responsible for forming photoreceptor cells. Opsin, a protein
within the retina, binds to cis-retinal and acts as a visual pigment. When visible light strikes cis-retinal in the
eye, it is excited and generates a nerve impulse causing cis-retinal to convert into trans-retinal, which in turn
dissociates the opsin. This reaction can be reversed with the enzyme retinal isomerase to reform cis-retinal.
orange in
β-Carotene
carrots
Oxidative cleavage to two

molecules of Vitamin A.
11 15
CH2OH
NADP Enzymatic oxidation

of the alcohol at C-15
and isomerization
NADPH + H
11
12
cis- retinal 15 CHO

(aldehyde) cis
Opsin
Light
Retinal isomerase cis-trans

Opsin isomerization
11 15 trans
CHO
trans- retinal
Vision is the result of absorbance of light by the cis-isomer, producing trans-retinal in the focal center
of the eye. This process is called photo-induced isomerization. This system is the antenna for our eyesight.
Production of the cis form switches the signal on—one photon at a time.
Opsin is the coenzyme carrier protein. In the absence of retinal, opsin is an apoprotein. Rhodopsin is
the [retinal·opsin] complex. Transfer of this signal to the optic nerve involves a G-protein-mediated
pathway involving the G-protein Transducin. (See the Signal Transduction chapter for details.)
Coenzyme Q
This coenzyme plays a crucial role in the mitochondrial electron transport process, as a mobile carrier of
reducing equivalents. The second important mobile carrier in that process is a small metalloprotein called
cytochrome c.
O
H3C O CH3
(dione) CH3 Ubiquinone (CoQ)

H
H3C O (CH2 C C CH2)6-10H (Oxidized)
O
+e e
O
H3C O CH3
Semiquinone anion (CoQ2 -)
CH3
H (Free radical anion)
H 3C O (CH2 C C CH2)6-10H
O
+2H 2H
+e e
OH
H 3C O CH3
(diol) Ubiquinol (CoQH2)

CH3
(Reduced)
H
H3C O (CH2 C C CH2)6-10H
OH
Ubiquinone functions in the oxidation-reduction reactions of cellular respiration. It is a stronger

oxidizing agent than both NAD+ and other flavin coenzymes, and can therefore be reduced by NADH and
FADH2. Because it has three oxidation states, the coenzyme can accept or donate either one or two
electrons.
The three exchangeable redox forms are:
(1) the oxidized CoQ (ubiquinone)
(2) the partially reduced semiquinone free radical anion. In electron transport, the free radical exists as the
dianion CoQ2-.
(3) completely reduced CoQH2, (ubiquinol).
Vitamin D
Vitamin D is a steroid that induces changes in gene expression by binding nuclear receptors.
24 24
H2C 25
HO 25 OH
1 1
3 3
HO HO
Vitamin D3 1, 25-Dihydroxycholecalciferol
(Cholecalciferol)
The active form is called 1, 25-dihydroxycholecalciferol. Two separate hydroxylation reactions

generate the active form. These compounds regulate the assimilation of Ca2+ by the body. They work by
modulating (1) intestinal absorption, and (2) deposition of the cation into bone.
Chapter 11
Carbohydrates and Glycoconjugates
11.1 Carbohydrates: Definition (saccharides, sugars)

The simplest sugar is ethylene glycol, the primary component of (old-school) antifreeze.
H2 C OH sweet &
deadly
H2 C OH
Be careful when storing it outside. It is both sweet and deadly to your children or pets.
11.2 Monosaccharides: Aldoses
Monosaccharides consist of one subunit. The structural designation is derived from L- and D-
glyceraldehyde. The letters D and L refer to the experimental direction of rotation of plane polarized light
in a polarimeter, dextrorotatory (right) and levorotatory (left), respectively. The designations R and S refer
to the absolute attachment of functional groups at the chiral carbon.
H O
( Aldoses )
D-glyceraldehyde C
C3 H C OH
CH2OH
H O
C
C4
H C OH Structural Definitions:
. D-erythrose Hydroxyl to Right = Down (in the ring form)
H C OH
Hydroxyl to Left = Up
CH2OH
β = -OH Up at C1'
C5 α = -OH Down at C1'
H O H O
C C
H C OH HO C H
H C OH H C OH
H C OH H C OH Note: The second C from
the bottom imparts the
CH2OH CH2OH 'D' stereoisomer designation
D-ribose D-arabinose
C3 C5
D-glyceraldehyde D-ribose: in coenzymes, nucleic acids
C4 D-arabinose
D-erythrose D-xylose
D-threose D-lyxose
94 Chapter 11 Carbohydrates and Glycoconjugates
D-gulose
C6 D-idose
D-allose D-galactose: half of Lactose
D-altrose D-Talose
D-glucose: fuel for Glycolysis
Aldoses:
D-threose (not shown)
D-xylose D-lyxose
(CHO CH2OH, xylitol) (not shown)
H O
C
C5 H C OH
HO C H
H C OH
CH2OH
.
D-talose
D-galactose
D-gulose
H O
D-idose
C
Ketose
H C OH
D-mannose
HO C H D-fructose
H O H
C HO C H
C6 H C OH
HO C H H C OH
C O
HO C H CH2OH
HO C H
H C OH
H C OH
H C OH
H C OH
CH2OH
CH2OH
11.3 Monosaccharides: Ketoses

Some important examples of ketoses and some derivatives are:
C3: Dihydroxyacetone is an intermediate formed by aldolase in the glycolysis pathway
C4: D- threose. Use of the derivative dithiothreitol (DTT) is described in the Disulfide Bonds section.
C5: D-xylulose can be converted to the sugar alcohol xylitol, a common sweetener in chewing gum.
D-ribulose is made in plants, where carbon is “fixed” by Ribulose Bisphosphate Carboxylase.
C6: D-fructose. The structure, shown above to the right, is a ubiquitous sweetener and food source.
11.4 Structural Features

(1) Glucose and galactose are epimers, which only differ at carbon 4, where the hydroxyl group is either
down and up, respectively.
Chapter 11 Carbohydrates and Glycoconjugates 95
(2) The L- and D- forms of a chiral compound are called enantiomers. These two mirror-imaged
compounds rotate plane-polarized light in levorotatory (left-handed) and dextrorotatory (right-handed)
directions in a polarimeter.
(3) Fischer Projections. The atoms are shown as a vertical chain with C1 at the top. Substituents are shown
to the right and left side of the main-chain atoms.
(4) Haworth projections are the standard way to present ring structures formed by carbohydrates. Some
defined parameters are shown in the Glucose Cyclization figure on the following page.
11.5 Intramolecular Cyclization

Hemiacetals, Hemiketals. The following cyclization reactions form two different structural classes.
aldehyde or ketone + alcohol → hemiacetal or hemiketal
Specific structural transformations are:
(1)
H+
O H
O
aldehyde R1 C H hemiacetal
R1 C H
O O
H R2 R2
alcohol
(2)
H+ H
O O
hemiketal
ketone R1 C R2 R1 C R2
O
O R3
H R3
alcohol
Glucose Cyclization
The following mechanism leads to conversion of linear glucose into the cyclic ring structure.
H+ bond formed
OH CH2OH
H O
C1 5
H C1 H O H
H
spontaneous H 1 α "anomer"
H C OH H C OH OH
cyclization HO (down)
OH
HO C H HO C H H OH
H C OH H C OH CH2OH
5
H C5 O H - H C O H O OH
OH 5 H
OH H 1 β "anomer"
CH2OH CH2OH HO H (up)
a "pyranose" H OH
(ring: C5, O)
Haworth projections
Ribose forms two structures. The furanose form is energetically less favored in solution than the pyranose
form, yet it is ubiquitous in nucleic acids, ATP, NADH, and so on.
bond formed
H
H O OH
H O H
H H β -D-ribopyranose
C HO H
OH OH
H C OH
H C OH bond formed
H C O H HO CH2
O OH
H2C O H H H β -D-ribofuranose
H H
OH OH
11.6 Conformations: Sugar Puckers

Pyranose undergoes a conformational transition from the chair ring pucker to the boat form.
6
4 4 6
1
5 O O
3 2
1 3 2
chair boat
The sugar puckers of a ribose or deoxyribose in RNA and DNA are different. They are referred to as
the C-2’ endo and C3’-endo sugar conformations. (For details, see the Nucleic Acids-DNA chapter.)
5
O
One O and three 4 2 "envelope" "Twist," "Pucker" - sugar
Cs form a plane. 1 conformation, torsion angles
3 This property is important to
distinguish different DNA and
One C is "puckered" out of the plane. RNA structures.
11.7 Sugar Derivatives

Sugar Alcohols
ATP ADP
sugar alcohol sugar phosphate

(e.g., glycerol) (e.g., glycerol phosphate)
R-O-H R-O-Pi
Sugar Acids
OH
H CH2
COO HO C
O
O O
OH
HO OH HO OH
HO
e.g., glucuronic acid Vitamin C
(ascorbic acid)
(in the lactone diol form)
.
Amino Sugars
Three major amino sugars exist in cell surface polysaccharides.
3
H3C O
CH2OH CH2OH C
O O NH O COO
OH
OH HO
HO OH OH OH
NH2 NH O
glycosidic H CHOH
bond C
O CHOH
α-D-glucosamine CH3
CH2OH
(GlcN) N-acetyl glucosamine N-acetylneuraminic acid
(NAG) (NAM)
NAG and NAM are enriched in brain tissue and ubiquitous in protein-linked carbohydrates, such as the
blood group determinant antigens. Do not confuse NAM with N-acetylmuramic acid (which is shown, for
example, in Moran et al. on p. 237).
Myo-inositol
This is a monosaccharide that is stabilized because it lacks a ring oxygen. It is released as inositol
triphosphate in response to a cellular “second messenger” signal transduction process.
OH OH OH
3 2 180o rotation 6
OH HO 5
OH 1 4 OH 1
4
5
HO 3 OH
6 2
OH OH OH
Sugar-X Derivatives
In these cases, X is a “natural product” compound.
Examples:
(1) rose pigment
(2) vanilla flavor
(3) salacin. This derivative of the pain relief medication aspirin was found initially in willow bark by
caveman-era humans.
CH2OH
β Note the ortho glucosyl and hydroxyl groups on
O benzene. Lignins in wood are enriched in
glucose polymers of such poly phenolic and benzylic
structures.
Aspirin (acetyl salicylic acid) is shown and described in the Eicosanoids section in the Lipids chapter. It
also has anti-inflammatory activity and is used to prevent heart attack.
11.8 Disaccharides
Two monomers linked by a glycosidic bond.
Sucrose. An unusual structural feature is the presence of two anomeric linkages. The formal chemical
name of sucrose is: O-α-D-glucopyranosyl-1, 2-β-D-fructose (or fructoside).
CH2OH
O
glucose OH
HO
α
HO glucosyl (α1 β2) fructose
O
CH2 O β
fructose HO HO
CH2OH
OH
Lactose. galactosyl β(1 → 4) glucose.

CH2OH
CH2OH O
galactose HO O OH glucose
O OH
OH
OH
OH
b
11.9 Polysaccharides
Carbohydrate polymers made from the same or mixed monomers.
Purposes
(1) Energy precursor storage: compact way to preserve the carbohydrate resources Example:
Glycogen produces glucose-1-phosphate, which can be converted to glucose-6-phosphate, the fuel for
glycolysis.
(2) Decreases diffusion rates of molecules located near membrane surfaces, which are composed of
glycolipids, proteoglycans, glycoproteins and ribose-enriched nucleic acid components.
(3) Regulate osmotic pressure: High molecular weight carbohydrates attract H2O because it tries to
dilute the carbohydrate enough to establish equilibrium concentrations with that of the bulk fluid (serum,
cytosol, organelles, etc.), which is usually much lower. Because the subunits in the polymer are
interconnected, they cannot be diluted. As a result, the cell remains wetted and “slippery.” This is a key to
the structure of many biocoating materials.
Starch
This polysaccharide contains both amylose and amylopectin. Starch is readily degraded to glucose units by
the hydrolase Amylase in saliva, the first stage of food digestion.
(1) Amylose (unbranched).
The structure is linear. It only contains α (1→4) linked glycosidic bonds.
CH2OH CH2OH
O O CH2OH
R2 Glucose
OH OH
R1 O O subunits: O
OH
HO HO
HO OH
α (1 4) linkage HO
(2) Amylopectin is branched because it contains both α (1→6) and α (1→4) linkages.
CH2OH
O 1
OH
R1
O O α (1 6)
α (1 4)
OH CH2
CH2OH 6
O O
R2 OH OH
R3
O O O
OH α (1 4) OH
Glycogen
This polysaccharide is essentially the same as amylopectin, except that it contains more branches. The
glucose–glucose bonds are cleaved by the enzyme Glycogen Phosphorylase a. Branched polymer has more
exposed glucose “ends,” it can supply them more efficiently than in the linear form.
Cellulose. This polysaccharide constitutes 50% of the carbon in the biosphere.
OH OH
R1 CH2 OH CH2
O O HO
O O
HO
O O HO O
OH CH2 OH R2
OH
(i) All of the linkages are β (1→4). This leads in part to the solid nature of wood.
(ii) Note that the oxygens of every other sugar flips with respect to its neighbors.
Chitin. Present in insect and crustacean exoskeletons.
CH3
C
H O
CH2OH N
O O
R1 OH OH R2
O O O
N CH2OH
O H
C
CH3
(i) Contains only β (1→4) linked NAG subunits.

(ii) Note that the sugar oxygens flip with respect to each other.
11.10 Carbohydrate-Protein Conjugates

Peptidoglycans
(1) This cross-linked material forms the structural support in bacterial cell walls. The peptides are (ala-
isoglu-lys-ala) and (gly)5.
NAG NAM NAG NAM
peptide 1 peptide 2 peptide 1 peptide 2
(2) Penicillin inhibits formation of the linkage between peptides 1 and 2. Lack of bond formation kills
the bacterial cell. The drug, which was discovered by Alexander Fleming, was the first known antibiotic.
(3) Lipid-containing examples of peptidoglycans also exist.
Glycoproteins. Three types of linkages exist between protein- and carbohydrate-based species.
(1) O-linked: Three amino acids are involved: Ser, Thr, hydroxylysine (in collagen).
HO
CH2 protein
O chain
O
OH C
α e.g., NAG ser
HO O CH2 CH
protein
. N H
N O
H
C
protein
CH3 chain
(2) N-linked: Generally linked to asparagine via an amide linkage.
HO
protein
O
chain
H O C
CH2
O N C CH2 CH
OH
N H
HO O
N protein
C CH3
H chain
(3) P-linked: for example, a phosphatidyl inositol diacylglycerol linked sugar. Note that the lipid
anchor is connected to asparagine through the following sequon (i.e. a small conserved sequence): asn – x –
ser/thr.
Gal
Ser H
protein Asn X or N (CH2)2 O Pi Man Man Man Gal Gal Gal
Thr
NAG
ethanolamine
a
"Sequon" H2C O Pi inositol
O
C O CH phosphatidyl
inositol
C O CH2 Lipid
O
Proteoglycans
These components of the extracellular matrix contain approximately 5% of the reinforcing structural
proteins collagen and elastin. They also consist of unbranched disaccharides, such as glucuronyl – NAG.
(1) Hyaluronate and Keratin Sulfate. Hyaluronate is a disaccharide-repeat unit in a glycosaminoglycan

polymer found in our joints. The polymer is very viscous and hydrated, so it makes an excellent shock
absorber and lubricant. Keratin sulfate is a major component of cartilage.
(2) Heparin, a sulfate-modified carbohydrate, is used to inhibit blood clotting.
Since heparin is highly anionic it competes with DNA-binding proteins and dissociates protein-DNA
complexes. Heparin-Sepharose affinity chromatography is used to purify DNA-binding proteins, such as
the restriction endonucleases.
(3) Cartilage. Cushioning material in skin and joints composed of bottle-brush shaped proteoglycans.
(4) Chondroitin sulfate: High molecular weight carbohydrate (Mr ~ 2x106 Da) that is an effective cushion
between bones, muscle, keratin, skin, etc. It modulates compressibility. It is a major component of
cartilage.
(5) Lysozyme is an antibacterial agent because it hydrolyzes peptidoglycans in bacterial cell walls.
General Physiological Functions of Carbohydrates (CHO)
(1) Attachment: CHO is added in the Golgi apparatus.
(2) Export: moving proteins out of the cell. Carbohydrates are typically added when the protein is to be
exported (e.g., on antibodies).
(3) Clearance: destruction of used proteins. The length and degree of oxidation of the carbohydrate are
indicator of the status of the structure. These characteristics determine whether the attached protein is
preserved in circulation or cleared/destroyed. The components are dismantled and reabsorbed by the cell.
11.10.5 Carbohydrate-Specific Binding Proteins

Lectins are plant proteins that recognize and bind carbohydrates. Recognition depends on the specific
monosaccharide or sequence. Purified lectins are used to isolate and characterize carbohydrate chains in
CHO-protein structures, CHO-lipid complexes, cells, viruses, and so on.
Example: Ricin
Two proteins from castor beans, Ricin A and B. They form a heterodimer complex in which each
subunit has a distinct activity.
Related proteins occur in corn seeds, beans, cucumbers and other plants. The idea is that they are
intrinsic pest management complexes, which inactivate translation of an invading mold or bacterium, yet
maintain protein synthesis requirements of the seedling.
Ricin B is a lectin. Lectins binds specific carbohydrate sequences. They are available as commercial
tool to differentiate amongst a variety of carbohydrate analytes. Ricin B attaches to the carbohydrate chains
of cell surface antigens, directing the catalytic ricin A subunit to the malignant cells. Once there, the
ribosome inactivating protein (RIP) Ricin A inactivates protein synthesis in the targeted cell, which kills it.
Ricin A cuts ribosomal RNA at a specific nucleotide and thereby inactivates catalysis of protein synthesis
by the damaged ribosome.
The activities of Ricin A, and other RIPs, have been investigated as possible medical reagents. The
premise is that the RIP can be connected covalently to a targeting molecule, such as an antibody or nucleus-
directing protein, and the connected protein will escort the RIP into the targeted cell. The RIP will stop all
protein synthesis of that targeted cell and thereby stop the cancer.
11.11 Synthesis and Structural Characterization

(1) Synthesis. It is possible to synthesize carbohydrates by machine, although it is not done as a wide-
spread practice. Their intrinsic ability to hydrogen bond inter- and intramolecularly make them stick
together. This is useful in their many biological niches, but makes working with them difficult.
(2) Chemical Reagents. The reagent periodate reacts with aldehydes, reducing them to red iodoalcohols.
Many other functional group tests exist; for example, the DNS test for ‘reducing ends,’ the Benedict test,
and so on.
(3) Biophysical Characterization. A lot of analytical work has been done to characterize the structures,
motions, interactions and reactions and so forth of carbohydrates. Especially useful information has come
from circular dichroism (CD), mass spectrometry (MS) and nuclear magnetic resonance (NMR) studies.
Chapter 12
Lipids
12.1 Structural Overview

Key Property. Water Insolubility. The key feature of lipids is their solubility in non-polar solvents, and
cellular environments, and their insolubility in polar solvents such as H2O. They have limited or no water
solubility. As a result, they block the movement of polar substances.
Structural Diversity. A wide variety of structures exist.

(1) Fats and Oils: Amphipathic alkane and/or alkene chain structures with Cn-length tails
(2) Cholesterol: an isoprene-derived four-ringed steroid structure
(3) Eicosanoids: Prostaglandins thromboxanes and leukotrienes. hormone-like molecules
(4) Waxes: Cn - alcohols; derivatives
(5) Terpenes: ubiquitous covalent compounds composed of 2 or more isoprene subunits
(6) Cerebrosides, Gangliosides, Sphingomyelins: all enriched in nerve and brain gray matter
12.2 Saturated and Unsaturated Fatty Acids

Table 1. Correlation Between Micelle Formation and Structure
Selected Saturated and Unsaturated Fats. 1
Number Number of Structural Common Tm (°C)

of Cs double bonds Abbreviation Name
12 0 *laurate 44
(like in SDS)
14 0 *myristate 52
16 0 *palmitate 63
16 1 palmitoleate - 0.5
18 0 *stearate 70+
18 1 C18:1∆9 *oleate 13
18 2 C18:2∆9, 12 linoleate -9
18 3 C18:3∆9, 12, 15 linolenate - 17
20 0 arachidonate 75+
20 4 C20:4∆5, 8, 11, 14 *arachidonate - 49
22 0 behenate 81+
24 0 lignocerate 84+
1
See p. 255 in Horton et al., Principles of Biochemistry (4th ed.), 2006.
12.2.1 Stability Measurements: Melting Profiles

The melting temperature (Tm) is used to quantify the capability of a lipid to form a gel-like complex, called
a liquid crystal, from a solution containing small aggregates of dispersed lipid molecules. The Tm is
proportional to the enthalpy change (∆H°d) for the transformation between dispersed and liquid crystal
states. A higher Tm means the complex is more stable; lower Tm means it is less stable.
Chapter 12 Lipids 103
(1 double bond) ("saturated" lipids)
dissolved 16 ∆9 18 ∆9 12 14 16 18 20 24
(dispersed) small
A605
lower Turbidity
(Measured as a lower A605
due to less light scattering) increased
A605
ordered gel
liquid crystal
(membrane-like) -20 0 20 40 60 80
T (oC)
Melting Temperature (Tm)
Double Bonds Destabilize Lipid Assembly into Aggregates

The trend of Tm as a function of carbon numbers is plotted below. It indicates that longer chains form more
stable complexes. These complexes are called monolayers, bilayers, vesicles, and micelles. They are the
types of structures from which membranes are constructed.
90
saturated
80 lipids
Tm (oC) 70
60 unsaturated
lipid Tms are
50 lower by ~ 60oC
40
12 14 16 18 20 22 24
n (carbon atoms)
The explanation for this trend is that introducing a double bond produces a kink in the chain. This kink
prevents the chain from forming strong surface-surface interactions with the neighboring lipids in a
membrane or other liquid-crystal type systems, for example, vesicles, micelles, and lipid rafts.
O
C O-
O
O
C O-
C O-
Bend obstructs
No bend: forms membrane packing
packed membranes
more effectively
C12 C9
C9 C10
Stearate Oleate Linolenate

(saturated) (cis ∆9) C15 (cis ∆9, 12, 15)
The unsaturated chain is less prone to ordered liquid crystal formation. They gel less easily.
104 Chapter 12 Lipids
12.3 Functions
(1) Lipids are the major structural components of biological membranes. Some are not synthesized by
humans, so they are essential in our diets. They are a means of energy storage and they form protective
barriers that facilitate sequestration of materials and processes.
(2) Energy Storage. Lipids store about 9 kcal per gram. Carbohydrates only store about 4 kcal/gm.
(3) Vitamins. Examples: vitamins A (retinal), D (cholecalciferol), E (α-tocophorol),
and K (phylloquinone).
(4) Novel functions. Examples include the pain response (prostaglandins); intracellular “hormones” (signal-
transduction, phosphoinositides); nerve cell membranes (sphingomyelins), lemon scent (limonene), and
many others.
12.4 Diacylglycerol Lipid Derivatives

These structures are the predominant components of biological membranes. Diacylglycerol consists of
glycerol esterified to two fatty acids. The components are two H2O-insoluble hydrocarbon chains, a
glycerol backbone; and a water-soluble head group.
O
C O CH2
O
C O CH
O
H2C O P O X
Chain lengths depend on
which fatty acids are present.
O-
Some typical head group structures are:

R - (CH2)2 - N+ (CH3)3 Phosphatidyl choline —causes platelet coagulation
+
R - (CH2)2 - NH3 Phosphatidyl ethanolamine—involved in lung tissue maintenance;
also a major constituent of bacterial
membranes
R - CH2CH(NH3+)(COO-) Phosphatidyl serine
R - CH2 (CHOH)CH2OH Phosphatidyl glycerol — involved in lung surface maintenance
Other examples (to look up) are:
Phosphatidyl cardiolipin
Sphingolipid — key component of lipid raft elements
Myelins — enriched in the myelin sheaths of nerves
12.5 Structural Motifs

Monolayers consist of a single ordered layer of lipid molecules, aligned with the polar head groups at one
end and the apolar hydrocarbon tails at the other.
H2O soluble head groups
H2O-soluble
Monolayers are also
head head head illustrated as::
group group group
H H2 H H2 H H2
liquid-monolayer H 2C C C O H2C C C O H2C C C O
interface O O
O O O O
O C C O O C C O O C C O
hydrocarbon
chains (not
H2O soluable)
Micelles. Monolayer spherical complex composed of 20 to 100 lipids. The inside chains are dynamic and
somewhat disordered. A hydrophobic molecule, for example, greasy dirt, can be trapped inside the central
hydrophobic core. This is the concept behind soap and detergent action. For example, many shampoos
contain sodium laural sulfate (aka. SDS).
Bilayers. These elements are the basis of membrane structure.

outer surface
inner surface
Vesicles. These spherical structures can sequester solutes or solvents within the central cavity.
.
Central
cavity
Membranes allow effective sequestration of charged species. Charged molecules cannot cross the
hydrocarbon layers, which act as a nonpolar barrier to transport between compartments. This phenomenon
is critical in the functions of nerve conduction, export of natural products, proper control of transmembrane
concentration gradients, signal transduction, mitosis, and so on.
12.6 Assembly
Energetics: Hydrophobic Effect Revisited
As with proteins, the hydrophobic effect stabilizes lipid assemblies. Enthalpy accrued by hydrogen bonding
between solvent water molecules drives the process, offsetting the large entropic cost of trapping the lipids
in the organized configuration(s). As with proteins, the assemblies denature. In this case, the lipids disperse
rather than unfold.
Bilayer stability is affected by the local water concentration, so lipid assembly reactions are strongly
subject to reagents that modify the hydrophobic effect. Recall that different lipids impart different
stabilities to these liquid crystal-type assemblies, depending on length, degree of saturation, and
polar/nonpolar properties. See the Stability Measurements: Melting Profiles section for details.
Osmolytes Modify Bilayer Stability

Osmolyte molecules change the osmotic pressure of a solution, which can, in turn, affect the stability of
adjacent lipid bilayer surfaces. For example, the unusual amino acid taurine is made in large concentration
within salt-water clam tissues. The purpose is to offset the osmotic pressure induced by the high
concentration of salt in sea water. Other osmolytes include glycerol, glucose, guanidinium, urea, and
metabolites such as the polyamines (e.g., spermidine) which are enriched in cell nuclei, and
neurotransmitters ( e.g., γ-aminobutyric acid; GABA), which function in nerve conduction.
Induced Assembly In Vivo

Bilayer structures are assembled in vivo by lipid chaperonins called snaps, snares and annexins. The
tendencies of the lipids to self-assemble to form membranes are enhanced by the proteins.
Natural vesicles are made from cholesterol and sphingolipid-enriched lipid rafts, which initiate the
process of packaging proteins intended for delivery from the golgi apparatus to the extracellular space. (For
details, see My Life On A Raft by Kai Simon in The Scientist, vol. 24, pp. 24–29, 2010.)
12.7 Structural and Dynamic Characterization

A few of the tools that have been used extensively to study lipids and their assemblies are:
(1) Freeze-fracture Electron Microscopy. This approach has been used extensively to produce suitably
dissected views of the structure of biological membranes.
(2) Atomic Force Microscopy. A microscopic stylus is scanned across a molecular sample at molecular
dimensions. This reveals the surface topography of the assembly.
(3) Fluorescence Affinity-Labeling and Time-Dependent Tracking. The flourescence quenching imaging
techniques allow scientists to follow the movement, assembly, and so forth, of lipids in membrane systems.
(4) Nuclear Magnetic Resonance. Nuclei such as 1H, 13C, 31P and 2H can be attached to lipids in specific
positions, allowing researchers to follow them structurally using various NMR techniques. Peak-width and
shape measurements and analyses allow characterization of the motional characteristics of the side-chains
and head groups, respectively, in the same molecule.
(5) Electron Spin Resonance. The nitroxide spin label can be attached to the head group, allowing a
researcher to study the motions of the chain structure, and many other specific details regarding the
dynamic nature of the lipid or assembly.
The following dynamic properties have been characterized:

Bilayers Are Charge Impermeable. Charge separation between the inner and outer surfaces of a bilayer
leads to a voltage. This potential energy is captured and used to power cells and drive cross-membrane
movement of metabolites. When the transmembrane potential is obstructed, the membrane leaks and these
functions are lost. Diarrhea is caused by cross-membrane potential decoupling. It is an important example
since it is responsible for more deaths worldwide (as a result of dehydration) than any other disease symptom.
Surface Diffusion. Lipid head groups diffuse laterally around the surface of the layer leaflet. The lipid
molecules move laterally via two-dimensional planar diffusion.
. lipid-solvent
interface surface
randomly
wandering
lipids
Cholesterol Solidifies Membranes. Lipid movement is modulated by the stability of liquid crystal
structure, which is affected by the lipid properties of the admixture that is present. The well-known lipid
cholesterol induces solidification of lipid assemblies, a necessary property to retain healthy pliable
membranes.
In dietary excess, cholesterol forms plaques in human arteries, leading to atherosclerosis, cardiac
arrest and strokes. The medical approach to controlling the level of cholesterol in human serum is
discussed in the Regulating Cholesterol Levels section.
12.8 Eicosanoids
These molecules regulate inflammation, pain, sensitivity, swelling, reproductive processes and are the
target of aspirin and similar pain medicines. The precursor fatty acid arachidonate is converted into a
prostaglandin by a cyclization reaction. The product can be converted to a variety of bioactive structures.
Biosynthetic Pathways
A variety of prostaglandins as well as prostacyclin and thromboxane A2 can be formed via the
prostaglandin H synthase pathway. Arachidonic acid is oxidized to form prostaglandin H2 (PGH2), which
is converted to the other products. The Prostaglandin H Synthase Cyclooxygenase (COX) step is inhibited
by aspirin. Such pharmaceuticals are called COX inhibitors or NSAIDS.
O
8 5
C O
Arachidonate OOH O
C O
11 14
2 O2
Prostaglandin H
(COX) synthase cyclooxygenase Inhibited
activity by aspirin 5-Hydroperoxy-∆6, 8, 11, 14
eicosatetranonate
O
O C O
Prostaglandin G2
O H2O
OOH
H O O
Prostaglandin H synthase
hydroperoxidase activity C O
O
O C O Leukotriene A4
Prostaglandin H2
O
OH
O
O C O
O
C O
O
OH
O Prostacyclin
Thromboxane A2
OH
OH
(Adapted from Horton et al., Principles of Biochemistry (4th ed.), 1996, p. 489.)
Biological Activities
These are potent molecules. Thromboxane A2 regulates confined changes in blood flow in areas where the
eicosanoid is produced. This leads to localized platelet aggregation, blood clots, and constriction of smooth
muscle within the arterial walls. In another important role, prostaglandins induce contractions of the uterus
during labor.
Plants produce linoleate, which is essential to humans to fuel arachidonate and eicosanoid
biosynthesis. Linoleate is required for survival.
O
H 3C C
CH O O CH3 O
C
O O O
C O NH
C O O
CH3 CH2 S
CH CH3
O
H3C CH3 OH
Aspirin Ibuprophen Acetaminophen Rofecoxib

(Vioxx R )
Aspirin (acetylsalicylic acid) inhibits the enzyme Prostaglandin H Synthase Cyclooxygenase (COX-1)
activity. Aspirin acetylates a serine hydroxyl near the active site, which irreversibly prevents arachidonate
binding. As a result, subsequent reactions in the eicosanoid pathway cannot occur.
COX-1 is expressed constitutively at low levels in a variety of cell types. It regulates mucin secretion
in the stomach, thereby protecting the gastric (stomach) wall. The result is that pain, inflammation and
stomach health are related. The connection really does depend on how one’s tummy feels.
Two isozymes of COX exist. The second one COX-2 controls the extent of inflammation, pain and
fever in a tissue. The omega-3 fatty acids, which are enriched in fish and flaxseed oils, are used to regulate
the onset of atherosclerosis. Ingestion of one aspirin per day is advised because it regulates the
prostaglandin pathway, and thereby inhibits cardiac arrest.
12.9 Phospholipases
These enzymes catalyze selective removal of fatty acids from the glycerol group of the phosphatidyl group.
For example, Phospholipase C functions in the Inositol-Phospholipid Signal Transduction Pathway (IP-
STP) by liberating inositol-1, 4, 5-triphosphate from the remaining diacyl glycerol. Each component
functions as a second messenger. (Details are described in IP-STP section of the Signal Transduction
chapter.)
12.10 Phosphoinositides
This molecule is a second messenger in signal transduction that contains the inositol group in the 1, 4, 5-
triphosphate form. Phosphatidylinositol (PI) is phosphorylated at C1. The other forms are 4, 5-
diphosphoinositol (PIP) and 1, 4, 5-triphosphoinositol (IP3).
O
C O CH2 Phospholipase C cleavage
O
C O CH O
O
O
- O
-
P
-
(DAG) H2C O P O
H O
O 6 5
H
1 HO OH HO H 4 O
Inositol-1, 4, 5-triphosphate
O-
H
hydroxyl pattern 2 3 O P
H H
1 2 3 4 5 6 (PIP2) O-
up-up-up-down-up-down
12.11 Steroids
The following examples range from (1) the much vilified membrane component cholesterol to (2) two
gender-specific hormones to (3) a physiological detergent called a bile salt.
(1) (2) (3) COO

+
OH OH OH Na
isoprenoid
R group
HO O HO OH
HO
Cholesterol Testosterone β -Estradiol Sodium cholate
Cholesterol
(1) Cholesterol makes membranes more rigid, modulating their pliability.
(2) Cholesterol is incorporated into chylomicrons in the gut after ingestion. It is stored within cells in
plasma lipoproteins.
(3) It can suppress transcription of specific genes.
(4) By decreasing serum cholesterol levels, one can decrease their risk of coronary heart disease. The statin
drugs are often used to treat excessive serum cholesterol levels, called hypercholesterolemia.
Testosterone and β-Estradiol: These are the male and female sex-determining hormones.
12.10.3 Sodium Cholate: This bile salt mediates lipid absorption by increasing their solubility.
12.12 A Potpourrié of Lipids

Vitamins (Coenzymes). Vitamins A, D, E, and K are all isoprenoid compounds made by plants. As
discussed in the Coenzymes section, these compounds are important in visual perception, calcium
metastasis, and scavenging free radicals.
(1) Vitamin A
(trans-Retinol) CH2OH
(2) Vitamin D
(Cholecalciferol)
CH2
HO
(3) Vitamin E
(α -Tocopherol) CH3
O
HO
The hydroxyl group forms a stable free radical by binding an electron. It's a free radical scavenger.
O
(4) Vitamin K
(Phylloquinone)
O
Vitamin K and Blood Clotting

This compound is required to convert glutamates into γ-carboxy-glutamate in several blood clotting factors.
These modified amino acids line Ca2+-binding grooves that bind a set of Ca2+ to create a positive site,
which binds strongly to electronegative proteins such as Fibrinogen.
O O
R1 NH CH C R2 Vitamin K- R1 NH CH C R2
dependent
carboxylase
CH2 CH2
CH2 CH
OOC COO
COO
γ-carboxyglutamate
Waxes
Myricyl palmitate is an example of a wax. Formed from a long-chain mono-hydroxylic alcohol, it is a fatty
acid ester.
Myricyl
alcohol CH2 CH2 CH2 CH2 CH2 CH2 CH2
CH2 CH2 CH2 CH2 CH2 CH2 CH3
O 9
O C CH2 CH2 CH2 CH2 CH2 CH2 CH2

CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3
Palmitate
Natural Products. This classification encompasses a huge cross-section of biologically active polyprenyl
compounds. Three examples are shown below:
CH2OH
9
Bactoprenol
(Undecaprenyl alcohol)
Limonene
(lemon odor, "zest") O
CH3
O
O
Juvenile hormone 1
Limonene is a lipid found in lemons that contributes to their acidic smell.

Bactoprenol is a lipid found in bacteria that is important in cell wall formation.
Juvenile hormone I regulates larval development in insects, that is, molting.
Chapter 13
Membranes
13.1 The Fluid Mosaic Model

Biological membranes contain a variety of components, including lipid-embedded proteins, glycoproteins,
proteoglycans, and so on. The lipid and protein content differs on the inside and outside surfaces. The
following figure shows the structure of a typical eukaryotic plasma membrane. Typical components are listed.
Extracellular space
Oligosaccharide chains
of glycosphingolipids
Oligosaccharide chains Lipid-anchored
of glycoproteins protein
Peripheral
membrane
protein Peripheral
membrane Integral
protein membrane
protein
Integral
membrane Cytosol
proteins
Membrane-bound Proteins
(1) Anchor proteins. These are linked to carbohydrates via the N- and P-type linkages described in the
Carbohydrate section.
(2) Integral proteins. O-linked; CHO outside; 20 amino acid tail (hydrophobic; membrane-embedded)
13.2 Detergents
These molecules are used to solubilize membrane proteins when purifying a biomolecule or fractionate
from cells. Though all of these detergents are not ionic, they are all amphipathic, possessing both polar and
nonpolar regions.
OH COO Na CH2OH
H O O (CH2)7 CH3
CH3 CH3 H
H3C C CH2 C O CH2 CH2 O H HO OH H H
CH3 CH3 n
H OH
HO
Triton X-100
(Polyoxyethylene p-t-octyl phenol) Sodium deoxycholate Octyl β-D-glucoside
112 Chapter 13 Membranes
(1) Triton X-100: This is the most frequently used detergent. 9 or 10 oxyethylene units (n) are attached.
(2) Sodium deoxycholate: similar to the compound CHAPS. The bile salt cholic acid is the protonated
species.
(3) Octyl β-D-glucoside: a lipid-carbohydrate bioconjugate.
13.3 Distribution of Lipids in Biological Membranes

The phospholipid contents are asymmetrically distributed with respect to the inner and outer leaflets of the
human erythrocyte membrane. Patterns found in erythrocyte membranes, as well as that of the bacterium
Micrococcus luteus, which also shows the asymmetry, are shown below.
60 TPL
Human red blood Micrococcus luteus
TPL cell membrane
50 membrane
40
Outside DPG
the cell 30 Sph PC PG
20
10
Percent
lipid 0
composition .
-10
PI
PS
-20
Inside PE
the cell
-30
-40
-50
The abbreviations are:

TPL total phospholipids Sph sphingomyelin
PC phosphatidylcholine PE phosphatidylethanolamine
PS phosphatidylserine DPG diphosphotidylglycerol
PG phosphatidylglycerol PI phosphatidylinositol
13.4 The Hydropathicity Scale

Seven-Helix Transmembrane Proteins. The protein bacteriorhodopsin from Halobacterium halobium is
a classic integral membrane protein, with seven polypeptide chains that span the membrane. The seven α-
helices are bundled and interconnected by consecutive loops that project into the solvent/cytoplasm from
both the inner and outer surfaces. The center of the complex contains a channel that transports protons from
one side of the membrane to the other.
NH3+
Extracellular
space
Plasma
membrane
Cytosol Seven membrane-spanning

α-helices
-OOC
Chapter 13 Membranes 113
The protein binds the light-harvesting coenzyme retinal, which it uses as an energy source to drive protons
through the membrane. Experimental demonstration of the use of this proton pump to drive ATP synthesis
is described in the Oxidative Phosphorylation section.
Measuring the Hydropathicity of an Amino Acid

One can determine the preference of an amino acid (aa) to distribute between water and a solvent that
mimics the environment within a generic membrane, that is, octanol. This involves placing equal amounts
water and octanol in one tube. Because they are immicible (do not mix), the added aa will distribute into
one or both of the solvents. One determines the partitioned percentage of the aa in each layer analytically.
This information can then be used to calculate equilibrium constants (and Gibbs free energies) for the
partitioning equilibrium aawater ↔ aaoctanol for each of the amino acids.
The results allow one to assess the hydropathicity of the molecule, which gives us a sense of their
preference for the aqueous environment. More hydrophobic amino acids prefer the interior of the lipid
bilayer. The free-energy changes for the partitioning process are listed below:
Table 2. Polarity Scale for Amino Acid Residues Based on H2O-Octanol Partition
Coefficients
Amino acid ∆G for transfer Amino acid ∆G for transfer
(kJ mol-1) 1 (kJ mol-1)
Isoleucine 3.1 Proline -0.29
Phenylalanine 2.5 Threonine -0.75
Valine 2.3 Serine -1.1
Leucine 2.2 Histidine -1.7
Tryptophan 1.5 Glutamate -2.6
Methionine 1.1 Asparagine -2.7
Alanine 1.0 Glutamine -2.9
Glycine 0.67 Aspartate -3.0
Cysteine 0.17 Lysine -4.6
Tyrosine 0.08 Arginine -7.5

1
Calculated as ∆G = -RT ln K,
The Hydropathicity Plot

The hydropathicity plot for bacteriorhodopsin is shown below.
2 Hydrophobic
residues
Free Energy Change
for transfer to H2O
(kJ/mol)
0
Hydrophilic
residues
-2
0 50 100 150 200 250

First Residue in Window
.
114 Chapter 13 Membranes
(1) Positive free energies in the hydropathy plot indicate that the plotted amino acid prefers the
hydrophobic environment associated with: (i) the protein interior, or (ii) being embedded within a
membrane, in an integral membrane protein.
(2) Negative free energies indicate that the amino acid will project into the aqueous environment. It is
hydrophilic.
If one squints, the pattern in the plot has seven positive peaks, indicating the presence of seven
membrane-spanning α-helices. The five clear hydrophilic regions (seven are expected) are the intervening
loops. Pattern tending toward more hydrophilic sequence are evident in the intervals where they do not
cross over into hydrophilic regime per se.
13.5 Lipid-Anchored Membrane Proteins

The following figure shows three types of lipid-anchored membrane proteins:
(2)
Protein
O C
NH
Phospho- CH2
ethanolamine CH2
residue
O Man GlcN Ins
O P O
O O
H2N
O P O
O
1 2 3
H2C CH CH2
O O
O C C O
Outer
Leaflet
Inner
Leaflet
C O
O S
CH2
Protein
(1) Protein
(3)
Fatty Acyl Proteins. The inner leaflet of the membrane in anchored to a myristoylated protein.
Glycosylphosphatidyl-Inositol Proteins. The parasitic protozoan Trypanosoma brucei has a surface
glycoprotein that is anchored by this protein. It is bound covalently to a phosphoethanolamine residue
which is also bound to a glycan. Phosphoethanolamine attaches onto the mannose residue of the glycan.
The inositol group of phosphatidylinositol is attached to the glucosamine residue of the glycan. The protein
is anchored to the membrane by the diacylglycerol portion of the phosphatidylinositol.
Prenyl-Anchored Proteins. This is an example of anchoring via the farnesyl group of a membrane protein
that has undergone prenylation. A covalent bond joins the isoprenoid chain to the protein membrane with a
thiol group of a cysteine residue close to the protein’s C-terminus.
These three anchor types can be found in the same membrane, but it does not resemble the structure
illustrated. The following compounds are abbreviated: glucosamine (GlcN), mannose (Man).
Chapter 13 Membranes 115
13.6 The Erythrocyte Cytoskeleton

Lipid bilayers are essential in the formation of a cell, however, they are very thin and do not contribute
much stability to the structural support network. The cytoskeleton acts like a protein skeleton within the cell
to provide the necessary strength to maintain cell shape, allow cellular movement, aid in intracellular
transport and provide protection. The cytoskeleton is distributed throughout the cell, attached to the inside
face of the membranes.
The inside face of the erythrocyte membrane is linked to a protein cytoskeleton network composed of
actin and a fibrous polypeptide, spectrin. These components are organized into a meshwork, which is
linked to the membrane-bound protein ankyrin.
Erythrocyte
Erythrocyte
membrane
Cytoplasmic
Actin surface of
membrane
Band
4.1
Glycophorin Ankyrin Spectrin

Anion
exchange
protein
The anion-exchange protein, Band 4.1, stabilizes the spectrin-actin association. Ankyrin anchors the
mesh-like pattern by binding the cytoplasm region of band 4.1, which binds to the cytoplasm region of
glycophorin A. Notice how this molecular framework resembles the re-bar reinforcement in cement
structures.
Chapter 14
Transport Through Membranes
14.1 The Transmembrane Potential
An electric potential (∆ψ) is formed by the separation of charges across the membrane of most living cells.
The typical standard ∆ψ across a plasma membrane is -60 mV, with an excess of negative charge inside the
cell. When a charged solute is moved across the membrane, equation 1 must be altered by adding one term
to take into effect the membrane polarization.
∆G = R T ln (c2/c1) + z F ∆ψ (1)
where z is the unit charge on the solute that is transported, F is the Faraday constant (96.48 kJ V-1 mol-1),
and ∆ψ is the transmembrane electric potential, measured in volts. Free energy is released (accrued) as a
positively charged solute moves from the positively charged extracellular side of the membrane to the
intracellular negative side. In contrast, transporting a negatively charged solute to the intracellular side of a
membrane costs energy.
14.2 Active Transport

Active transporters are driven by many different types of energy sources, for example, ATP, light, ion
gradients. A direct source of energy is required to run primary active transport. Proton or sodium
concentration gradients drive secondary active transport. Some active transporters are fueled by light, for
example, bacteriorhodopsin, which is described in detail in the Oxidative Phosphorylation section.
All organisms have a number of ATP-driven ion transporters, called ion-transporting ATPases.
Active transporters, such as the Na+, K+ and Ca2+, H+ ATPases, create and preserve the ion concentration
gradients across cellular membranes.
Table 3. Energy Sources for Active Transport
Type of Transport Energy source Transporter Transported species
Primary Light Bacteriorhodopsin Protons

ATP ATPase Ions
ATP P-Glycoprotein Nonpolar compounds
Substrate oxidation Electron transport Protons
(electron transfer) proteins
Secondary Proton gradient Lactose permease Lactose
Sodium gradient Active glucose Glucose
transporter
Chapter 14 Transport Through Membranes 117
14.3 Ionophores
Valinomycin and gramicidin D are potent antibiotics that cause cations to leak across membranes,
destroying the transmembrane cation gradients. Since these gradients are required to produce ATP and
drive secondary active transport, ionophores kill cells.
Valinomycin
This is an extremely selective ionophore, which binds K+ 1000-fold more strongly than Na+. The complex
is lipid soluble because the ionophore has nonpolar side chains that interact with the acyl chains of
membrane lipids. As a result, the [valinomycin·K+] complex can diffuse across the membrane and release
the ion on the other side.
The secondary structure of valinomycin and the tertiary structure of the [valinomycin·K+] complex are
shown in the following figure.
H H
H H
C O C C N
H C C
N O H3C O H C
H C HC O O H
C H O CH CH3 C
3
O C CH3 C
O
N H
H3C C O CH3 H3C H
C C H C C H
H3C H H3C O
C
H N
O
C O H3C
CH3 C
C H
H H3C O
O O CH 3
C
C HC H 3C O
CH3
C CH3 O
O C
N CH O H C H
H C
C O C C N
H
H CH
H3C CH
3 (K+ is in the center)
. .
Gramicidin D
This compound has ion channels that are continuously dissociating and reforming every second, allowing
and inhibiting ion conduction with each alteration.
Hole diameter = 0.4 nm
D-Leu O
H O D-Leu C NH
C L-Ala L-Ala
L-Trp L-Trp L-Trp CH2
HN Gly D-Val
D-Val D-Leu CH2
D-Leu
L-Val1 L-Val OH
L-Trp
.
Unlike mobile carriers who need to diffuse through the lipid bilayer, channel-forming proteins do not.
Therefore, the rate of transmembrane ion diffusion by gramicidin D is 104-fold faster than that of
valinomycin.
118 Chapter 14 Transport Through Membranes
14.4 The Acetylcholine Receptor Ion Channel

Recalling from the Enzyme Inhibitor section, acetylcholine esterase degrades the neurotransmitter
acetylcholine, which terminates synaptic transmission and resets the open receptor for another nerve
impulse. For proper nerve conduction, the appropriate type of membrane polarization must be present. The
following steps are necessary:
Acetylcholine
Vesicle-
membrane
fusion
Plasma
membrane of
presynaptic
neuron
Acetylcholine
release to cleft
The plasma membrane of the postsynaptic neuron is depolarized by the released neurotransmitter, which
binds the port protein and activates ion channel opening.
(polarized) (depolarized)
(1) At rest, the channel is closed and the inside of the postsynaptic neuron has more negative charge than
the outside, creating a negative membrane potential.
(2) The presynaptic neuron releases acetylcholine which then binds to its receptor, allowing an influx of
sodium ions. The entrance of sodium depolarizes the membrane, reducing the electric potential.
14.5 Lactose Permease and Secondary Active Transport

In the electron transport chain of E. coli, oxidation-reduction reactions produce oxidized substrates (Sox)
from reduced substrates (Sred). During this process, the proteins of the chain pump a proton across the
membrane with each formation of a redox species, forming a proton concentration gradient.
Extracellular
space H+
Sred
Cytosol Lactose H+
Sox
As these protons move down their concentration gradient, energy is released, which causes one lactose to
be translocated into the cell through a symport, along with one H+, by the enzyme lactose permease.
Chapter 14 Transport Through Membranes 119
14.6 Mechanism of Transport by Na+, K+ ATPase

Na+, K+ adenosine triphosphatase (ATPase) pumps three sodium ions out of the cell as two potassium ions
enter the cell. Energy must be supplied by the hydrolysis of ATP because both ions are moving up their
concentration gradient. The mechanism following mechanism is adapted from Moran et al. Biochemistry (2nd
ed.), 1994, pp. 12–31; Fig. 12.35.
Extracellular
space Start
5. The three Na+ sites of the ATPase 1. Three Na+ in the cytosol
are again exposed, and the process bind to the exposed Na+
can begin again. sites on the ATPase.
Extracellular OH
space
Start 3 Na+
(loaded
Extracellular
Extracellular for export) space
space
Cytosol
Pi ADP
Cytosol OH ATP
Pi
H2O
2 K+
in
2. Phosphorylation of an aspartate
residue of the ATPase induces a
4. Upon dephosphorylation, the conformational change that results
ATPase changes conformation in the release of Na+ into the
and releases K+ into the cell. extracellular space.
2 K+
+ (loaded
3 Na
for import)
out
Extracellular
space
Extracellular
space
Pi
Cytosol
3. Two K+ outside the cell bind to Cytosol
Pi
the exposed K+ sites on the ATPase.
14.7 Ion Channel Blockers

The compounds ouabain and digitoxigenin are extracted from the purple foxglove. The mixture is used as a
heart medication called digitalis. These cardiotonic steroids are extremely toxic because they inhibit Na+,
K+ ATPases. Upon binding to the extracellular domain of the ATPase, the compound freezes the
mechanism at the phosphorylated state, channel opened outward.
O
O O
O
OH CH3
Digitoxigenin Ouabain HO
CH3 HO
CH2
CH3
H OH
OH HO O O
CH3 OH
HO H H
H H
OH OH
Digitalis works by increasing sodium influx into the heart muscle cells, which effectively activates the
Na+, Ca2+ antiport system. Sodium leaves the cell as calcium enters, which increases the strength of the
heart muscle contractions
Chapter 15
Signal Transduction
15.1 Signaling Pathways: Hormones, GTPases, Second Messengers and

Intracellular Regulation
Signal Transduction occurs when a molecular signal is transferred across the plasma membrane of a cell.
The signal is passed to the inside of the cell by the following chain of events:
(1) Extracellular first messenger molecules (typically hormones) bind to a receptor, which is closely
associated with a transducer enzyme, within the membrane. Each receptor is activated or repressed by the
signaling/binding molecule, which is designed physiologically to affect its activity in a certain way – to
control the intended cellular function.
(2) The transducer then transmits the signal that instigates production of the second messenger within the
cell. Transducers are commonly Guanine Triphosphatases (GTPases), which control a variety of biological
functions.
(3) Second messengers can induce:

(i) a cytosolic enzyme to modify their substrate protein,
(ii) a membrane-bound port complex to allows entry of a particular ion, for example, Ca2+,
(iii) and many other responses.
Hormones and growth factors are examples of first messengers that have specific binding sites on particular
cellular receptors.
(4) A protein within the membrane transports the signal to the cytosol-side membrane-bound effector
enzyme.
(5) The effector enzyme catalyzes formation of the second messenger, typically a small molecule (e.g.,
cAMP, IP3, DAG).
(6) The signal is then transferred to its final destination by the second messenger to produce the
intracellular response, for example, regulation of some reaction or pathway in the cytosol, nucleus,
mitochondria, and so on.
External stimulus
(first messenger)
Outside
Membrane Effector Plasma

receptor Transducer membrane
enzyme
Inside
Cytosolic
Second messenger and nuclear Cellular
effectors response
In contrast to the large number of ligands, membrane receptors, and transducers that are known, only
a few effector enzymes and second messengers have been found. Therefore, in response, each cell has a
Chapter 15 Signal transduction 121
specific detecting ability to notify when an extracellular signal resides at the surface but use familiar
pathways to carry these signals through to the inside of the cell.
15.2 The Adenylate Cyclase Signaling Pathway

Activation Pathway. When (1) an extracellular hormone binds to a stimulatory transmembrane receptor
protein (Rs), (2) a stimulatory G protein (Gs) is activated. The Gs protein functions by (3, 4) activating the
membrane-embedded enzyme adenylate cyclase, which (5) produces cyclic AMP. Cyclic AMP (6) activates
protein kinase A which leads to (7) phosphorylation of targeted cellular proteins, and (8) the intended
intracellular response.
Extracellular
Hormone-Stimulated space
Control Phase GTP-dependent
GTP-dependent
activation lobe
inhibition lobe Inhibitory
Stimulatory hormone
(1) hormone Adenylate
Cyclase Hormone-Inhibited
Control Phase
Rs
α Ri
GDP Gi
β γ GTP GTP GDP
(3)
(2) (4)
Gp
(5)
GDP GTP GTP GDP

ATP cyclic
AMP
Protein kinase A
(inactive) AMP
(6) Phosphodiesterase
Kinase Activation (second messenger Cytosol
or Inhibition
Phase
destroyed)
Protein kinase A
(active)
(7)
ATP ADP (8)
Protein OH Protein Pi Cellular
Response
Metabolic Functional
Control Phase
Inhibitory Pathway. This enzyme can also, in specific cases, be inhibited. This is variation on the G-
coupling theme is mediated by an inhibitory G protein (Gi). When a hormone binds to an inhibitory
receptor (Ri) in the membrane, it binds adenylate cyclase through the G protein, thereby inhibiting the
enzyme. The basic pathway is the same as activation. The specific signal molecules and intracellular targets
are different, depending on what is being controlled.
Some hormones or cellular conditions stimulate the stimulatory pathway, others induce the inhibitory
route. Cellular development, disease progression and many means of physiological control depend on this
type of peripheral membrane signaling system.
122 Chapter 15 Signal transduction
15.3 The Inositol-Phospholipid Signaling Pathway

The steps are summarized below:
(1) When a hormone binds to its transmembrane receptor (R),
(2, 3) it activates the G-protein Gp.
(4) Membrane-bound Phospholipase C (PLC) is stimulated by the activated Gp to catalyze hydrolysis of the
phospholipid PIP2, which is located on the inside wall of the plasma membrane.
The act of splitting PIP2 into

(5) inositol-1, 4, 5-triphosphate (IP3) and
(5’) diacylglycerol (DAG) begins the process of using second messengers to transport/diffuse the signal to
their targets in the cell interior.
(6) IP3 travels and diffuses into the endoplasmic reticulum, acting to open Ca2+ channels in the membrane
that can only be accessed by IP3.
(7) This releases the stored Ca2+ into the cell to produce cellular functions, specifically PKC (see 6’).
(6’) Diacylglycerol is used in the plasma membrane to activate the calcium- and phospholipid-dependent
enzyme Protein Kinase C (PKC).
(7’) The phosphorylated target proteins go on to catalyze, regulate, or otherwise affect various metabolic
processes, thereby producing a wide variety of cellular responses.
Hormone-Stimulated or
Inhibited Control Phase
PLC (Activated to cleave the
(1) Stimulatory inositolphosphate-
hormone modified Extracellular
phospholipid) space
R
α
GDP
GDP α
GTP
(3)
(5') Cytosol
Gp β γ
(2) (4) PIP
2
GDP Gp DAG ATP
GTP PKC
IP3-Dependent (bound and IP3
Activation activating PLC)
Protein O H
Phase γ (6')
β (5)
Kinase
Protein O Pi Activation
Phase
(7') Cellular responses
Metabolic Functional
(7) Control Phase
(6)
Ca2+ Cellular responses
Lumen IP3 - gated Ca2+

channel
Chapter 15 Signal transduction 123
15.4 Phorbol Myristyl Acetate

The plant Croton tiglium stores the toxin phorbol ester in its leaves. This natural product mimics
diacylglycerol (DAG), and thereby activates PKC.
CH3
(CH2)12
CH3
O C
C O
O
H3C O
CH3
HO
H3C CH3
O HO
CH2OH
One difference between DAG and phorbol ester is that the toxin is metabolized inefficiently, and therefore
over-activates PKC. Phorbol myristate acetate is a carcinogen and strong tumor promoter. This may be
attributed to the extended activation of PKC, which leads to unrestrained growth of cancerous cells.
15.5 The Insulin Receptor
This insulin receptor complex is composed of two parts. The extracellular portion is made of two α chains
which both have an insulin-binding site. The transmembrane portion is composed of two β chains, which
each contain a cytosolic Tyrosine Kinase domain. Glycogen metabolism involves regulation by the activity
of both insulin and the counteracting protein glucagon.
Insulin
(1)
Insulin
(2)
Dimerization α α
α S S S
Extracellular S S
space S
S S S
β β β
Gs Phospho-
Cytosol
protein lipase C
Tyrosine Kinase
domains
(3) (4) (5)
other
IP3 DAG
phosphorylations
and responses (6) (7)
Ca2+ Phospho-
release kinase C
(8) Cellular responses
(Adapted from Moran et al., Biochemistry (2nd ed.), 1994, p. 12-46; Fig. 12-51.)
124 Chapter 15 Signal transduction
Binding of insulin to the α chains causes autophosphorylation of tyrosine residues in the intracellular
domains of the insulin receptor. Tyrosine kinase also phosphorylates other cellular proteins, creating a
cascade of results that produce the overall response to insulin binding.
Prior to use, the insulin monomers are transported through the circulatory system as a hexameric zinc
complex. For details regarding (1) the medical connection between insulin and diabetes, and (2) the post-
translational steps in the maturation of insulin, see the Protein Maturation section.
15.6 Glucagon
The action of glucagon, which ultimately is to stimulate glucose biosynthesis is discussed in the Glucagon
and Fructose-2, 6-bisphosphate section of the Gluconeogenesis chapter. The protein is interesting because
it illustrates the multi-faceted binding capability of a single polypeptide. Glucagon is a symmetric dimer
that contains: (1) a site that binds glycogen or oligosaccharides, (2) a catalytic site that binds glucose-1-
phosphate or glucose, and (3) an allosteric site that binds glucose-6-phosphate, AMP and ATP.
Table 4. Summary of Signals and Target Functions in Signal Transduction

Pathway Second Messenger(s) Function Activated
cAMP Cyclic AMP (cAMP) Protein Kinase A
Inositol-P-lipid G-proteins Phospholipase C (PLC)
Inositol-P-lipid PIP2 (IP3) IP3-Gated Ca2+ Channel 1
Inositol-P-lipid DAG, G-proteins Protein Kinase C (PKC)
2+
Inositol-P-lipid Ca release Blood clotting, glucose metabolism, etc.2
Insulin tyrosine autophosphorylation Tyrosine phosphorylation
Glucagon Fructose-2, 6-bisphosphate Gluconeogenesis
1
For example, the released calcium can inhibit glycolysis. Calcium released from the lumen of the ER activates
Glycogen Phosphorylase Phosphatase. This traps Glycogen Phosphorylase a in the dephosphorylated inhibited
form and stops the release of glucose-1-phosphate from glycogen. As a result, the IP3 signal can turn off the
glucose fuel supply.
2
Calcium also activates Isocitrate Dehydrogenase allosterically, thus stimulating the Krebs Cycle.
15.7 G-Proteins
Range of Occurance. G-proteins mediate the control of cellular responses to external stimuli. They mediate
functions such as intermediary metabolism, cell growth and division, secretions, epidermal platelet
function, nerve growth, movement toward or away from a chemical stimulus and immune cell (interleukin)
function. Some specific examples are:
(1) Transducin (Gt) mediates visual excitation, after the retinal isomerization reaction occurs.
(2) Gk regulates potassium channels in heart muscle.
(3) The β- and α-adrenergic receptors in heart muscle stimulate Gk protein.
(4) Somatostatin regulates cell growth.
(5) Epinephrine (adreneline) regulates growth and “fight or flight” responses
(6) The insulin response
Targeting Agents. G-proteins are targeted by a variety of malicious and benign effectors. Examples
are:
(1) Cholera. The toxin produced by cholera catalyzes ADP-ribosylation of an arginine in GTPases,
rendering them inactive in signal transduction.
(2) Whooping cough (pertussus) toxin operates via the same mechanism.
(3) Caffeine, theophylline and theobromine are various N-methylated analogs of guanine. Since they mimic
GTP, they bind and stimulate G-proteins. The result is a set of natural stimulants that are remarkably
benign in humans.
(4) Phorbol esters promote tumor formation.
Chapter 16
Nucleic Acids: DNA
16.1 DNA and RNA

Discovery. DNA was first isolated and characterized by Frederick Meischer using pus collected from battle
wound dressings from the Prussian war.
Structure. Watson and Crick used x-ray diffraction data from Rosalind Franklin, and A•T , G•C ratios
from Irwin Chargaff, to surmise that DNA is composed of two intertwined strands, the famous double helix
structure. They proposed that DNA is stabilized by hydrogen bonding between A and T, and between G
and C in the respective A·T and G·C base pairs.
(1) These polymers are composed of ribose or deoxyribose, alternating with phosphate linkers. Strand-to-
strand binding is mediated by base pairing, that is, hydrogen bonding between the centrally located bases.
5'
3' A U O
5' end
5' end O
O O P
O
P H (or -CH3) O
O O O H
H N N H
CH2 CH2
H
O N O H
H H H N H N H
N H
H N O O
O O
O O H H
O P
P O
H (or -H) (or -H) H O
O O 3' end
3' end
3'
. 5'
(2) Hydrogen bond interactions occur between the ring substituents to form A•T and G•C base pairs.
A. Purines B. Pyrimidines
Adenine H H Thymine (Thy)

. (Ade) N O
or CH3 in Thymine
N H
N Uracil (Ura) H N
H
in Uracil
H N N O H
N
R R
Guanine Cytosine
(Gua) O (Cyt) H H
N
H 6 5 N 5
N1 7
H
N 4
H 3 R is ribose in RNA or
H 8
deoxyribose in DNA
N 2 N 4 N9 O 2 6 H
3 N1
H R R
(3) The deoxyribose or ribose C1’ atom is attached to the base ring nitrogen to form the glycosidic bond.
126 Chapter 16 Nucleic Acids: DNA
Functional Roles
(1) DNA is a stable reservoir of genetic information. Hershey demonstrated that DNA carries the genetic
information. Implanting cells with DNA, transforming them, can impart the new genetic phenotype.
(2) In RNA, the 2’-hydroxyl group catalyzes alkaline hydrolysis, making the chain readily subject to
breakage. As a result, RNA turns over quickly. It is a transient messenger source derived from the DNA.
This allows regulation of mRNA levels. The cell can make a lot of enzyme when the mRNA is initially
made then can degrade the mRNA easily when it’s served as the template for translation.
16.1.4 DNA Sequence Presentation

Watson-Crick double helices have a 5’ to 3’ strand polarity. The best way to show a DNA sequence
depends on what type of information is to be conveyed. Five common variations are shown below
5'
DNA sequence 5' - A C G T A T T G G G G T T G G G G T T - 3' O
O
P
Complementary O
RNA sequence
3' - u g c a u a a c c c c a a c c c c a a - 5' O CH2
O base
(lower case letters) H H
H H
deoxy- O O H
O
G A T C ribo- P
G A T C O
O
OH OH H OH 3'
HO Pi Pi Pi OH
Some others common representations are: (1) as letters: GATC, and (2) as the 5’-phosphorylated
pGpApCOH. (3) Purines are abbreviated as Pu or R; pyrimidines are Py or Y. Using these codes, a particular
sequence can be written as: PuPuPyPY or RRYY.
Combinatorial DNA/RNA Synthesis

This nomenclature can be used to show a set of sequences that vary at specified positions. Combinatorial
permutation sequences are really more than one DNA. They only vary at a single position. This can be
accomplished using synthetic DNA primers in the Polymerase Chain Reaction (PCR) technique. When the
primers are made, one programs the DNA synthesizer to insert all of the intended nucleotides into a
particular step. Since one draws from more than one bottle of reactant, the next position in the synthetic
product attached to the solid support matrix is more than one nucleotide (* below).
Coding DNA sequence 5' - A C G T A T T G G G G T T G G G G T T - 3'

*
Product DNAs 1 3' - T G C A T A A C C C C A A C C C C A A - 5' wild type
2 3' - T G C A T A A C C C C T A C C C C A A - 5'
mutants
3 3' - T G C A T A A C C C C G A C C C C A A - 5'
When the population of molecules is cleaved from the synthetic column, more than one molecule, varying
only at the intended position, is recovered. If three bottles were drawn from during one synthetic step, three
sequences are made, and so on.
Chapter 16 Nucleic Acids: DNA 127
16.2 Physical Properties

Ultraviolet Absorbance of the Bases. Light is absorbed strongly by nucleic acid bases at 260 nm. Their
absorbance occurs with a large molar extinction coefficient, so they can be detected with high sensitivity.
This property has been used in many ways.
(1) Absorbance approaches provide a convenient way to measure and study the equilibrium poise between
single- and double-stranded nucleic acids.
Ka
2 complementary single-stranded DNAs 1 duplex DNA
(2 ss DNAs) [higher A260] (ds DNA) [lower A260]
5' 3' 5'

3'
3'
3' 5'
5'
Other names for the reversible process are duplex-to-single-strand equilibrium, denaturation-renaturation
equilibrium and duplex melting reaction. Duplex formation is called annealing and hybridization in many
molecular biology procedures, for example, PCR. Duplex dissociation is typically called denaturation.
(2) The Beer-Lambert Law (Beer’s Law) allows one to convert measured A260 values to molar nucleic acid
concentrations.
A260 = ε260 b c (1)
The parameters are: A260 absorbance at a wavelength of 260 nm (A260)

ε260 the ‘molar extinction coefficient’ (units: M-1 cm-1)
b the cuvette pathlength (which is typically 1 cm)
c the molar concentration
Duplex Stability Measurements are used to study the denaturation of double helix (duplex) structures. This
approach can be used to determine equilibrium association constants (Ka) for different designed duplex
structures. By measuring Kd values at a series of temperatures and DNA strand concentrations, one can
determine the ΔG°, ΔH° and ΔS° values for duplex denaturation with the specified molecule or molecules.
(1) The stability of a duplex DNA varies with the percent G + C. To do this, chromosomal nucleic acid are
sheared to 200–1000 base pair lengths for measurements. The stability can be calculated in terms of the
melting temperature (Tm), the denaturation-renaturation midpoint, as follows.
Tm = 69.3°C + 0.41 (% G + C) (2)
A larger G-C content is correlated with a higher Tm.
(2) Cooperative Melting. When a duplex dissociates, loss of the first base pair makes the next one
dissociate more easily. This cooperativity makes many duplex dissociation events very rapid. Conversely,
formation of the first base pair association nucleates subsequent base pairing. Duplex formation is also a
cooperative process.
(3) To reinforce the points of similarity, the following plots compare melting curves obtained with two
different classes of biomolecules:
(i) Nucleic acid: duplex-to-single-strands dissociation-association equilibrium of a simple DNA
.
2 single strands
(10-20% more absorbance)

A260
double helix Melting Temperature (Tm)
(10-20% less absorbance)
0
-20 0 20 40 60 80
T (oC)
(ii) Lipid: liquid crystal-to-dispersed equilibrium of a fatty acid
.
liquid crystal
(more scattering)
Tm
A605 dispersed
(less scattering)
0
-20 0 20 40 60 80
o
T ( C)
This effect has been used to characterize the energetics of defined nucleic acid structures. Details are
described below in the Secondary Structure Predictions section.
16.3 Secondary Structure
Secondary Structure Maps
The base pairing pattern, the secondary structure, formed by one long RNA strand is shown below as: (1) a
three-dimensional view, and (2) a two-dimensional secondary structural map.
Renaturation
Single stranded
Denaturation
('Melting')
Complex
5' secondary
3' structure
5' 3'
a hairpin
a two-way junction a pseudoknot

.
The large secondary structures of, for example, ribosomal RNAs and virus genomes, look like complicated
city roadmaps.
Empirical Predictions. The data base obtained from duplex melting studies can be used to calculate the
best predicted secondary structure, as well as close next-best variants. In mathematical models, a given
secondary structure is represented by placing the backbone of the structure on an arc. Base pairs are
represented as lines (chords) that connect the two respective nucleotide positions on the arc. Different
secondary structural variants have different specific chord patterns, which represent the base paired
secondary structure, when folded together. RNA secondary structure analysis programs can be downloaded
from the Web.
16.4 Backbone Structure

The following tetranucleotide structure shows the hydrogen bonding sites. The phosphodiester phosphates
and ribose C1’ atoms link the base sequence and backbone together.
hydrogen bonded
to T (or U)
5' end A H N
H
N N
hydrogen bonded
O H to A
N H
N
O
P
O CH2 O O
H H
O H H3C
H H (or -H) H
O (or -OH) H
N T hydrogen bonded
O
N
O
(or U) to C
P
CH2 O
O H H O
O H
H
N 5 6
H H 7 N
1
O (or -OH) H
H 8
N 4 N 2 N
O P 9 3
H
O
O CH2
H
O
H H G H
N H
H 5 4
H H
O (or -OH) N hydrogen bonded
H 2
3
to G
6
O P N
1 O
O CH2 O
O H H H
H H
C
O (or -OH)
O P
O CH2
O
3' end
A nucleoside consists of a base and sugar, deoxyribose in DNA and ribose in RNA. A nucleotide is
the base, the sugar and one or more phosphate(s)—connected to any of the hydroxyl positions. The
following figure compares a carboxyester functional group, which is a monoester, with the phosphodiester
bond.
O O
R1 R2 R1 O P O R2
C O
O
-
Carboxymonoester Phosphodiester
The phosphate is attached to the sugar at the 3’ and 5’ oxygen atoms to produce the interconnected
series of phosphodiester bonds. In some cases, the 2’ oxygen also binds a phosphate to form a
phosphodiester bond. In DNA, the 2’ position contains hydrogen, not a hydroxyl group. This makes the
chain resistant to breakage via base-catalyzed hydrolysis, which happens easily with RNA (see the Alkaline
Hydrolysis of RNA section for the mechanism).
16.5 Counterions
Nucleic acids are highly electronegative (polyanionic), however the charge is substantially neutralized by
bound counterions, most commonly Mg2+, Na+, and K+. The pKa for protonation of the phosphodiester
oxygen is reduced to below 6 in the polymer, so one negative charge is present per nucleotide residue.
There are two negative charges per base pair. Counterions are atmospherically bound, exchanging with
solvent cations multiple times each millisecond. They are an intrinsic, but transient, part of the nucleic acid
structure. Manning-Record Counterion Condensation theory is used when considering the charge-
dependent properties of nucleic acids.
16.6 Chemical Synthesis

DNAs are synthesized using phosphoramidite chemistry via solid phase synthesis. Chemical synthesis of
RNA is more difficult but can be done, however enzymatic synthesis is usually the method of choice when
making specific RNAs.
16.7 Watson-Crick Base Pairs

A·U and A·T. The hydrogen bond patterns in the A·U and A·T pairs are shown below. The RNA version is
shown. In DNA, the C5 atom of deoxyuridine (dU) is methylated to form deoxythymidine (dT).
5' end
5' end
O
A U O
O O P
O
P
O
O O H
H N O H (or -CH3)
CH2 N H CH2
O H H
H H N N O H
H N H
N H
H N
O O O O
O O H H
O P
P (or -H)
H (or -H) H O
O O O
Not hydrogen
bonded
3' end 3' end
Characteristics are:
(1) Two hydrogen bonds form between (i) the amino H and carbonyl O, and (ii) the imino H and the ring N
lone pair.
(2) A·T and A·U are more hydrophilic than C·G.
(3) The A·U in RNA is more hydrophilic than A·T in DNA for two reasons:
(i) the 2’-hydroxyl occurs in uridine, while -H occurs in deoxythymidine
(ii) the C5 atom on U is hydrogen; it is a CH3 in dT.
Drawing Proper Strand Polarities. DNA looks like a ladder. Drawings of base pairs are flawed because
they don’t convey the true geometry. The base pairs shown here are shown in the plane of the page. Unlike
what is shown, the 5’ end of one strand projects directly into the page, while the other 5’ end projects out of
the page. This type of strand polarity is called “antiparallel.” In some special cases, certain DNAs can form
parallel-stranded structures.
G·C. This base pair is shown as an RNA fragment.

.
5' end
5' end
O
O G C
O
P
O O 8 H O P O
H N
CH2 7 5 O H N
4 H O
O N 6 5
H H H 9 4 N H N3 H
1 2 6 CH2
H N 2
O O 3 N1 O
O
P N H O H H H
H (or -H)
O H H
O O O
O
P
(or -H) H
3' end O O
3' end
Characteristics are:
(1) Three H-bonds form: (i) carbonyl O to amino H, (ii) imino H to ring N, (iii) amino H to carbonyl O.
(2) The G·C pair is more hydrophobic then A·U.
Molecular Recognition Patterns. Note the differences in H-bonding interfaces in the AT and GC pairs.
Specific charge complementarity and proper spatial apposition of the functional groups occurs, and it’s
different in the two cases. This represents an extended encoded structural message. The details are
“written” in terms of charge and shape surface architecture. This is literally what is “read” by proteins in
the midst of genetic recognition, decoding, and maintenance in a given biological niche.
16.8 Structural Modifications

Modified Bases in DNA
A number of base modifications occur in DNA. In one remarkable case found in bacteriophage DNA, an
entire glucose is connected to the C5-methyl group of thymidines!
The C5 position of cytidine in many eukaryotic chromosomal DNAs is methylated within GpC Islands
sequences. The presence of this nucleoside, 5-methylcytidine (m5C), is correlated with epigenetic gene
inactivation in developmentally specialized cells.
The Hoogsteen Base Pair: A Rare Tautomer of Adenosine

This base pair contains (1) the imino tautomer of adenine and (2) the unusual synclinal conformation about
the glycosidic bond (see below).
.
Normally used in
5' end G-C base pairs
5' end
O O imine H
O
H
P O
P
N N O
O O H O H
N N H
O
CH2 N
O CH2
H N
H H N H N O
N N H H
H H
O O H
O H
H O O
P H (or -H) O P
O O
H O O
imidazole (or -H)
nitrogen
3' end
3' end
imino A syn G
The Hoogsteen variant is an unusual, but biochemically significant, base pair. It illustrates how a rare
tautomer can lead to the insertion of a mutation. Since imino A decodes in replication like C, a G would be
inserted into newly replicated DNA, instead of the correct T.
The guanosines in left-handed Z-DNA also adopt the syn G conformation, in which the base is rotated
to a position above the sugar. The syn G conformation is not as stable as anti G due to repulsion between
the partially negative charges of N3 and G O1’.
Modified Bases and Base Pairing in RNAs
Many examples of modified base pairs, triples and other assemblies occur in Transfer Ribonucleic Acids
(tRNA), the adapter molecules used to synthesize proteins in translation. Some common examples of
modifications are 5, 6-dihydrouridine, pseudouridine, N6-isopentenyladenosine, 7-methylguanosine, and
uridylate-5-oxyacetic acid. One particularly complicated modification found in the phenylalanyl tRNA in
yeast is called wyebutosine, a fluorescent 3-ringed base.
Ribose Methylation. Ribose is sometimes modified to produce 2’-O-methylribose, which short-circuits
alkaline hydrolysis of the RNA backbone. Two examples are the cap structure in mRNA and a common
modification located adjacent to the anticodon sequence in tRNAs.
16.9 Three-Dimensional Structures

Three canonical duplex DNA structure, the B, A and Z forms, are shown and described below.
B-DNA. The following figure shows side and top views of B-DNA, in ball-and-stick, space-filling, and line
formats. This is the classical structure proposed by Watson and Crick base on Rosalind Franklin’s X-ray
fiber diffraction data.
Right-handed B
form DNA helix
helix axis
pitch
width
(Adapted from Saenger, W., Principles of Nucleic Acid Structure, Springer-Verlag, 1984, p. 262; Fig. 11-3.)
Characteristics:
Most DNA sequences prefer the B form.
Characteristics of B-DNA are:
(1) 10 base pairs (bps) per turn; 3.4 Å per base pair (bp) rise.
(2) The bps are almost perpendicular to the helix long axis.
(3) The B helix is slightly narrower and more stretched out than A-form.
(4) Unlike the A-form, the B duplex has no central axial cavity.
Sugar Pucker. Deoxyribose has a C-2’ endo sugar pucker. The glycosidic torsion angle
between the sugar and base is anti.
5'
O
CH2 H
base
H
O C2' endo
longer than in H
the A form H H
O
3'
The following figure shows sugar puckers formed by deoxyadenylate in B- and A-form DNA, respectively.
5' 5'
N O O
H H N
O
CH2
O H N N H O
CH2 N N H
o H O
o
7 A H H N
N H
6 A H
H N
H H N H
H O
O H H
H
C2' endo C3' endo 3'

3'
(Adapted from Structure and Conformation of Nucleic Acids and Protein-Nucleic Acid Interactions, (M. Sundaralingam and S. Rao,
Eds.), 1980, University Park Press, p. 487.)
A-DNA. The canonical A-form structure appears below. Note the shapes of the major and minor grooves; the
major groove is more deep and narrow.
Right-handed A form
DNA or RNA helix
helix axis
tilt = 19o
pitch
width
Characteristics: The A form is preferred in RNA, which does not adopt the B form.
(1) There are 11 bps per turn; with a 2.3 Å per bp rise.
(2) The helix is wider than B form; not as stretched out (slightly more stout).
(3) The bps are tilted 19° from perpendicular to the helical long axis.
Sugar pucker. The ribose has a C-3’ endo sugar pucker.
5'
O H
CH2
flatter than in base
B-form O
H O H H
O
3'
H
C3' endo
Z-DNA. The molecular structure of left-handed poly(dGdC)·poly(dGdC) is based on coordinates for Z1-
DNA derived on the basis of the hexanucleotide d(CGCGCG) in side and top view. The zig-zagged
alternating phosphodiester backbone conformations give the molecule its name.
helix axis
pitch
width
Characteristics:
(1) The duplex is left-handed.
(2) Z-DNA has 12 bp per turn and a 3.8 Å per bp rise. The helix is more stretched out than B-DNA.
(3) It is longer and narrower than B- and A-DNAs. The width is 18.4 Å. Those of the B and A forms
are 23.7 Å and 25.5 Å.
(4) The base pair tilt angle (9°) is half-way between those of A- (19°) and B- (1°) DNAs
Sugar Pucker. The Gs adopt syn glycosidic torsion angles, while the cytidines are in the anti conformation.
The alternating base-sugar juxtapositions are accommodated by alternating sugar puckers and the Z-formed
backbone.
Zig-zagged Backbone
Two different phosphodiester conformations exist. The Z-DNA is stabilized by > 2.7 M NaCl and >10 mM
MgCl2, both of which relieve charge repulsion between closely juxtaposed backbone phosphates.
Structure of a G•C base pair in Z-DNA is shown below. The sugar adopts a C-2’ endo conformation,
and the base is in the anti structure, as in B-DNA.
syn G anti C 5' end
syn strong interaction

O
conformation
H H O P O
N O H N H
3' end O
H N
H N H N H CH2
H N N
O O
O N H H H H
O H O
P H
O H
O H O O
CH2
H
anti P
O
glycosidic bond O O
P O conformation
O
O
5' end 3' end
(Adapted from Saenger, W., Principles of Nucleic Acid Structure, New York, Springer-Verlag, 1984; p. 287.)
(1) In solution, the anti conformation usually predominates in free nucleosides. Guanine nucleotides can
adopt the syn conformation because favorable electrostatic attraction occurs between the C-2 amino group
and the 5’-phosphate.
(2) Synthetic alternating purine-pyrimidine sequences form Z-DNA at elevated salt concentrations. In
contrast, the presence of negative supercoils in plasmid DNA that contains an alternating CG motif can
induce Z-DNA formation at in vivo salt concentrations.
The Structural Parameters of A-, B- and Z-DNA are summarized below.
Property A-DNA B-DNA Z-DNA

Helix handedness Right Right Left
Repeating unit 1 base pair 1 base pair 2 base pairs
Rotation per base pair 32.7° 34.6° 30°
Base pairs per turn + 11 + 10.4 - 12
Inclination of base pair 19° 1.2° 9°
to the helix axis
Rise per base pair 0.23 nm 0.33 nm 0.38 nm
along the helix axis
Pitch 2.46 nm 3.40 nm 4.56 nm
Diameter 2.55 nm 2.37 nm 1.84 nm
Glycosidic bond Anti Anti syn
Conformation
Sugar Pucker (Ring C-3’ endo C-2’ endo C-2’ endo at C
Torsional Conformation) C-3’ endo at G
(Adapted from Moran, et al., Biochemistry (2nd ed.), 1996, p. 24-22; Table 24-3.)
16.10 Recognition of Sequences

Sequence Microheterogeneity. Structural studies have shown that DNA is really a mixture of B and A
forms. An example is the dodecamer sequence from the lac repressor binding site characterized by Richard
Dickerson and colleagues using x-ray crystallography.
Sequence Microheterogeneity refers to the presence of many subtle structural handles on one DNA
sequence. The grooves contain different functional groups, which are read when proteins bind to their
recognition sequences. The surfaces and charge characteristic encode the recognition information.
To recognize a sequence, a DNA-binding protein must be able to tell how it differs from the structure
of another incorrect potential binding site sequence. This means it must be able to differentiate correct and
incorrect DNA surface architectures in terms of charge, shape, hydrophobicity, and so forth.
Bent DNA. The detailed structure depends strongly on local sequence details. For example, specific
adenine-enriched sequences can make DNA form a structure called Bent DNA. Adopting the bent form
involves formation of a bound spine of waters in the grooves.
16.11 Genetic Mutations and Antisense Nucleic Acids

Genetic Mutations
Incorrect nucleic acids are rarely made by replication. Despite the proofreading ability of DNA Polymerase,
mutations occur regularly in human cells. Rare tautomers, e.g., the Hoogsteen base pair described above,
occur at a rate of ∼ 108s-1. We have extensive DNA repair systems to remove and replace incorrect or
structurally damaged nucleotides.
Some mutation is useful. Mutations can lead to three outcomes, (1) evolutionary improvement, (2) no
effect (a silent mutation), and (3) genetic damage that is functionally apparent (e.g., disease, temperature-
sensitive mutation). The latter can be inherited. Cancer produces the disease after sufficient time has passed
for the essential mutations also accumulate and produce their effect. For example, a series of specific
mutations occurs in the pathway that leads to colorectal cancer.
Antisense Duplex RNA

The typical mRNA is translated to form a protein. The strand produced for this purpose has as its encoding
progenitor the sense DNA strand, which produced the sense mRNA strand.
Antisense RNA is the sequence that is complementary to the normally transcribed mRNA. If antisense
RNA’ is made, it can bind the mRNA and form a [mRNA·antisense RNA] duplex. This renders the mRNA
unusable in translation, which prevents formation of the encoded protein. The antisense RNA inhibits gene
expression. The antisense approach is used naturally by bacteria to limit the number of copies of plasmid
DNA it makes, which is called copy number control.
Antisense Triplex DNA

This idea is used in techniques designed to bind a third DNA (or RNA) strand to a duplex DNA, thereby
rendering the duplex sequence incapable of use in transcription. Triple helix (triplex) structures can form in
three ways:
Watson-Crick (WC) WC base pair WC base pair

strand 1 base pair strand 1 strand 1
A T strand 2 G C strand 2 GC strand 2
T Hoogsteen CH+ base

C-C +
G G-G
base
base pair pair
strand 3 strand 3 pair
strand 3
Gene Therapy involves using designed nucleic acids to targeted a specified gene function for control by
binding the antisense nucleic acid. The advent of Small Interfering RNA technology is an extension of this
idea. The latter approach is described in the named section in the RNA chapter.
16.12 Unusual DNAs

Triplex DNA
Duplex DNAs can bind a third strand via base pairing to form a triplex DNA. Sequences that form
“triplexes” are common in the upstream control regions of eukaryotic genes. Triple helices always have
both parallel and antiparallel strand-strand pairing interactions.
Base pairing schemes are shown in the Antisense Triplex DNA section. Complexes can form from
three strands, two strands, where one is a hairpin duplex structure, and from a single strand that has two
hairpin turns. The three base triples [T-A~T], [C-G~C+] and [C-G~G] have been studied in triplex DNAs.
The ability of a given triplex to form depends on sequence, cation type, and temperature.
.
3 strands 5' one continuous
3' 5' or 3' 3' strand 5'
5'
5' or 3'
hairpin
duplex
1 strand
5' 3'
3' 5' or 3' 5' or 3'
Quadruplex DNA
These structures contain strands arranged in 4-fold symmetry about the central axis.
.
Hydrogen
Bonds
Central
Ion
H H
N N O
H
Hydrogen
N Bonds
N
backbone N
H
deoxyribose
backbone
.
Variants. Synthetic quadruplex DNAs can adopt either parallel or antiparallel structures, depending on the
sequence, cation conditions, temperature, and the presence of certain proteins.
.
4 strands 2 strands 1 strand
5'
5' 5'
5'
3' 5' 3'

3' 5' 3' 5'
3'
3' 3'
Certain G-rich strands can form both triplex and quadruplex structure, depending on the sequence and
ionic conditions. (For details, see Hardin et al., Biopolymers, Nucleic Acid Sciences, 2001, 56, 147–194.)
Biological Importance
Quadruplex DNA has received attention in its role as a protein-binding aptamer. A specific quadruplex
binds the blood clotting factor Thrombin. This interaction inhibits binding of thrombin to fibrinogen,
thereby inhibiting blood clotting. The structure of the protein-quadruplex complex has been determined by
x-ray crystallography.
Guanine-rich sequences are very common in the telomeres of chromosomes (see below), the GpC
Islands in transcriptional promoters, and immunoglobulin gene-switching sequences. An HIV coat protein
binds HIV RNA and makes it form a quadruplex.
Cruciform DNA
This structure is formed from an extended duplex DNA fragment composed of a palindromic DNA, which
is also called an inverted repeat. The two-step extrusion process is shown in the figure below.
(1) The central sequences extrude outward

from their original base-paired positions. hairpin 1
A B
5' A B 3' 5' 3'
3' B A 5' 3' 5'
(2) The inverted repeats extrude and form B A

hairpin on either side of the DNA. hairpin 2
This sequence is cut by a cruciform DNA-dependent nuclease. More complicated extra-duplex bonding
arrangements called Gierer trees can also form.
16.13 Stabilization of Nucleic Acids

Contributions. The following factors contribute to the stability of nucleic acids:
(1) The hydrophobic effect forces bases inward. It constitutes about two-thirds of the duplex stability. A
large gain in stability occurs when the H2Os surrounding the strands maximize hydrogen bonding, which
squeezes the strands together.
(2) Base pairs provide ≤ 1/3 of the stability.
(3) The order of hydrophilicity of the bases is: U (T) > C > A > G; the latter is the most hydrophobic.
(4) About 10–20% of stability is due to van der Waals forces (London dispersion forces). These forces are
the result of dipole/induced dipole interactions between the nearby atoms.
Adjacent base pairs undergo stacking interactions, like coins in a roll. H
H ribose-5',3'-
N diphosphate
stack
formation H
N
N O
H
H
N ribose-5',3'-
The charges on the electronic lobes N N diphosphate
H
change and track with each other as
time progresses. H
N
H N
London dispersion forces occur between π electron orbitals on the lobes of atoms in adjacent stacked
bases. The electron densities in the orbitals switch back and forth, correlated in time with each other, to
produce a bond that is negotiated transiently. They are transient dipole-dipole interactions.
Phosphate Groups. Metastability

(1) Buckminster Fuller developed the concept tensegrity, in which stability is gained at the expense of a
tense structural standoff. For example, many modern mountain tents are held up by the tense tentpoles, held
taut against the reinforced pockets they fit into.
The phosphodiester phosphates on each strand are polyanionic, so they naturally repel each other.
This repulsion is held in check by counterions, as we’ll see below, so this stability of the DNA is a tense
standoff. DNA has tensegrity. This is also a case of metastability. The helix is stable, but given the chance
to break, it might dissociate.
(2) The strands are fairly easily separated. If that were not true, transcription and replication would be more
difficult because the polymerases could not separate the strands easily. Strand-strand repulsion in DNA
supports transcriptional gene expression. Specialized single-stranded binding proteins actually help the
duplex separate to single-stranded form.
(3) Counterions offset this electrostatic repulsion between the strands. Physiological salt concentrations
nearly neutralize the negative charges on the strands, but not completely. About 10% remains per
phosphate. This result is predicted using Manning-Record counterion-condensation theory.
Hydration
(1) The grooves of DNAs are filled with water molecules, bound to the atoms of the backbone and base
functional groups. The minor grooves in B-DNA are densely packed with water, which form bonds with
adjacent water molecules as well as the exposed atoms of the bases. Water molecules in the major grooves
hydrogen bond to phosphate oxygen atoms and the keto and amino groups on the bases. Approximately
three water molecules bind to each phosphate group and about 20 total water molecules are present per
nucleotide in DNA. B-DNA is stabilized as a result of the rigidity induced by these hydrogen bonds.
(2) Double-stranded DNAs are stabilized by hydrophobic interactions, base stacking, hydrogen bonding,
and electrostatic repulsion.
(3) The hydrophobic effect is especially important. The purine and pyrimidine rings are typically buried
inside of the double helix, inaccessible to the aqueous solvent. Similar to proteins, the entropy of DNA is
increased by the decreased order (larger number of combinations) of water molecules in the vicinity of
exposed hydrophobic functional groups of the bases. This high entropy is counteracted by binding of highly
ordered water molecules, which hydrogen bond with base functional groups in the grooves.
(4) The structure of nucleic acids is not rigid. DNA is constantly breathing. a process in which the
hydrogen bonds between base pairs constantly break and reform, allowing small degrees of
“conformational heterogeneity” to occur in the chain.
16.14 Secondary Structure Predictions

Duplex Melt Measurements
Thermodynamics for association of DNA duplexes and RNA secondary structures have been assessed by
methods that involve determining the melting temperature as a function of [strand]. The ΔGa values
calculated for the neighboring base pairs of a specific RNA secondary structure are shown below. Such
calculations are used to predict the most stable structures.
Example: The following figure shows an application of data to evaluate a possible secondary structure for a
55 nucleotide fragment from R17 virus. The net calculated stabilizing ΔG is -21.8 kcal/mole at 25°C. All of
the energies are in kcal mol-1.
- 1.8 - 5.0 - 1.8 - 1.8 - 2.2 - 5.0

- 1.2 - 2.2 - 1.2 + 2.0 - 2.2 - 1.2 + 2.0 0
- 2.2 - 1.2 - 3.2 - 2.2
U C 3'
C A U U U UC G U OH
A U U C C A U A C G A A C C C G
A
U A A G GU U A A A UU C A U G C U U G GG U 5'
A C Pi
A U
+6 - 2.2 + 3.0
- 1.2 + 3.0
Verification of Predicted Structures

The predictive approach involves using a computer to look at how well evolutionary conservation occurs at
specific sequences.
(1) A large number of chemical modification, and chemical- and nuclease-cleavage methods, have been
used to study nucleic acids. The techniques allow one to verify the sequences and structures involved in
particular secondary and tertiary structures.
(2) Gel electrophoresis-based chemical protection techniques and DNA footprinting have been used to
determine which functional groups bind to binding proteins. Chemical modification and nuclease cutting
experiments allow one to determine protected atoms on the nucleic acid, and thus the protein binding
surface.
(3) Alignment of sequences is used on a daily basis to look for matches between one sequence and a set of
sequences in some data base. Many applications have been developed in crime analysis, lineage
determination, and so on.
Example: The DNA left in fecal deposits of Artic wolves has been used to track the territories of
individuals and populations. Single wolves range over hundreds of miles, within days, looking for food.
G·U and U·U Base Pairs

The mismatched G·U base pair is a sturdy base pair. It is special and occurs as a wobble base pair in
codon·anticodon interactions between mRNA and tRNAs. The ΔGa of G·U is negative; it is a legitimate
base pair. When evolutionarily conserved sequences are compared in the context of the known secondary
structure the pair occur within, G·U is found to be conserved. Evolution knows and uses the energetic fact
that G·U forms a stable base pair to accomplish biologically important pairing interactions.
Stacking Energies for the Ten Possible Dimers in B-DNA

DNA has different ΔGa values than the analogous RNA. The following table gives numerical values for all
possible ways in which two bases can pair in the context of defined nearest neighbor base pairs.
Stacked Dimers a Stacking Energies Stacked Dimers Stacking Energies

(kJ mol-1) (kJ mol-1)
3' 5' 3' 5'

C G -61.0 T A
G C A T
-27.5
5' 3' 5' 3'
3' 3' 5' 3' 5'

5' 3' 5'
C G G C A T
T A
A T G C -44.0 T A C G -27.5
3' 5' 3' 5' 3'
5' 5' 3'
3' 5' 3' 5'

3' 5' 3' 5'
C G G C T A
A T -28.4
T A G C
-41.0 A T C G
5' 3' 5' 3' 5' 3'
5' 3'
3' 5' 3' 5'
3' 5' A T T A
G C T A
C G -40.5 A T 5' 3' -22.5
5' 3'
5' 3'
3' 5' 3' 5' 3' 5'

G C C G A T
G C C G -34.6 T A -16.0
5' 3' 5' 3' 5' 3'
a
Arrows designate the direction of the sugar-phosphate backbone and point from C-3’ of one sugar to C-5’ of the next.
[Adapted from Ornstein, R. L., et al. (1978), An Optimized Potential Function for the Calculation of Nucleic Acid Interaction
Energies: I. Base Stacking, Biopolymers 17: 2341-2360.]
16.15 Chromosomes
The genetic state of a chromosome and levels of gene expression are maintained by many factor proteins.
These proteins are regulated by modification of amino acids, binding of specific proteins and nucleic acids,
and many other moderating influences. The basic ideas are described below. For details, see books such as
Molecular Biology by Weaver and The Cell by Alberts et al.
Required Features
Chromosomes must contain the following elements:
(1) Histones are protein complexes that bind chromosomal DNA like beads on a string.
(i) They are enriched in lysine and arginine.
(ii) The four types of proteins form an octameric core around which about 200 base pairs of native DNA
wraps within chromosomes.
(iii) Control of the transcriptional activities of particular genes involves enzyme-catalyzed reactions that
cause histone methylation, acetylation, phosphorylation, and others modifications. This is called the
Histone Code. (Details are described below.)
(2) Centromeres. These structures are the site of spindle fiber attachment and contain specific unique
sequence motifs.
(3) Telomeres. These are complex protein DNA complexes at the chromosomal ends.
(i) Telomerase. This RNA-protein replicase (a reverse transcriptase) carries its own C-enriched template
RNA to make the complementary G-rich strand of telomeric DNA. Details are described below.
(ii) The enzyme maintains the ends of the chromosomes, which are required for long-term survival.
(iii) Telomerase is required for telomere maintenance, and is involved with cell aging and cancer.
(4) Autonomous Replication Sequences (ARS) are the internal start sites for the discontinuous replication
bubble form of DNA synthesis found in eukaryotes.
(5) Replicases. The DNA polymerase complex makes DNA in a template-requiring process that produces
two daughter duplexes from one maternal duplex sequence.
(6) Transcription Promoters. These are sequences such as the classic TATA box in prokaryotes; and
typical GC-enriched sequences in eukaryotes.
(i) Transcription Repressors. In the operon model, they bind the operator DNA and thereby repress
transcription
(ii) Transcription Activators. Also called transcription factors and transactivator proteins.
(7) Translation. Protein synthesis involves three types of RNA: ribosomal, messenger and transfer. Details
are discussed in the RNA chapter.
Propagating DNA
The following example show a cross-section of the contexts in which DNA is propagated.
Yeast Artificial Chromosomes

YACs contain the necessary information to operate as chromosomes in yeast cells, providing a minimal
model for chromosomal structure-function studies.
Plasmid DNA and Supercoils

A plasmid is a closed-circular DNA that can be replicated within a host cell. It typically encodes an
antibiotic resistance gene and has an “insertion site” in which one inserts specifically designed DNA
fragments, such as gene sequences of interest.
When the DNA is internally connected end-to-end, the possibility of forming different topological
isomers exists. These species can have a different number of twists in the overall ring structure, called
supercoils. A plasmid with more twists is considered to have a more negative helical density. A relaxed
plasmid has a more positive helical density.
Supercoils have different intrinsic energies. The number of supercoils can be changed by enzymes
called Topoisomerases. Relaxing superhelical stress is accomplished by type 1 Topoisomerases. The other
type, called DNA Gyrase, uses ATP as an energy source to impart negative superhelical density, that is, add
more supercoils.
The mechanism of the Type 1 enzymes involves making a nick, winding or unwinding of the duplex
strand, then resealing the nick.
5' 3'
3'
5'
Cut the strand to produce

a nick with exposed 5'
and 3' ends.
Transpose the 5' end
under the other strand
then re-attach it.
5' 3' 5' 3'
3' 5' 3'
5'
Re-attach the 5' end,

producing a single
"superhelical turn"
between the strands.
5' 3' 5' 3'
3' 5' 3' 5'
The type 2 enzyme (1) wraps a strand around itself, (2) breaks one of the two adjacent strand segments, (3)
passes the other strand between the broken stranded gap, and then (4) reseals the gap.
Viruses
These species consist of a nucleic acid genome, which is circular or linear DNA or RNA, depending upon
the virus. Some viruses require two circular species, for example, the plant Geminiviruses. Some require
helper viruses to carry out infection. Some have several different functional forms. Details the complicated
propagation system of HIV are discussed in the Virus section of the Cell Biology Review chapter.
“Rolling-Circle Replication” involves production of multiple connected genomes, which are later cut apart.
Viruses usually have very few genes. They encode sufficient RNA or DNA, coat proteins, control factors,
and so forth to integrate themselves into the host cell and occupy their genetic niche.
Telomeres
Telomerase Catalyzes Chromosome End Extension. The steps in the telomere buildup process catalyzed
by telomerase are shown below.
.
Step:
5' 3'
TTGGGG TTGGGG TTGGGGTTG
(1) Initial telomere
AACCCC
3' 5'
Rest of
chromosome
5' 3'
TTGGGG TTGGGG TTGGGGT (2) RNA template bound in the
AACCCC ACCCCAAC reverse transcriptase site of
3' 5' protein telomerase
template RNA
synthesized DNA
5' (3) Telomerase synthesizes the

TTGGGG TTGGGG TTGGGGTTG GGG TTGGGG TTG 3' G-rich strand. As shown, two
AACCCC ACCCCAAC terminal transferase copies are
3' 5' protein
template RNA added.)
5' 3'
TTGGGG TTGGGG TTGGGGTTG GGG TTGGGG TTG (4) DNA polymerase fills in
AACCCCAACCCC AACCCC AACCCC 5' the C-rich strand
3'
second synthesized DNA
(5) DNA ligase seals the gap
For details see Osterhage, J. and Friedman, K. (2009) Chromosome End Maintenance by Telomerase, J.
Biol. Chem. 284, 16061–16065.
The Telomerase [Protein·RNA·DNA] Complex. Chromosomal DNA is bound by the telomerase RNA
template within the protein, which is analogous to a protein hand grasping the duplex RNA·DNA complex.
Rest of the
telomerase
RNA
"fingers"
(protein)
Telomerase
template RNA
Newly synthesized
telomeric DNA
Rest of "Palm" of the

chromosome "thumb" telomerase
(protein) active site
(Adapted from Alberts et. al. Molecular Biology of the Cell (4th ed.), 2002, p. 264; Fig. 5-43.)
Nucleosomes
Two molecules of each of the four histone proteins, H2A, H2B, H3, and H4, make up an organized
octameric complex called the nucleosome. Each one is wrapped with about 200 base pairs of DNA for
about 1.75 turns. This coiling of DNA around the core nucleosome causes positive supercoils to form in the
remaining DNA which are relieved by eukaryotic topoisomerase II, an enzyme that binds to chromatin. Of
the wrapped DNA, about 146 base pairs are closely associated with the histone octamer and make up the
nucleosome core particle. After the removal of the nucleosides, the naked eukaryotic DNA has about one
negative supercoil per original nucleosome.
+ + +
H2A H2B H3 H4
dimer dimer dimer dimer Histone Octamer
Linker DNA is found between each core particle and is usually about 54 base pairs in length. In
higher-order chromatin structures, illustrated in the beads on a string structure, the fifth histone, H1, binds
to both the linker DNA and the nucleosome core particle.
Nucleosome
(core particle + linker)
Histone
Octamer
30 nm
30-nm
chromatin fiber
H1 Histones
16.16 Some Protein Nucleic Acid Binding Motifs

Zinc Finger Proteins
These proteins bind the grooves of DNA. Two or more fingers are typically found per protein. Each finger
has a central Zn2+ and four ligands composed of His and/or Cys residues.
C-terminus C-terminus C-terminus

His His Cys His Cys Cys
2+
Zn Zn 2+ Zn2+
N-terminus His His N-terminus His Cys N-terminus
Cys Cys
Leucine Zippers
Protein-protein binding can occur between two Leucine Zipper Proteins. This allows two DNA-binding
proteins to use their back sides to bind to each other while binding two separate DNA strands with the
DNA-binding site.
Two different proteins could bind through their zipper domains to a single protein. The two dimers
differ by which DNA sequence the interchanged protein binds. The net effect is that the dimer can select
two different genetic activation situations. Only three proteins are required instead of four. The common
protein plays the role of two proteins, depending on which partner it binds. This allows DNA binding
proteins with different genetic functions to mix and match in order to bind different genes. This is an
example of a combinatorial binding system.
Transcription factor
heterodimer 1 DNA
binding site
Zipper structure
Leucine zippers
interacting to
stabilize the dimer
TF heterodimer 2
DNA binding site
.
Transcription Factors (TFs) bind via hydrophobic interactions to each other to form heterodimers. This is a
necessary step to activate transcription of the receptive promoter sequence. Different combinations of
proteins can bind together: TF-1 to TF-2, TF-3; TF-4; and many others.
TF-1 Different TF-1 TF-2

sequence-specific
helix-helix
interactions
TF-2 TF-3 TF-3

.
16.17 Recombination
This process is mediated by protein complexes. A well-studied example is the RecA complex. The process
occurs at a four-stranded structure called a Holliday Junction. Such structures have been modeled using
synthetic DNA fragments. They are similar to cruciform DNAs.
The V(D)J Recombinase mechanism is used to produce the diverse array of antibodies made by the
humoral immune system. See Alberts et al. for detailed descriptions of each of these areas.
Chapter 17
RNA
17.1 Cells Contain a Variety of Types of RNA
DNA molecules act as the storehouse for genetic information. RNA molecules, in contrast, operate in
several different forms to express the genetic information. Inside of a cell, RNA can be found in many
copies for a particular purpose specific to its type. There are four major classes of RNA:
(1) Ribosomal RNA (rRNA) is incorporated into the ribosome. Ribosomes are made up of protein and
RNA and are the site of protein synthesis. Ribosomal RNA makes up the majority of the RNA of a living
cell, accounting for 80% of the total cellular RNA. It is the active catalytic focus of the ribosomal reactions.
Most of the proteins can be removed yet the individual activities are retained.
(2) Transfer RNA (tRNA) transport activated amino acids to the ribosome to add to the growing
peptide chain of the protein being made. Transfer RNA molecules are short in length, usually about 73 to
95 nucleotides, and make up about 15% of the total cellular RNA.
(3) Messenger RNA (mRNA) are responsible for coding the DNA into amino acid sequences to be
transported to the ribosome for protein synthesis. They are the least stable of the RNA molecules and
account for about 3% of the total cellular RNA.
(4) Noncoding Small RNA molecules also exist in all living cells. Most are found in the nucleolus
portion of the nucleus. Some have catalytic functions dealing with proteins. After RNA is synthesizes,
some small RNA molecules are active in the processing for modification of these. Some examples are small
nuclear (sn) RNAs, small nucleolar (sno) RNAs, micro (mi) RNAs and silencer (si) RNAs.
17.2 RNAs Have Stable Secondary Structure

The difference between an RNA polynucleotide and a DNA polynucleotide is the presence of 2’-hydroxyl
group and the replacement of the base uracil for thymine in DNA. These bases only differ slightly, with
replacement of the methyl group at the 5 position of thymine by a hydrogen in uracil. The backbone of both
RNA and DNA linked by 3’ to 5’ phosphodiester bonds.
RNA polynucleotides fold back on themselves to form hairpin structures composed of complementary
base pairs in duplex regions and intervening loops. This is the essence of RNA secondary structure. One of
these is a hairpin, or stem-loop, which is made when the folding causes short regions of complementary bases
pair with one another. This is displayed in the cloverleaf secondary structure of transfer RNA.
3' The dashed lines represent the Watson-Crick
OH
5' A base pairs between nucleotides. Some
Pi C
C
nonstandard nucleotides are present and listed. R
is a purine nucleotide and Y is a pyrimidine
Acceptor nucleotide.
stem
m1A, 1-methyl-adenylate;
D arm TΨC arm m6A, N6-methyladenylate;
DR
A U A
Cm, 2’-O-methylcytidylate;
G Y
G R D, dihydrouridylate;
R G C
A Y T Ψ Gm, 2’-O-methylguanylate;
m1G, 1-methyl-guanylate;
7
Variable m G, 7-methylguanylate;
Anticodon arm
arm I, inosinate; Ψ, pseudouridylate; and
m7G T, thymidylate.
Y Ψ
Cm
U R
m6A, m1G
I
(and other
modified Anticodon
nucleotides) trinucleotide
Chapter 17 RNA 147
17.3 Tertiary Structure: Transfer RNA

X-ray crystallography of tRNAphe. X-ray crystallography has been used to determine a number of transfer
RNA structures. The best characterized structure is that of phenylalanyl transfer RNA (tRNAphe). A tube-
format structure is shown below.
TΨC arm
1
5' 3'
75
Complicated tertiary
structural interactions
Acceptor
stem
D arm Variable arm
Anticodon
arm
Anticodon
The anticodon loop and arm are located across the “bay” from the acceptor stem. Each arm of the L-
shaped tRNA is double-stranded and forms a short stacked right-handed helix that looks much like typical
A-form DNA.
Aminoacylation of tRNA
The amino acid acceptor stem is attached covalently to an amino acid by a specific catalytic Aminoacyl
tRNA Synthetase. These acceptor-specific proteins catalyze the attachment of each specific type of amino
acid to its tRNA. These enzymes recognize both the codon loop region and amino acid acceptor stem.
The amino acid is linked to the tRNA, via its carboxylate. Attachment can occur at either the 2’ or 3’
hydroxyl group on the ribose of the 3’-terminal adenylate. The primary transcript is processed, by adding a
CCA trinucleotide at the 3’ end, to produce the matured tRNA, by a CCA terminal transferase. The 5’
nucleotides of tRNAs are phosphorylated.
Decoding mRNA The anticodon trinucleotide sequence forms a duplex structure with the codon of the
messenger RNA. The recognition patterns for codons with specific amino acid isoacceptor tRNA species
are characterized by the Genetic Code. More than one tRNA exists for most of the 20 amino acids.
The code is not entirely universal. For example, some codons encode a different tRNA in
mitochondria. A second level of information control is called codon usage bias, in which one compartment
or cell type will favor certain codon(s) to encode a given amino acid, other will favor a different choice of
codon(s).
Table 2. Predicted base pairing between the 5’ (wobble) position of the

anticodon and the 3’ position of the codon.
Nucleotide at 5’ (wobble) position of Nucleotide at 3’ position of codon

anticodon
C G
A U
U A or G
G U or C
I* U, A or C
Modified Nucleotides
Transfer RNAs are decorated with a large number of chemical modifications. Specific modified nucleotides
are always present in two arms of tRNA: (1) the invariant ribothymidine (T or rT) – pseudouridine (ψ) –
cytidine sequence in the TψC loop, at the end of the TψC arm. The D arm is named after its invariant
dihydrouridine. Each tRNA has a variable arm, which can be anywhere from 3 to 21 nucleotides in length,
located between the anticodon arm and the TψC arm. Most tRNAs are 73 to 95 nucleotides in length. In
one odd case, smaller tRNAs occur in mitochondria that are missing most of their D-arm.
The secondary structure of a tRNA is affected by each covalent modification.Two examples are:
(1) The modified nucleotide dihydrouridine is a non-planar, non-aromatic ring, which is chemically
unstable. Yet, for subtle structural reasons, it is there.
(2) The modified nucleotide pseudouridine (Ψ) occurs in the conserved TΨCG loop of tRNAs and is
structurally odd. The ribose C1’ is attached to a base ring carbon, not to nitrogen, as in the conventional
uridine-ribose glycosidic bond. The result is that the carbonyl oxygens are “presented” at a very different
angle from those in the unmodified nucleotide. This change supports the hydrogen-bond pattern that
stabilizes the central domain of the tRNA tertiary structure.
17.4 Messenger RNA (mRNA)

Most students have had a genetics course and are aware of the complicated nature of protein synthesis
(translation). The ribosome reads the mRNA and the tRNA transfer amino acids, one by one, onto the
growing nascent protein chain.
Coupled Transcription-Translation. A prokaryote-specific coupled transcription-translation mode of
protein synthesis is shown.
DNA
RNA polymerase
holoenzyme
"Sense" mRNA strand
70S ribosome
initiation complex
1-2 hairpin
primer
mRNA DNA
5'- (Pi)3
During transcription of the trp operon in
E. coli, RNA polymerase pauses at the 1-
2 hairpin.
Meanwhile, the ribosome assembles at the

initiation codon on the mRNA that encodes
the leader peptide.
(Adapted from Moran et. al. Biochemistry (2nd ed.), 1996, p.30.34; Fig. 30-30.)
Coupled transcription-translation makes the pausing mechanism described below possible. Two things
are occurring simultaneously:
(1) the mRNA transcript is being produced by the RNA polymerase complex, and
(2) the ribosome has actively engaged the mRNA in the decoding process.
As the nascent polypeptide is synthesized, it emerges from the ribosome through the exit tunnel, as
indicated by the arrow.
Note that eukaryotic mRNAs are not transcribed while being translated. Eukaryotic mRNAs are
transcribed in the nucleus, transferred to the cytoplasm, and extensively processed on the way. They are
Chapter 17 RNA 149
then translated in the cytoplasm, typically on rough endoplasmic reticulum. The mechanism shown in the
figure typically involves the use of a polycistronic mRNA.
Polycistronic mRNA Prokaryotes make mRNAs that contain more than one gene sequence. These
polycistronic genes encode more than one protein or RNA product, sometimes some of each. These
proteins or RNAs are typically involved in the steps of one metabolic pathway. As a result, production of
all of the enzymes and components required to support and regulate the pathway is controlled by regulating
one combined gene, one transcription process and one translation process, if they all occur. It provides a
way to coordinate the production schedule of all of the components, and regulate everything en masse,
through one set of switches.
Polycistronic mRNAs can encode several enzymes, several rRNAs, several tRNAs, and combinations
of two or more of these species. Transcription from these genes is regulated coordinately (all at once) by
protein repressors and activators, as well as by other mechanisms.
The best known example of an operon that is encoded by a polycistronic mRNA is the Lac operon,
which is involved in uptake and metabolism of lactose by bacteria. Since most students have encountered it
at this point, we do not recount the many details that govern the regulation of transcription in that system.
Pausing. The Tryptophan Operon

The tryptophan operon encodes several enzymes and transcriptional repressor proteins. All of the
components are involved in the biosynthesis of tryptophan, and are all co-expressed from the same mRNA.
This example of regulation requires coordination between the availability of tryptophanyl tRNAs, the
position of the mRNA on the ribosomes, and the structure adopted by the mRNA. As a result, protein
synthesis can titrate the availability of tryptophan.
The availability of aminoacylated (“charged”) transfer RNA, tryptophanyl-tRNAtrp depends on
whether or not sufficient trp is available for the aminoacyl tRNA synthetase to aminoacylate the tRNA. The
mRNA can form two alternative secondary structures. Each leads to a different outcome.
(1) When charged tRNA is in short supply, a particular hairpin structure can form in the leader sequence
called the pause hairpin.
(2) When the sufficient tRNA is available, that is, when the amino acid is available, a different hairpin
arrangement forms, the termination hairpin.
(3) When amino acid and charged tRNAs are in short supply, the pause hairpin forms and the ribosome
pauses. As a result, the termination hairpin is blocked from forming, and translation of the polycistronic
mRNA produces the encoded proteins. A shortage of the amino acid assures that the enzymes are made,
which replenishes amino acid via biosynthesis.
(4) When the concentration of amino acid is high and pausing does not occur, the termination hairpin
forms, which stops production of the enzymes.
Structural details of the hairpin-forming sequences in the two configurations are shown, for example,
in Figure 22.28 on p. 690 of Moran et al., 2012.
The Shine-Dalgarno Sequence

This is a conserved sequence in prokaryotic mRNAs that directs their recognition by ribosomes. It operates
by forming a duplex between the mRNA and the 3’-end of the 16S ribosomal RNA sequence.
The Literature. Many more details about related subjects such as operon structure, TATA boxes,
transcriptional termination sequences, the details of translation, and so on, can be found in Molecular
Biology of the Cell by Alberts et al., 2002, the pair of books entitled Molecular Biology by Weaver 2001
and Cox, Doudna, and O’Donnel 2011, the latest edition of The RNA World, the Cold Springs Harbor Press
monographs, and the Current Protocols series.
17.5 Eukaryotic Messenger RNA

Post-transcriptional Modifications
The following structural elements typically occur in eukaryotic mRNA.
5' to 5' Triphosphate

CH3 Linkage Exon (coding) Exon 3'-NTR
O
H
N +
N 5'
O Intron(s) ... AAA(A) OH
H2N N N O 5'
O P base 2
1' O
O poly(A) tail
O 1'
3 Coding "reading frame"
base 1 O OH O
O
(-H or -OCH3)
m7G
(-H or -OCH3)
base 3
This nucleotide contains a 3' end

5'-Nontranslated Region
at what is structurally the 5' end.
(NTR)
The components are:
(1) The Cap Structure contains a 5’-terminal m7G that is covalently linked to the second nucleotide through
a 5’-to-5’ phosphodiester bond. Note that this resembles the case in NADH, except that NADH contains a
5’-to-5’ diphosphate linkage, unlike the monophosphate linkage in the mRNA cap structure.
(2) Introns and Exons. Eukaryotic RNAs are initially transcribed as genes in parts. The spliceosome, which
catalyze intron removal, is composed of a set of proteins and small nuclear RNAs (snRNAs). The
remaining mRNA is composed of two or more exon sequences.
(3) Poly(A) Tail. A poly(A) tail is added to the 3’ end of mRNAs.
(i) The poly(A) sequence forms a cyclic structure with the 5’ end of the mRNA. This system regulates
the lifetime of the mRNA molecule. Shortening of the tail is analogous to a train ticket being punched by
the conductor. When the tail is too short, the mRNA is degraded and turned over.
(ii) The poly(A) tail is not a universal feature of all mRNAs. For example, histone mRNAs contain a
Watson-Crick hairpin structure instead.
(4) One mRNA per Gene Sequence. Eukaryotes typically encode one gene per mRNA, they are
monocistronic.
Exceptions to the Eukaryotic One-Gene-per-mRNA Scenario
(1) Intron-Based Genes. The typical one gene per mRNA scenario in eukaryotes does not occur when the
mRNA contains an intron (intervening sequence) that encodes an RNA or protein other than the normal
exon-derived product. These intron-derived genes are sometimes used to make a functional product RNA
or protein.
(2) Alternative Splicing. Another exception occurs when an intron is removed at one of two different alternative
splicing sites. This produces two different mRNA products. The products are partially the same and have
different lengths and nucleotide sequences. The two alternative gene products will have different 3’-ends. The
two proteins will differ in that the shorter sequence will produce a truncated (shortened) gene product.
(3) Minus-strand Product Synthesis. Transcription can occur from the minus strand as well, leading to a
mechanism that produces two different complementary RNAs from the same gene.
Intron Removal from Precursor mRNA
Introns are removed from mRNA by a process that involves catalytic participation by specific functional
groups in the pre-spliced mRNA. The spliceosome is a multiprotein, multi-RNA complex that assembles on
the transcript and removes the intron in a site-specific manner. The basic reaction is:
spliceosome
5'
-exon-G--GU-intron-AAG--G-exon-3' 5'
-exon-G-G-exon-3'
.
splice
sites intron
removed
Chapter 17 RNA 151
The steps in the mechanism are shown below.

(1) Precursor mRNA
Intron
portion to be
removed
O
O
P
U O
O
CH2
A
Pi H
H N H
H O
O H N N
H O H
O
H G P
N O OH H N N
N
O
G N N
H
(2') H
N Pi
H H
O H
H
OH A
5' H2C Pi
O
O O G
P H O
O P
O O 3'
Exon 1 O Pi
G
Exon 2
(2) Intermediate structure
Intron
portion to be
removed
O
O
U O
P
O A
Pi CH2
H
H N H
H O
H
H
G O
O
H N N
H
O P H
N O O N N
N
Pi
G N
H O
H
N
N Pi
H H
O H
5' H
OH
A Pi
H2C
O
P
O H
OH G
O (3') 3'
O Pi
Exon 1 G
Exon 2
(3) Spliced mRNA
O
H O
H P
O N
N U O
G N
H O
CH2
A
N Pi H
H H
N N
H O
H H + G O
O H N N
H
O H OH Intron P H
O O N N
5' H Pi
O H 2C "Lariat" O
O O H
P H Pi
O 3' Pi
Exon 1 O G
A Pi
Exon 2 G
(Adapted from McKee and McKee, Biochemistry the Molecular Basis of Life (4th ed.), 2009, p. 716; Fig. 18-42.)
17.5.4 Autoimmune Antibodies. Characterizing Splicing Complexes

The body sometimes (incorrectly) makes antibodies that recognize substances we’re supposed to have. This, in
turn, wrongly tells the cells, which typically eliminate foreign substances by the immune response, that these
self components are not self. The incorrect immune response against the self antigens of the patient is called
autoimmune disease. Examples are Systemic Lupus Erythromatosis (SLE), arthritis and Crohn’s disease.
The splicing apparatus proteins and snRNAs have been extensively characterized because they are
common antigens for the autoimmune antibodies produced by SLE patients. Many of the early discoveries
regarding the assembly pathways in mRNA splicing were characterized using immunochemical techniques
and the unique autoimmune antibodies they make. These reagent antibodies have been captured as
monoclonal antibodies, and can be produced in large amounts from hybridoma cells in tissue culture for
experimental analyses.
17.6 Alkaline Hydrolysis of RNA

Hydroxide ions in alkaline solution can remove a proton from the 2’-hydroxyl group of any accessible
RNA nucleotide. The nucleotide is converted to an alkoxide, a strong nucleophile, which attacks the
phosphorous atom of the 3’-terminal backbone phosphate. This results in two steps: (1) release of the 5’
oxygen of the next adjacent nucleotide and (2) formation of a 2’-, 3’-cyclic nucleoside monophosphate
intermediate. The 2’-, 3’-cyclic intermediate is unstable in alkaline solution, so cleavage occurs. Another
hydroxide ion from the solution attacks the intermediate and catalyzes hydrolysis, forming either a 2’- or
3’-nucleoside monophosphate.
2', 3'-Cyclic nucleoside monophosphate R1

R1
CH2 O B CH2 B
O
R1 H H 2'-Nucleoside
H H CH2 O B
H H H H H monophosphate
O H H HO O
O O H H
H O
O P O H O O O H O P O
P or
O H O H O
O
O H H2C O B +
H H H R1
H H H CH2 O B
O
H H 3'-Nucleoside
O OH H2C O B H H monophosphate
O O
R2
H
H H
H
+ OH
H O P O
O OH
O
R2
(1) When the polymer reacts, rapid breakdown of the chain occurs because internal phosphodiester
bonds are cleaved, leading to many subfragment products.
(2) Since the methyl group cannot be extracted from the 2’ oxygen, methylation of the 2’ hydroxyl
oxygen renders the phosphodiester linkage resistant to alkaline hydrolysis.
See the DNA Preparation: Phenol-Chloroform Extraction section in the Biotechnology chapter for
use of this principle in the preparation of RNA-free DNA in practical lab situations.
Chapter 17 RNA 153
17.7 Small Interfering RNA

Small interfering RNAs (siRNA) molecules target an Argonaut siRNA-dependent ribonuclease complex to
cut a specific RNA sequence. These siRNAs are encoded in the genomes of many, if not most organisms
(including humans) as micro RNAs. They are not translated and interfere with protein expression by either
directing the destruction of a transcript, or preventing its use in translation.
(1) Bases 2-8 of the guide RNA form an A-form double helix with the target RNA. Prior to target RNA
binding, bases 2-6 in the siRNA RISC complex are exposed as an “activated” probe called the “seed region.”
PIWI Mid
siRNA Seed Region

of the
siRNA
PAZ
N-terminal
Argonaut Target RNA RISC-siRNA

protein complex
Target RNA
(e.g., the fas mRNA)
RISC-siRNA
complex
Cut target RNA
(2) The RNA-induced Silencing Complex (RISC) can find and cleave its target almost 10-fold faster than
the lone guide RNA can anneal to its target. For details, see Pratt, A. and MacRae, I. (2009) The RNA-
induced Silencing Complex: A Versatile Gene-Silencing Machine, J. Biol. Chem. 284, 17897–17901.
(3) This technique was used in a medical application to destroy the mRNA for the fas ligand on the surface
of T-lymphocyte cells. This treatment prevents the worst damage to the liver caused when Hepatitis A or C
virus infection occurs.
The human RISC contains a siRNA that is complementary to the fas mRNA cut site. When the target
RNA binds, the argonaut ribonuclease cuts it, destroying the fas mRNA so the fas ligand protein is not
made by the T-lymphocytes.
With no fas ligand made, they cannot bind the fas receptors on the liver, so they do not enter it. If this
occurred, it would initiate an apoptosis (programmed cell death) response. The ability to cut the fas mRNA
allows one to prevent the most deadly and acute damage to the liver. See Lieberman, J., Master of the Cell
in The Scientist, Apr. 2010, pp. 43–48.
(4) In another application, a siRNA was designed to silence one of the genes of the respiratory syncytial
virus (RSV), which is the leading cause of infant hospitalization, yet is fairly harmless in healthy adults.
The siRNA was administered in a nasal spray and only 44% of those who received the RSV siRNA spray
became infected with RSV, compared with 71% in the placebo group. This approach is also being tested as
a means to protect lung transplant patients. See Holmes, B., Gene Silencing Prevents Human Disease, in
The Scientist, May 2010, p. 10.
Chapter 18
Biotechnology
Biotechnology involves combining the concepts biochemistry has uncovered with the natural inclination
scientists have to tinker with their systems to make new tools.
For example, Wendell Lim and colleagues were investigating the light-sensitive plant protein
Phytochrome and its binding partner Phytochrome Interaction Factor (PIF). In response to red light, they
found that the two bind and then translocate into the nucleus. The researchers decided to connect PIF to a
cytoskeletal protein. They had to do some genetic engineering with both phytochrome and PIF first, but
with the right constructs, when they turned on the red light, the cytoskeletal protein was activated and
reshaped the cell. They had created a way to turn on cytoskeletal protein-protein interactions using light.
The researchers have made the two plasmid DNAs that encode the mutant protein constructs available on a
nonprofit basis. (See Lim et al., Nature 461, 997–1001, 2009.)
The route to their discovery involved first characterizing their idea then deciding it would be
interesting to try the light-dependent PIF function in a non-natural task, reshaping the cell. They had to
modify their natural proteins to accomplish their task, but were able to develop an engineered light-
sensitive intracellular switching system. Biotechnology is the playing field where biology meets
nanotechnology.
The essential tools used to accomplish cloning are: (1) restriction endonucleases, (2) single-nucleotide
resolved gel electrophoresis, (3) the ability to propagate DNA sequences and test their in vivo functions
using plasmid DNA, and (4) the ability to replicate plasmid DNA after transforming it into a bacterial cell.
Each is described below.
18.1 Restriction Endonucleases

This class of enzyme catalyzes DNA restriction by recognizing the DNA sequence and cleaving both
strands to produce two duplex fragments containing staggered ends, also called sticky ends.
.
restriction
endonuclease 3' 5'
5' - NN-GAATTC-NN - 3' 5' - NN-G AATTC-NN - 3'
3' - NN-CTTAAG-NN - 5'
+ G-NN - 5'
3' - NN-CTTAA
5' 3'
5' - GAATTC - 3' Inverted two-fold

symmetry axis sticky ends
3' - CTTAAG - 5'
180o
.
The NN sequences indicate the rest of the strands on either side of the restriction site.
(1) In most circumstances, four- to eight-base pair segments operate as recognition sites with a 2-fold axis
of symmetry. The paired sequences read the same forward and backward on the complementary strand.
These kinds of sequences are known as palindromes. Examples are BIB, DEED, RADAR, MADAM I’M ADAM,
and A MAN, A PLAN, A CANAL, PANAMA.
(2) When placed at the ends of full-length DNAs from chromosomes, plasmids, and so on, these staggered
ends act like Velcro that only binds the specific complementary staggered end. The specificity in splicing
two pieces of DNA together offered by this technique is one basis of cloning technology.
Restriction Inhibition
The host bacterium can evade its own restriction enzymes by protecting its restriction sites with
methylation. The host bacterium DNA is methylated at the restriction enzyme recognition site. Foreign or
invader DNAs are cut because they are not methylated at the restriction endonuclease cutting sites.
Methylation distinguishes the host cell DNA from that of invaders.
Chapter 18 Biotechnology 155
The mechanism involves two steps:

(1) Following DNA replication, the GAATC site is hemimethylated.
(2) A methyltransferase catalyzes methylation of the second adenine residue in the recognition site.
.
H 3C H 3C H3C
replication methylation
5' - NNGAATTCNN - 3' 5' - NNGAATTCNN - 3' 5' - NNGAATTCNN - 3'
3' - NNCTTAAGNN - 5' 3' - NNCTTAAGNN - 5' 3' - NNCTTAAGNN - 5'
CH3 CH3
Restriction Mapping
Restriction enzymes can be used to map DNAs. Amino acid sequence-specific proteases were discussed in
the Protein section. Different proteases (e.g., trypsin, chymotrypsin) can be used to create overlapped
fragments. The same idea can be done using restriction enzymes. Computers can reassemble the fragment
information to make sense of it. As a result, one can use broken up preparations of DNA, yet still figure out
how they were linked together within the original unbroken DNA.
DNA shears apart each time it is ejected from or drawn into a pipette tip, so we are rarely working
with DNAs that are the size of full-length eukaryotic chromosomes. This shearing can be avoided by
preparing cells, nuclei, and chromosomes in gels directly then carrying out electrophoresis to analyze them.
The Alternating-Field Gel Electrophoresis method is used to study multichromosomal preps, such as in
chromosomal typing analyses.
A restriction map of Bacteriophage Lambda (λ) DNA is shown below. Five different “restriction
enzymes” cleave at the sites shown. Digestion, this enzymatic cleavage, according to the map shows that
the λ DNA is cut with the enzyme Apal, forming two fragments that are 10.00 kilobase pairs (kb) and 38.4
kb long.
KpnI KpnI
ApaI XbaI XhoI
Phage λ genome
(48.4 kb)
10.0 6.9 5.5 9.5 15.0
fragment sizes (kb)

The λ DNA genome is a linear, double-stranded DNA that consists of approximately 48,000 bp (48.4
kb). A restriction digest is made by treating this DNA with various restriction enzymes. By measuring the
sizes of the resulting fragments, it is possible to develop a map of the cleavage sites. The DNA fragments in
a restriction digest are separated by electrophoresis, as shown below.
48.8
38.4
33.4
30.0
larger 24.5 Size
(kb)
23.9
smaller
16.9
15.0
10.0
1 2 3 4 5
When λ DNA is subjected to gel electrophoresis, one can distinguish the DNA product fragment by size.
The smaller pieces have less molecular weight and therefore travel to the bottom of the agarose gel faster.
Lane 1: Apal digestion. Lane 2: Xhol digestion. Lane 3: KpnI digestion; we are unable to see the smallest
156 Chapter 18 Biotechnology
fragment (1.5 kbp). Lane 4: XbaI digestion; the two fragments are close in size and not well resolved. Lane
5: Intact λ DNA and an assortment of fragments from the other lanes.
Specificities. The following table lists the recognition sequences and cutting sites of many common
restriction enzymes:
Enzyme Recognition sequence Source
5’ ↓
BamHI G GATCC 3’ Bacillus amyloliquefaciens H
3’
C CTA↑GG 5’
BglII 5’
A↓GATCT 3’ Bacillus globigii
3’
TCTA↑GA5’
EcoRI 5’
G↓AAmTTC 3’ Escherichia coli RY13
3’
CTT↑AmAG 5’
5’ ↓
EcoRII CCmTGG 3’ Escherichia coli R245
3’
GGACmC↑ 5’
HaeIII 5’
GG↓CC 3’ Haemophilus aegyptius
3’
GG↑CC 5’
HindII 5’
GTPy↓PuAmC 3’ Haemophilus influenzae Rd
3’
CAmPu↑PyTG 5’ (Note that the sequence can
vary.)
5’
HindIII Am↓AGCTT 3’ Haemophilus influenzae Rd
3’
TTCGA↑Am 5’
HpaII 5’
C↓CGG 3’ Haemophilus parainfluenzae
3’
CCG↑G 5’
NotI 5’
GC↓GGCCGC 3’ Nocardia otitidis-caviar
3’
GCGGCC↑GC 5’
PstI 5’
CTGCA↓G 3’ Providencia stuartii 164
3’
G↑ACGTC 5’
SmaI 5’
CCC↓GGG 3’ Serratia marcescens Sb
3’
GGG↑CCC 5’
18.2 Cloning in a Nutshell

Basic Process. First, the gene you want to clone is cut with two restriction endonucleases then spliced into the
linearized target plasmid. That plasmid must first be prepared by cutting with the same two restriction
enzymes. This ensures that the upstream control sequences and the gene body sequences are aligned to produce
the mRNA, with all of the appropriate transcription and translation control sequence elements in proper order.
translation
transcription translation termination transcription
initiation initiation termination
Ori
Ori
e.g., EcoRI
e.g., EcoRI intended gene to clone
Selectable e.g., HindIII SMG

inserted
marker gene gene
(SMG)
(e.g, AmpR) Expression
Cloned gene inserted into e.g., HindIII
parent plasmid
the expression plasmid
(1) A selectable marker gene encodes a protein that destroys the antibiotic being used for selection
purposes. If the bacterium contains the plasmid, it will be able to live in the presence of the antibiotic. The
bacterium is antibiotic resistant.
(2) The gene expression cassette fragments are situated on upstream and downstream from the polycloning
site, where the gene to be expressed is inserted.
Ori Polycloning site (part 1) - consists of a set of
restriction endonuclease cutting sites.
The upstream DNA encodes sequences that

e.g., EcoRI (1) replicate the DNA (Ori) and
(2) initiate and transcribe the messenger
RNA sequence.
An expression plasmid will have these

SMG sequences upstream from the polycloning site,
inserted so one only has to insert the protein-encoding
gene
part of the gene. The plasmid takes care of the
rest.
e.g., HindIII
The downstream DNA encodes sequences
for termination of replication and required
mRNA sequences.
Polycloning site (part 2)

.
Transcription and Translation Sequences. The mRNA must contain sequences that initiate, elongate, and
terminate (1) transcription by the cellular RNA polymerase, and (2) translation by the ribosome, thereby
producing the encoded protein.
transcription translation transcription translation
initiation initiation termination termination
DNA
transcription
polycloning polycloning
site 2 site 2
RNA
translation
protein
18.3 DNA Preparation: Phenol-Chloroform Extraction

Manipulations involved in actually cloning a gene can be very difficult because, as a coworker once
lamented, “everything’s invisible!” Well, not always. Sometimes one can see the pellet when one ethanol-
precipitates a plasmid prep.
The phenol-chloroform DNA preparation method is very practical. One colleague tells me the
technique swept through the labs in Germany where he was working when it was first introduced. This
single-step method uses phenol/chloroform reagent, which is very effective in breaking apart most
membrane and cellular components by alkaline hydrolysis and then separating them from the DNA.
.
O H O δ δ+
H+ Cl
Cl C H
Cl
phenol phenolate
(1) The base phenol converts to phenolate, an effective nucleophile. Phenolate catalyzes alkaline
hydrolysis, which breaks down many types of bonds in membrane components. Proteins are denatured.
DNA is not susceptible to base-catalyzed hydrolysis under these somewhat moderate conditions. RNA will
only degrade after extensive incubation.
(2) Chloroform is polar enough to solubilize more polar materials liberated by breakdown of triacylglycerol
components and is effective in undoing the “hydrophobic effect,” which is a major stabilizer of membrane
structure.
Phospholipids
O O
O CH2
C + O CH2 O + O P O
δ
δ+
H2O O CH2
C O CH2 O
O
O δ+ + (= φ-Ο ) H2C O
Chain lengths depend on H2C O P O X + O X
which fatty acids are present.
O H
phenolate C O
+
O
Proteins
R2 R3 φ-O
O O H2O R2 R3
O O
δ+ δ+
δ+
R1 N Cα C N Cα C R4 R1 N Cα
δ+
C O + H3N Cα C R4
H H H H H H H H
RNAs
Base 1
R1 O
φ-O
OH H2O R1 O Base 1 Base 2
HO O
O O H
+
δ +
O P O
O O O OH
O Base 2 P
O H
O O R2
H
O OH
R2
For many more details about the practical techniques, ideas, and manipulations used in cloning,
expression, purification and detection of DNAs, RNAs and proteins, see Hardin et al., Cloning, Gene
Expression and Protein Purification, Oxford University Press, 2001.
18.4 Polymerase Chain Reaction (PCR)

Purpose
The goal is of doing PCR is to synthesize large quantities of a specified DNA sequence with precisely
correct ends.
Contrast with Cloning

Cloning and PCR are used to make large amounts of predetermined DNA sequences. Plasmids are used in
DNA cloning techniques to carry particular sequences in particular genetic contexts, allowing one to
produce mRNAs, proteins and other gene products. This can be done in the test tube (in vitro) but is most
easily accomplished in vivo. The unique advantage of a plasmid versus a PCR product is that one can
produce a desired product in the cell directly. This allows one to assess the biological effect of that product
in a sort-of-normal cell environment.
The difficulty with plasmids in producing large amounts of DNA (versus PCR) is that: (1) cells must
be grown, (2) the DNA must be extracted from the cells, (3) the plasmids must be separated and purified,
(4) the desired fragment in the plasmid must be cut out using restriction endonuclease enzymes, and (5) the
DNA fragment must be purified (usually on a gel). As a result, PCR has become the method of choice in
many, many types of experiments.
On the other hand, cloning is (1) relatively easy, (2) provides a permanent record of the DNA (when
stored as a glycerol stock solution), and (3) can generally be sequenced more easily than PCR fragments.
Mechanism of PCR
The technique involves three steps, which are repeated for a series of 20 to 30 cycles.
(1) Denaturation of the DNA template strands. This is typically done at 95 ºC.
(2) Anneal the two primer DNAs, one to the 5’ terminal point of the sequence to be copied on each template
strand. This is done at 55 ºC, but is varied to optimize product formation.
(3) Polymerization to produce the two product strands.
(The cycle is repeated)
DNA Polymerase Mechanism

DNA Strands with a 3'-terminal A DNA Strands with a 3'-terminal G
O O O O
P P
H H
O O H O O H
CH2 N N H
CH2 N N H
O N O N
H H H N H H H N
N H+ N
H H
H O H H O H
H H
O O
O
P P
dGTP O O O
O O O H8 O H8
N P N
P CH2 7 5 O CH2 7 5 O
O O O O O
O O O H H N 6
H H N 6
H 9 N H H 9 N H
4
1
P 4
1
P H N O H N
O O 3
2 O O 3
2
O H H
N H H N H
H H
Reaction temperatures are optimized to:

(1) denature the initial template sufficiently,
(2) allow the primers to bind, and
(3) assure that no competing secondary structures form.
The primer-template DNA-DNA binding temperature is kept high enough to assure that only
stringently selected primer-template double helices form.
Note. Two terms are used to refer to double helix formation: annealing and hybridization.
Reaction Components
(1) Primer DNAs: They “bracket” the sequence that is to be “amplified” (made in large amounts).
(2) Template DNA: Can be a plasmid, chromosome, or a biological sample. Chromosomes in blood and
saliva swab samples are used in CGI-type applications.
(3) Buffer: Required to support the DNA polymerase activity.
(4) Mg2+: This metal is required to assure, as much as possible, that the product has sufficient fidelity. The
3’ to 5’ exonuclease activity of the DNA polymerase carries out this assurance reaction/process.
(5) Deoxynucleotide Monophosphates (dNTPs): i.e. dATP, dCTP, dGTP and dTTP. Other nucleotides are
included in specific applications, such as body labeling the DNA probe molecule with digoxigenin (see
below).
(6) Taq DNA Polymerase. This enzyme is produced from the thermophilic bacterium Thermus aquaticus.
Taq DNA polymerase does not have the 3’ to 5’ exonuclease activity and is very error-prone. It has been
replaced by Vent and Pfu DNA polymerases.
PCR Reaction Steps

(1) First Cycle: Duplex Template.
Primer DNA binding sites
3'
3' 5'
Denature template
DNA at 95 oC
Anneal Primer DNAs

(2) Two Single-Stranded Templates.
at 55 oC
5' 3'
3' P1 5'
+
5' P2 3'
3' 5'
Replicate the template DNA

(Primer extension) at 55 oC
(3) First Cycle Products.
3'
5'
3' 5' P1 3'

+
5' P2 3' 3'
3' 5'
95 oC
Original template
(4) Second Cycle: DNA strands
3' 3' P1 5'

+
5' P2 3' 3'
Anneal Primer DNAs,

(5) Second Cycle Products.
55 oC
DNA Polymerase runs out of
5' P2 template, producing the
3' 3' intended ends
3' 3' P1 5'
+
5' P2 3' 3'
The 5' ends created by the 3' 3' P1 5'

Cycle 1 primers lead to correct
termini in Cycle 2
20 to 30 Cycles
5' 3' 3'
3' 3' 5'

N = 100,000s to millions
18.5 Probe DNA

A probe DNA is a known sequence one uses to search for the Watson-Crick complementary sequence, in
either RNA or DNA, depending on the goal of the experiment.
(1) One usually labels the probe with either radioactive [32P]-phosphate, a fluorescent molecule (a
fluorophore), the antibody-binding antigen digoxigenin, or the avidin-binding molecule biotin (see the
Coenzyme section).
(2) One then anneals the probe to the sample, meaning that the probe strands form duplexes with strands
from denatured duplexes in the sample.
(3) The purpose is to detect sequences that are complementary to the probe. If they are present in the
sample of interest, the probe binds to them. Since the probe is labeled, one also labels the fragment
containing the complementary sequence. Therefore, the third step involves detection using a piece of film,
a phosphorimaging apparatus, and so on.
This tool is used in a number of techniques, such as Northern and Southern Blot hybridization
analyses, Mobility Shift protein-binding assays, and Colony Hybridization. These methods have been used
extensively to study developmental gene expression, to determine the identity of a DNA in a plasmid, to
determine if a cell harbors a specific plasmid, and so forth.
Probe Construction: PCR from a Plasmid Template Using Digoxigenin-dUTP
Creating a probe involves using PCR. Body labeling of duplex DNA strands from a plasmid that contains a
specified Geminivirus DNA segment. The label digoxigenin (DG) is incorporated in each strand randomly
in place of some of the deoxythymidines.
P1 5' P1
3'
dNTPs,
P1, P2 DIG-dUTP DIG
P2 DIG
DNA Pol
DIG
3'
P2
5'
The structure of alkaline-labile Digoxigenin-11-Deoxyuridine Triphosphate (DIG-11-dUTP, aka DIG) is:

O
Digoxigenin O
OH
Deoxyuridine
triphosphate Linker
O O H O
H OH
N C N C
N C O
O H O
Pi Pi Pi N H
O
H H
HO H
Illustration of Probe DNA Use: Southern Blot Hybridization and Detection Using an Immunoconjugate
Prehybridization and Hybridization. To perform stringency control during duplex formation, one does a
prehybridization wash of the DNA on the blot filter. This is followed by hybridization of the probe to the
DNA on the blot filter.
5'
5'
3' 3'
Viral
'target'
DNA DIG
DIG
+ DIG
DIG
5' 5'
DIG
Blot DIG
filter 3'
3' X
X
DIG-labeled Blot filter-bound viral

probe DNA
Crosslink connecting the DNA hybridized to DIG-
viral DNA to the blot filter labeled probe DNA
Next, one washes the probe- DNA complexes on the blot filter using Standard Saline Citrate (SSC)
buffer containing sodium dodecylsulfate (SDS). Next one blocks the blot filter (i.e. the sites that do not
contain probe-viral DNA duplexes) with Bovine Serum Albumin (BSA).
.
5' 5'
3' 3'
DIG DIG
DIG + DIG
5' 5'
DIG BSA DIG
3' 3'
X X
Blocked blot filter

.
Immunoconjugate Detection of Bound Probe DNA. To detect hybridized DNAs, one then adds the
immuno-conjugate, a covalent antibody-enzyme complex, to the blot filter.
.
5' 5'
bound E
3' immunoconjugate 3'
immunoconjugate DIG
E
DIG
E
DIG + E DIG
5' 5'
DIG DIG
3' 3'
X X
Blocked blot filter BCIP
5'
E
3' BCIP (yellow soluble)
DIG
E
bound immunoconjugate product* (blue precipitate)
E
DIG
5'
DIG
3'
X
.
Colorimetric Label Detection is accomplished using the BCIP reaction as shown below. Appearance
of a blue precipitate on the blot membrane indicates that the DNA being sought with the probe is present at
that position on the gel pattern.
BCIP (5-bromo-4-chloroindoyl NBT (4-Nitroblue
phosphate, toluidine salt Tetrazolium cloride)
(colorless, soluble) (yellowish, soluble)
O O O-CH3 O-CH3
If the antigen is present, Cl P
N N
the immunoconjugate will Br
O O
N N
bind. 2 N
N N
N
N
O O
Alkaline Phosphatase 2 P
Anti-Digoxigenin O O NO2 NO2
Immunoconjugate
O-CH3 O-CH3
Cl
O H N N
Br N N N
N N
NH HN
N
Br
H O
Cl
* (blue precipitates) NO2 NO2

Chapter 19
Metabolism
19.1 Overview
The following map shows the general flow of components, the key intermediate species, the process names,
the energy sources produced by the catabolic processes and the sources of precursors used to make the four
groups of macromolecules. Many other important and sometimes subtle cross-connections are not indicated.
For example, the nucleotide bases are made using an intermediate from the urea cycle and an amino acid.
Membranes, assemblies
organelles,...
POLYPEPTIDES POLYSACCHARIDES LIPIDS
Proteins Glycogen Triacylglycerides
Translation (phosphorylase b) Esterification Lipolysis

(phosphorylase a)
Amino acids Fatty Acids

Glucose Glucose Phosphate NUCLEIC ACIDS
brain (G6P) Pentose
phosphate CO2 DNA
Glycolysis RNA
ATP pathway
NADPH
.
NADH Ribose-5-phosphate Replication Transcription
ATP
Gluconeogenesis
Nucleotides
Phosphoenolpyruvate (PEP)
Pyruvate
kinase
ATP Lactate
dehydrogenase
Pyruvate Lactate
CO2
Acetyl CoA
ketone bodies brain
Oxaloacetate
Citric
Urea Acid
Cycle Cycle CO2
GTP
NH4+ NADH
CoQH2
Oxidative
phosphorylation O2
ATP
H2O
(Adapted from Moran et al., Biochemistry (2nd ed.), 1996, p. 14-7; Fig. 14-7.)
164 Chapter 19 Metabolism
19.2 Metabolic Pathway Types

Linear Pathway. An example is the biosynthesis of proline, the product of each reaction in the pathway is
used as the substrate for the next step. Another key pathway of this type is glycolysis, which is described in
detail in that chapter.
Glutamate
γ-Glutamyl phosphate
Glutamate γ-semialdehyde
Pyrroline 5-carboxylate
Proline
Cyclic Pathway. A series of reactions form a closed loop. Each turn is completed by regenerating the
original intermediate. The Krebs Cycle metabolizes an acetyl group to form carbon dioxide and other
materials then reforms the initial component, oxaloacetate, in a series of ten reactions. Two other examples
described in later chapters are the Malate-Aspartate Shuttle and the Urea Cycle.
Acetyl CoA
CoA
Oxalo-
acetate
Citrate
Malate
Fumarate Isocitrate
CO2
Succinate α-Ketoglutarate
Succinyl CoA
CO2
Spiral Pathway. Fatty acid biosynthesis is the classic spiral metabolic pathway. The enzyme complex
lengthens the alkane chain by two carbon units with each repeated set of reactions, each turn of the spiral cycle.
.
O
S CoA
(C8)
O (Additional turns)
Turn 3 S CoA
(C6)
O
Turn 2 S CoA
(C4)
Turn 1
O
S CoA
(C2)
Chapter 19 Metabolism 165
19.3 Energy Conservation

Phosphorylation Energy. In catabolic reactions, conserved energy is stored in the form of nucleoside
triphosphates and reduced coenzymes. The relation between use and conservation of phosphorylation-
dependent energy in phosphate-containing compounds is:
.
Energy X + Pi
Energy
X P
where X is the molecule that accepts the phosphate.

For example, glucose is phosphorylated by hexokinase, which energizes the compound in preparation
for later reactions in glycolysis. In those reactions, the phosphate acts as a good leaving group. The energy
is provided by, for example, transfer of phosphate from ATP, an excellent leaving group. That is the
essence of energy transfer in many biochemical reactions.
Electron-Transfer (Reducing) Energy. In the same way, energy is captured by reduced coenzymes in
oxidation reactions.
Y
2H ,2e
2H ,2e
YH2
where Y signifies the oxidized component and YH2 its reduced component. For example, during the
reduction step in fatty acid biosynthesis, extra reducing power is “packed” into the alkene chain by
converting it to an alkane.
Linkage. These two modes of energy transfer and use are directly linked by events that occur in the
mitochondrion. ATP is used as an energy carrier. It is made, that is, receives its energy, from the reduced
coenzymes that feed electron transport. Reducing equivalent carriers NADH and CoQH2 provide the
reducing potential energy that drives the proton pumps, which, in turn, drive oxidative phosphorylation, the
process that makes ATP.
19.4 Key Pathways/Reactions

(1) Glycolysis is the universal pathway for glucose catabolism. A single 6-carbon molecule, glucose, is split
into two 3-carbon molecules, namely pyruvate, which can be further oxidized in the citric acid cycle.
Glycolysis functions without oxygen to generate ATP. This pathway produces three common products:
lactic acid, ethanol, and pyruvate.
(2) The Citric Acid Cycle metabolizes the acetyl CoA produced from pyruvate in glycolysis and requires
oxygen to run the cycle. Each acetyl CoA that enters the cycle is fully oxidized to carbon dioxide while
conserving energy in the form of NADH, CoQH2, and ATP or GTP.
(3) Glucose Metabolism comes in other forms as well including the synthesis and degradation of glycogen,
a polymer used to store glucose. The degradation of glycogen is not merely the reverse of its synthesis.
The enzymes involved in Glycogen Synthesis and Degradation act in response to metabolic signals in such
a way that the rates of these opposing reactions can be directly correspondent with the demands of the
entire cell or organism.
(4) Gluconeogenesis is the process responsible for the synthesis of glucose from three-carbon compounds
such as pyruvate, lactate, and glycerol.
(5) The Pentose Phosphate Pathway oxidizes glucose producing ribose instead of ATP, in preparation for
nucleotide and nucleic acid synthesis, and NADPH, which is needed for biosynthetic reduction reactions.
Because this pathway is used for both synthesis and reduction reactions, it is considered both an anabolic
and catabolic pathway.
(6) Electron Transport involves a series of electron-transfer reactions in which input NADH and CoQH2 is
eventually used to reduce molecular oxygen, the terminal acceptor of the reducing equivalents. Electron
166 Chapter 19 Metabolism
transport processes drive a set of proton pumps, which produce a proton gradient across the inner
mitochondrial membrane in eukaryotes, or cell membrane in the case of prokaryotes.
(7) Oxidative Phosphorylation. As the protons collect on one side of the membrane, a transmembrane
potential energy accumulates, which drives the membrane-spanning F0F1 ATP Synthase complex to
phosphorylate ADP and thereby produce ATP.
ADP + Pi → ATP + H2O (1)
(8) The Photosynthesis process captures light energy in the form of reducing equivalents, then uses them to
make ATP in a manner similar to the mitochondrial electron transport-oxidative phosphorylation pathway.
Chapter 20
Bioenergetics
20.1 Reaction Equilibria: Standard and Actual Free Energies
Reactions in most cellular niches occur at concentrations far from the 1 M, which is the case for all
reactants and products in the standard state value of the Gibbs free energy change (ΔG0′). Consider the
generic reaction:
A+B↔C+D (1)
The Gibbs free energy (ΔG) is calculated by subtracting the sum of the free energies of the products from
the sum of the free energies of the reactants.
ΔG = (GC + GD) - (GA + GB) (2)
Adding the actual free energy contribution to the standard (state) free energy:
ΔG = (GC0′ + GD0′ - GA0′ - GB0′) + RT ln ([C][D]/[A][B]) (3)
Defining ΔG°’ as GC ′ + GD ′ - GA ′ + GB ′, one gets:
0 0 0 0
ΔG = ΔG0′ + RT ln ([C][D]/[A][B]) (4)

(actual) (standard)
At equilibrium poise, the ratio of concentrations in eq. 4 is equal to the equilibrium constant Keq.
When the concentrations of products and reactants reach equilibrium (poise), the rates and extents of
forward and reverse reactions satisfy the ratio dictated by Keq. The result is that ΔG must equal zero. The
relation between free energy and equilibrium constant is:
ΔG0′ = -RT ln Keq (5)
If ΔG°′ is known, one can use eq. 5 to calculate Keq. One can also do the reverse calculation, use Keq to
calculate ΔG0′. A logarithmic relationship exists between ΔG°′ and Keq, so small change in ΔG0′ results in
large changes in Keq.
Free energy changes that occur under cellular conditions (ΔG) must be negative in order for an
enzymatic reaction to occur. The ΔG°′ values of many metabolic reactions have standard ΔG0′ values with
positive signs. The low substrate and product concentrations under cellular conditions produce a significant
difference between the ΔG and ΔG0′ values of many biochemical reactions.
Keq, is defined as the ratio of substrates to products when ΔG is 0.
Keq = ([C][D]/[A][B]); ΔG = 0 (6)
When the reaction components are not under standard state conditions, the ratio of products over substrates
changes. The change in free energy that corrects for the difference between ΔG°′ and ΔG is calculated as
follows:
ΔG = ΔG0′ + RT lnQ (7)
(actual) = (standard) + ΔΔG
Where ΔG is the steady state Gibbs free energy, ΔG0′ is the value for the same equilibrium under standard-
state conditions, Q is the mass action ratio, and ΔΔG is the perturbation to the equilibrium relative to the
standard state poise. This is the true driving force in a steady state situation, where a process is fed and
subject to a steady input at some given rate.
Q = ([C]′ [D]′/[A]′ [B]′) (8)
The value of Q determines the difference in free energy between standard and actual values, indicating the
difference between the reaction conditions and equilibrium poise if all of the components were present at 1 M.
168 Chapter 20 Bioenergetics
One determines the spontaneity of a reaction based on ΔG rather than ΔG0’. The degree of spontaneity
determines the predisposition of the reaction direction and potential to undergo concentration-dependent
changes in ratio of products to reactants. Remember that if ΔG is > 0, the reaction proceeds toward
reactants rather than products.
To partition means to shift a distribution, governed by a change in equilibrium poise, to establish a
new intended ratio of products over reactants, the actual state, rather than the standard state. This is
significant because biochemically important concentrations can span a wide range, from picomolar to
molar, with many in the µM to mM range.
The equations in this section were used to adjust the pattern of standard state free energies for the
reactions in glycolysis to the actual values (see below). This correction converts a seemingly meaningless
pattern into a very interpretable one, which reveals the principle that drives the flow of components through
the entire pathway, even through near equilibrium steps.
20.2 Metabolically Irreversible and Near Equilibrium Reactions

Reactions Types
Metabolic reactions can be separated into two types.
(1) A near-equilibrium reaction has a Q value close to Keq. This leads to the net steady-state ratio of
reactant to product concentrations in a living cell. These reactions have small actual free-energy changes
(ΔG) and are often strongly reversible.
(2) A metabolically irreversible reaction is strongly displaced from equilibrium, i.e. Q is far from Keq.
These differences are commonly up to 100- to 1000-fold. As a result, the ΔGs are large and negative for all
metabolically irreversible reactions.
Regulation
The amount of enzyme present in a cell to catalyze a metabolically irreversible reaction is insufficient to
drive a near-equilibrium reaction. Most pathways are regulated through enzymes that catalyze
metabolically irreversible reactions. In this sense, they act as a bottleneck to metabolic traffic that slows the
flux of metabolites through reactions later in the pathway.
Pathways are not well controlled by near-equilibrium reactions. Because they are so close to
equilibrium, the flux through this step cannot be easily increased. Large changes in substrate and product
concentration are the only control mechanism for near-equilibrium reactions. On the other hand, these
concentrations have virtually no effect on metabolically irreversible reactions, but are better controlled by
effectors that adjust the rate of catalysis.
When a pathway’s flux is altered, the concentrations of intracellular metabolites vary in a small range
of about 2- or 3-fold. Given the abundance of enzyme present to catalyze most near-equilibrium reaction,
the substrate and product concentrations restore readily back to equilibrium poise after perturbation.
Enzyme and Pathway Reversibility

The near equilibrium reaction enzymes can catalyze reactions in both directions. We will see that this is
useful, as illustrated with the glycolysis and gluconeogenesis pathways, which use the same near-
equilibrium reaction enzymes in both directions. As a result, gluconeogenesis only requires enzymes to
replace glycolytic enzymes to catalyze the metabolically reversible reaction steps. Four enzymes in
gluconeogenesis replace three enzymes use in glycolysis to accomplish this reversal of pathway flux.
Standard Free Energies of Hydrolysis for Common Metabolites

The following table compares the energies of some standard metabolic compounds.
Metabolite ΔG0′hydrolysis (kJ mol-1)

Phosphoenolpyruvate -62
1,3-Bisphosphoglycerate -49
Acetyl phosphate -43
Phosphocreatine -43
Pyrophosphate -33
Chapter 20 Bioenergetics 169
ATP to AMP + PPi -45 (kJ mol-1)

ATP to ADP + Pi -30
Glucose 1-phosphate -21
Glucose 6-phosphate -14
Glycerol 3-phosphate -9
(Adapted from Moran et al., Biochemistry (2nd Ed.), 1996, p. 14-21, Table 14-3.)
Note that phosphoenolpyruvate (PEP) is nearly twice as energetic as ATP. Two steps in gluconeogenesis
replace one step in glycolysis. These steps use two ATPs to reverse the step in glycolysis in which PEP
transfers its phosphate to make ATP. More insights are gained by looking at these values for the glycolysis
reactions, especially after correcting them to be consistent with intracellular conditions.
20.3 Energies and Regulation of Glycolysis

Comparison of ΔG and ΔG°′ for the Reactions of Glycolysis
The free energies of the glycolytic reactions, calculated for the standard and actual states, are listed in the
following table. One reaction, the interconversion catalyzed by triose phosphate isomerase, is not shown.
Enzyme ΔG0′ (kJ mol-1) ΔG (kJ mol-1)

1. Hexokinase -17 -33
2. Glucose-6-phosphate isomerase +2 near equilibrium (~0)
3. Phosphofructokinase-1 -14 -22
4. Aldolase +24 ~0
5. Triose phosphate isomerase +8 ~0
6. Glyceraldehyde-3-phosphate +6 ~0
dehydrogenase
7. Phosphoglycerate kinase -19 ~0
8. Phosphoglycerate mutase +5 ~0
9. Enolase +2 ~0
10. Pyruvate kinase -32 -17
(Adapted from Moran et al., Biochemistry (2nd Ed.), 1996, p. 15-21, Table 15-2 and p. 15-20, Fig. 15-23.)
The enzymes shown in italics catalyze the three metabolically irreversible steps. Each catalyzes a kinase
reaction. They drive the flux of components through the pathway.
The information is presented graphically in the following plots, which show ΔG and ΔG0’ for each of
the ten numbered reactions of glycolysis. The effect of the three kinase reactions is obscured when one
plots the standard state ΔG0’ values. The pattern is revealed when the adjusted actual free energies are
calculated.
0
Not considering
Standard State -50 cellular substrate and
ΔG0' product concentrations.
(kJ/ mol)
-100
1 2 3 4 5 6 7 8 9 10
0
Actual ΔG -72.3 kJ/mol
(kJ/ mol) -50
-100
1 2 3 4 5 6 7 8 9 10
Reaction Number .
170 Chapter 20 Bioenergetics
Some of the reactions of glycolysis have positive standard free-energy changes. This means that when
the enzyme is saturated with substrate the reaction will flow toward reactants, instead of products. Note
that the actual ΔGs are generally much smaller than the standard state values.
Regulatory Enzymes
(1) Hexokinase, phosphofructokinase-1, and pyruvate kinase all have large actual ΔG values, so they
are all subjected to stringent regulation. Regulatory enzymes commonly have oligomeric quaternary
structures. Of the ten enzymes in glycolysis, only phosphoglycerate kinase is monomeric. The others are
either dimers or tetramers. Each has sites for regulatory effector binding near the interface between the
subunits. The three regulatory kinases and two enzymes in glycolysis are subject to cooperative substrate
binding. Hexokinase is present in both monomer and dimer forms, but is more active as the dimer.
(2) Weak binding to a substrate increases the catalytic efficiency of an enzyme. To obtain the most
effective response to substrate changes and reestablishing equilibrium, the KM value for an enzyme should
be close to its cellular substrate concentration. This is not true for allosterically regulated enzymes. The
value for KM may change allosterically, so the cellular substrate concentration must be shifted away from
KM, in a direction that depends on whether up- or down-regulation is to be deployed.
Chapter 21
Bioelectrochemistry
21.1 Redox Reaction Principles

Reduction potential energies can be measured quantitatively using an electrochemical cells. This is
illustrated in the oxidation-reduction reaction where an electron pair is transferred from an atom of oxidized
zinc (Zn) to a reduced copper ion (Cu2+):
Zn + Cu2+ ↔ Zn2+ + Cu
(1) This reaction is performed using two solutions, which separate the anode from the cathode, and divide
the total reaction into two half reactions (see below). Two electrons are given up by each zinc atom, which
is the reducing agent or reductant, at the anode. A wire travels through a voltmeter, which connects the
zinc in one solution to the copper in the other. The electrons flow through this wire to the cathode, where
copper, the oxidizing agent or oxidant, is reduced to metallic copper.
(2) The two solutions are kept electrically neutral by a salt bridge, which is made of a tube filled with
electrolytes containing a porous partition. The salt bridge allows the nonreactive counterions to flow
through an aqueous conduit between the two solutions. This allows accurate voltmeter readings by
separating the ion flow from the electron flow.
Voltmeter
2e 2e
(Current, amperage)
Salt bridge
Zn2+
Cu2+ Cuo
o
Zn
SO42 - SO42 -
Zno Zn2+ + 2 e Cu2+ + 2 e Cuo
More negative potentials are assigned to reaction systems that have an increasing tendency to donate
electrons. Thus, electrons flow spontaneously from more negative to more positive reduction potentials.
21.2 Redox Energetics: The Nernst Equation

Electromotive Force
The current flows through the circuit indicating that Zn2+ is more easily oxidized than Cu2+. The purpose of
the voltmeter is to calculate the potential difference, the difference between the reduction potentials of the
left and right reactions. The electromotive force is the potential that is measured.
The reference half reaction corresponds to the oxidation of hydrogen (H2). Hydrogen’s reduction
potential under the standard condition (E0‘) is subjectively set at 0.0 volts (V).
172 Chapter 21 Bioelectrochemistry
The standard reduction potential of any given half-reaction can be determined using a reference half-
cell and a sample half-cell to create an oxidation-reduction couple. The reference half-cell is composed of a
solution of 1 M H+ and 1 atm of H2 (g). The sample half-cell includes 1 M each of the oxidized and reduced
species. When standard conditions are present, the concentration of the hydrogen ion in the sample half-cell
is 10-7 M (pH 7). The voltmeter measures the difference in reduction potential between the reference and
sample half-reactions. Because the standard reduction potential of the reference half-reaction is 0.0 V, one
determines the value of the sample half-reaction.
Reaction systems that are more likely to donate electrons have more negative potentials. Therefore,
electrons spontaneously flow towards more positive reduction potentials.
Standard Reduction Potentials and Gibbs Free Energies

The standard reduction potential associated with electron transfer can be used to calculate ∆G0′ using the
following equation:
∆G0′ = -n F ∆E0′ (1)
where n is the number of electrons transferred, F is Faraday’s constant (96.48 kJ V-1 mol-1); and ∆E0′ is the
difference between the standard reduction potentials of the oxidized and reduced species (in V).
∆G0′ = -RT ln Keq (2)
∆E0′ = - (RT/n F) ln Keq (3)
Standard reduction potentials for some important biological half-reactions are given in the following table.
Reduction half-reaction E0′ (V)

Acetyl CoA + CO2 + H + 2 e- → Pyruvate + CoA
+
- 0.48
Ferredoxin (spinach), Fe3+ + e- → Fe2+ - 0.43
2 H+ + 2 e- → H2 (at pH 7) - 0.42
α-Ketoglutarate + CO2 + 2 H+ + 2 e- → Isocitrate - 0.38
Lipoyl dehydrogenase (FAD) + 2 H+ + 2 e- → Lipoyl dehydrogenase (FADH2) - 0.34
NADP+ + 2 H+ + 2 e- → NADPH + H+ - 0.32
NAD+ + 2 H+ + 2 e- → NADH + H+ - 0.32
Lipoic acid + 2 H+ + 2 e- → Dihydrolipoic acid - 0.29
Glutathione (oxidized) + 2 H+ + 2 e- → 2 Glutathione (reduced) - 0.23
FAD + 2 H+ + 2 e- → FADH2 - 0.22
FMN + 2 H+ + 2 e- → FMNH2 - 0.22
Acetaldehyde + 2 H+ + 2e- → Ethanol - 0.20
Pyruvate + 2H+ + 2 e- → Lactate - 0.18
Oxaloacetate + 2 H+ + 2 e- → Malate - 0.17
Cytochrome b3 (microsomal), Fe3+ + e- → Fe2+ 0.02
Fumarate + 2 H+ + 2 e- → Succinate 0.03
Ubiquinone (CoQ) + 2 H+ + 2 e- → CoQH2 0.04
Cytochrome b (mitochondrial), Fe3+ + e- → Fe2+ 0.08
Cytochrome c1, Fe3+ + e- → Fe2+ 0.22
Cytochrome c, Fe3+ + e- → Fe2+ 0.23
Cytochrome a, Fe3+ + e- → Fe2+ 0.29
Cytochrome f, Fe3+ + e- → Fe2+ 0.36
NO3- + e- → NO2- 0.42
Photosystem P700 0.43
Fe3+ + e- → Fe2+ 0.77
½ O2 + 2 H+ + 2 e- → H2O 0.82
Photosystem II (P680) 1.1
(Adapted from Moran et al., Biochemistry (2nd Ed.), 1996, p. 14-27, Table 14-4.)
Chapter 21 Bioelectrochemistry 173
The Nernst Equation

In most cases, the concentrations of the species under consideration will not be 1 M, so one must adjust to
the conditions of interest using the Nernst Equation. Similar to the relation between ∆G and ∆G0′, the
actual reduction potential (∆E) can be calculated from the change in standard reduction potential (∆E0′)
and the partition coefficient (Q) for the redox reaction.
∆E = ∆E°′ – (RT/n F) ln ([Aox][Bred]/[Ared][Box]) (4)
At 298 K, equation 4 reduces to:

∆E = ∆E°′ - (0.026/n) ln Q (5)
where Q is the ratio of actual concentrations of reduced and oxidized species.
When a reaction is not under standard conditions, one can calculate the electromotive force using the
Nernst equation and actual reactant and product concentrations. Remember that a positive ∆E signifies a
spontaneous reaction.
Example Calculation
(1) Consider the net reaction for electron transport, a redox reaction between NADH and O2. The oxidized
half-reaction will have a more negative standard reduction potential. Here, NADH will be oxidized and
oxygen will be reduced.
NAD+ + 2 H+ + 2 e- → NADH + H+ E°′ = - 0.32 V
½ O2 + 2 H+ + 2 e- → H2O E°′ = 0.82 V
The net reaction is:

NADH + ½ O2 + 2 H+ → NAD+ + H2O E°′ = 1.14 V
Using equation 1:
∆G°′ = -n F ∆E°′ = -(2)(96.48 kJ V-1 mol-1)(1.14 V) = -220 kJ mol-1
The ∆G°′ for ATP synthesis from ADP and Pi is 30 kJ mol-1, so the energy released during oxidation of
NADH under cellular conditions is sufficient to drive formation of several ATP molecules.
(2) A larger set of comparative calculations are presented in the Review problems. They compare the
redox capabilities of NAD+ and FAD, and use selected values from this table. The conclusion agrees with
the concept presented in the Coenzyme chapter, that FAD is used to accomplish more difficult redox
reactions than NAD+.
21.3 Electron Transport Chains

A series of coupled redox reactions occur in the electron transport chains of both mitochondria and
chloroplasts. NADH and CoQH2 are oxidized by the electron transport chain in each case. The energy is
captured and converted by trans-membrane proton pumps into a proton gradient. These gradients drive
ATP synthesis by F0 F1 ATPases.
Oxygen is the terminal electron acceptor of the reducing equivalents that are delivered to electron
transport by NADH and CoQH2. Due to the involvement of O2, they are called respiratory electron
transport chains.
Changes in ∆G and ∆E produced across a combined pair or series of reactions can be calculated as
the sum of the ∆G or ∆E values for the individual reactions in the chain. Since the values are additive, the
total energy for the chain is the sum of each individual sub-reaction.
Two examples of composite plots that illustrate the patterns of potential energies in redox chains are
shown in the Electron Transport section, for mitochondrial electron transport, and the Photosynthesis
chapter, for the Z-Scheme in chloroplasts.
Chapter 22
Glycolysis
22.1 Reactions 1 Through 10

The following reactions occur. Pyruvate can be used in three different pathways, producing the three
indicated products.
Glucose → Pyruvate → Acetyl-CoA, Lactate or Ethanol
Glycolysis consists of 10 reactions that can be separated into two phases called the hexose and triose
stages. The hexose stage makes up the first four steps. At step 4, a C-C bond of fructose 1, 6-bisphosphate
is cleaved and all subsequent pathway intermediates are triose phosphates. The two triose phosphates
formed from fructose 1, 6-bisphosphate in step 4 undergo interconversion to produce the single triose
phosphate, glyceraldehyde-3-phosphate, which continues through the remainder of the pathway.
HOCH2
H O H
H
OH
HO OH
H OH
Glucose
1a. Hexokinase ATP

1b. (anabolic direction): Glucokinase
ADP
[Negative regulation (-)]
2-
O3PO-CH2
H O H
H
OH
HO OH
H OH
Glucose-6-phosphate
2. Glucose-6-phosphate Isomerase
2-
O3PO- CH2 OH
O
H HO
H CH2OH
OH H
Fructose -6-phosphate
3. Phosphofructokinase-1 ATP
(Regulation:
ATP (-) ADP
Citrate (-)
AMP (+)
2-
Fructose-2, 6-bisphosphate (+) O3PO-CH2 OH
(See Regulation section.) O
H HO 2-
H CH2OPO3
OH H
Fructose-1, 6-bisphosphate
4. Aldolase
2- O H
CH2OPO3 C
5. Triose phosphate Isomerase C O (Step 6)
(converts DHAP to G3P)
H C OH
CH2OH 2-
CH2OPO3
Dihydroxyacetone phosphate Glyceraldehyde-3-phosphate

Chapter 22 Glycolysis 175
(continued)
O H
C
H C OH
(Step 5)
2-
CH2 OPO3
Glyceraldehyde-3-phosphate
Note. NAD+ is produced by
the Alcohol and Lactate
Dehydrogenase reactions. .
6. Glyceraldehyde-3-phosphate NAD + Pi It is required to drive further

dehydrogenase
NADH + H glycolysis.
O OPO3 2-
C
H C OH
2-
CH2OPO3
1, 3-Bisphosphoglycerate
7. Phosphoglycerate kinase ADP

ATP
COO-
H C OH
2-
CH2OPO3
3-Phosphoglycerate
8. Phosphoglycerate mutase
COO -
H C OPO3 2-
CH2OH
2-Phosphoglycerate
9. Enolase H2O
COO -
2-
C OPO3
CH2
Phosphoenolpyruvate
10 Pyruvate Kinase ADP

- Regulation:
ATP
Fructose 1, 6- bisphosphate (+)
COO-
C O
CH3
Pyruvate
176 Chapter 22 Glycolysis
The reactions and enzymes that catalyze them are:

Reaction Enzyme
(1) Glucose + ATP → Glucose 6-phosphate + ADP + H+ Hexokinase, glucokinase
(2) Glucose 6-phosphate ↔ Fructose 6-phosphate Glucose 6-phosphate isomerase
(3) Fructose 6-phosphate + ATP → Phosphofructokinase-1

Fructose 1, 6-bisphosphate + ADP + H+
(4) Fructose 1, 6-bisphosphate ↔ Aldolase

Dihydroxyacetone phosphate + Glyceraldehyde 3-phosphate
(5) Dihydroxyacetone phosphate ↔ Glyceraldehyde 3-phosphate Triose phosphate isomerase
(6) Glyceraldehyde 3-phosphate + NAD+ + P Glyceraldehyde 3-phosphate

↔ 1, 3-Bisphophoglycerate + NADH + H+ dehydrogenase
(7) 1, 3-Bisphophoglycerate + ADP ↔ 3-Phosphoglycerate + ATP Phosphoglycerate kinase
(8) 3-Phosphoglycerate ↔ 2-Phosphoglycerate Phosphoglycerate mutase
(9) 2-Phosphoglycerate ↔ Phosphoenolpyruvate + H2O Enolase
(10) Phosphoenolpyruvate + ADP + H+ → Pyruvate + ATP Pyruvate kinase
ATP is consumed then produced during glycolysis. These gains and losses are:
ATP consumed per glucose: - 2 hexose stage
ATP produced per glucose: +4 triose stage
Net ATP produced per glucose: +2
The steps in the triose stage of glycolysis occur twice per molecule of glucose metabolized, so all yields
must be multiplied by two.
22.2 Regulation: Activation and Inhibition

Allosterism.
The following allosteric regulators affect the indicated sites: (+) is activation; (–) is inhibition.
Glucose Fructose-1, 6-bisphosphate
Hexokinase (-)
Glucose-6-phosphate
Fructose-6-phosphate
Phosphofructokinase-1
Phosphoenolpyruvate
AMP (+) ( -) ATP
Fructose-2, 6-bisphosphate (+) (- ) Citrate (+) Pyruvate Kinase
Pyruvate
Glucagon
This protein hormone offsets the action of Insulin. Instead of causing glucose usage, Glucagon limits
it and promotes the storage of glucose as polymeric Glycogen in the liver. Glucagon action involves the
following steps.
(1) When the blood glucose concentration is low, glucagon release triggers an increase in the activity of
Protein Kinase A, as described in the Signal Transduction chapter.
(2) Protein Kinase A catalyzes phosphorylation of Phosphofructokinase 2 (PFK-2), which inactivates the
enzyme and slows Glycolysis.
(3) The resulting decreased concentration of fructose-2, 6-bisphosphate, a potent activator of PFK-1,
decreases the activity of PFK-1. The result is a net activation of Gluconeogenesis.
2- 2- 2-
O3PO-CH2 OH O3PO-CH2 OPO3
O O
H HO 2- H HO
H CH2OPO3 H CH2OH
OH H OH H
Fructose-1, 6-bisphosphate Fructose-2, 6-bisphosphate
22.3 Four Fates of Pyruvate

Lactate
In mammals, the production of lactate in muscle cells is naturally followed by its subsequent reconversion
into pyruvate. During exercise, lactate builds up in the muscle cells and is transported out, carried through
the bloodstream, then into the liver where the enzyme lactate dehydrogenase converts lactate into pyruvate.
Oxygen is required for further metabolism of pyruvate. When the oxygen supply in the tissues is low,
glycolysis occurs under anaerobic conditions to produce lactate. Lactate can accumulate in the blood to
sufficiently high levels to produce a condition called lactic acidosis, which can cause a dangerous drop in
the pH of the blood.
COO COO
NADH, H NAD
C O HO C H
CH3 CH3
Pyruvate Lactate Dehydrogenase Lactate
The net reaction for the breakdown of glucose to lactate is:
Glucose + 2 Pi + 2 ADP → 2 Lactate + 2 ATP + 2 H2O
As with NAD+, glycolysis requires that ATP be regenerated to continue flux through the pathway. Adding
the reaction for ATP consumption to the reaction above, one obtains:
2 ATP + 2 H2O → 2 ADP + 2 Pi + 2 H+
Note that lactic acid, not lactate, is the product.
Glucose → 2 Lactate + 2 H+ → 2 Lactic Acid
Metabolic and Food Applications

Lactic acid is the cause of the Ache in Muscles during and after Exercise.
During Fermentation of milk sugars to lactic acid by bacteria, the acid denatures the proteins. As a result,
proteins such as β-lactoglobulin, the main component of whey protein, form curds. Curdling is required in
the production of cheese and yogurt.
178 Chapter 22 Glycolysis
Ethanol
Anaerobic Metabolism
Pyruvate can be anaerobically metabolized to produce ethanol in yeast. The net reaction for conversion of
pyruvate to ethanol is:
Glucose + 2 Pi + 2 ADP + 2 H+ → 2 Ethanol + 2 CO2 + 2 ATP + 2 H2O
Under anaerobic conditions, pyruvate is converted to ethanol and CO2 by yeast cells during oxidation of
NADH. Two reactions take place. First, the enzyme pyruvate decarboxylase functions to decarboxylate
pyruvate into acetaldehyde. Secondly, acetaldehyde is reduced to ethanol by the enzyme alcohol
dehydrogenase and the cofactor NADH.
O 2-
O Glyceraldehyde OPO3 O
H 3-phosphate O
C C (several C
dehydrogenase
steps)
Sugar H C OH H C OH C O
2- 2-
CH2OPO3 Pi
NAD NADH + H CH2OPO3 CH3
Glyceraldehyde 1, 3-Bisphosphoglycerate Pyruvate
3-phosphate
Pyruvate
decarboxylase CO2
H Alcohol H O
Dehydrogenase C
drunken
behavior H C OH
CH3
CH3 NADH + H Acetaldehyde
NAD
Ethanol
Used to drive
glycolysis
Metabolic Purpose
Reduction of NAD+ to NADH occurs, so NAD+ is required as a reactant for glycolysis to proceed
continuously. The metabolic purpose of making ethanol is to regenerate more NAD+, so more glycolysis
can occur under anaerobic conditions. When oxygen is present, NADH oxidation occurs by oxidative
phosphorylation, an aerobic process. In the absence of oxygen, the production of ethanol or lactate
consumes NADH, regenerating the essential NAD+ to drive more glycolysis. This makes metabolic sense
because:
(1) The conditions are anaerobic, so, the yeast cell sacrifices the NADH. It cannot cash it in anyway.
Because no O2 is available, and it is the terminal acceptor of reducing equivalents in electron transport,
that process cannot occur. Since electron transport drives formation of the proton gradient that fuels
oxidative phosphorylation, the latter is also shut down.
(2) The anaerobic alternative is to squeeze more metabolic energy out by making more NAD+ and driving
more glycolysis.
Commercial Applications
These reactions are practical in their commercial role of beer and bread manufacturing. In the brewery, the
conversion of pyruvate to ethanol produces carbon dioxide. This CO2 is captured and used to carbonate the
alcoholic brew, producing the foamy head and effervescent tingle. In the bakery, carbon dioxide causes the
bread dough to rise.
Acetyl CoA
Pyruvate Translocase. Getting Acetyl CoA Into the Mitochondrion. Glycolysis and the Krebs cycle are
linked through the bridge reaction, in which pyruvate is converted to acetyl CoA. Pyruvate from glycolysis
is transported from the cytosol to the mitochondria, where the Krebs cycle occurs, by a symport called
pyruvate translocase, which also requires H+.
Pyruvate translocase
(a "H+ symport") CoA-SH Pyruvate Dehydrogenase
complex
Pyruvate Pyruvate
HS-CoA CO2
H H CO2 COO S CoA
Acetyl CoA C O C O
Cytosol Mitochondria (mobile C2 carrier) CH3 CH3
NAD NADH, H+
Pyruvate Acetyl CoA
Oxaloacetate
CoA-SH
(C4)
Citrate
(C6)
Citric
Acid
Cycle
CO2
(C5)
(C4) CO2
Pyruvate Dehydrogenase: Coenzyme Paradise

The pyruvate dehydrogenase complex converts pyruvate to acetyl CoA. Five coenzymes are involved.
Exercise: Identify them and review the details of each individual redox reaction. What is oxidized and what
is reduced? What are the products?]
The pyruvate dehydrogenase complex is a heterotrimer, with each successive enzyme catalyzing the
next reaction in the cyclic sequence. The product of the first reaction is immediately used as substrate by
the next. Metabolites are directed through the system by covalent binding to a flexible prosthetic group
bound to the enzyme E2.
.
O
Thiol to thiol transfer:
HS CoA H3C C S CoA Acetyl CoA
O 2
H3C C Dihydrolipoamide
SH 3
S Acyltransferase
H SH
O E2 SH
TPP
H3C C COO
FAD
Pyruvate
Pyruvate bound NADH + H
Dehydrogenase dihydro- Dihydrolipoamide
lipoamide Dehydrogenase
E1
E3 NAD
CO2 TPP
S FADH2
H3C CH OH S 1
H3C R2
Hydroxyethylthiaminepyrophosphate
R1 N S
(HETPP) C
H3C CH OH
.
Oxaloacetate. This compound is produced in the first step of gluconeogenesis by Pyruvate Carboxylase.
See the Biotin section of the Coenzymes chapter for details.
Chapter 23
The Krebs Cycle

23.1 Pathway
This pathway, which is also called the Citric Acid Cycle and Tricarboxylic Acid (TCA) Cycle, involves the
eight consecutive reactions shown below. Each turn of this cyclic pathway begins with the linkage of the
two-carbon acetyl group from acetyl CoA with oxaloacetate. The overall process releases two molecules of
CO2, produces three NADHs and one CoQH2, transfers a phosphoryl group to GDP or ADP, and
regenerates the original catalyst oxaloacetate.
.
S CoA
Acetyl CoA
C O
COO CH3
C O Entry of substrate
by condensation
CH2
with oxaloacetate
COO
Oxaloacetate
8
NADH + H 1 H2O
Malate
III. Oxidation Dehydrogenase Citrate
NAD Synthase HS CoA + H
COO
COO
HO C H
CH2
CH2
HO C COO
COO
CH2
L-Malate
COO
7 Citrate
II. Hydration
H2O Fumarase
2
Aconitase Rearrangement
COO
H C COO
C H CH2
H C COO
COO
Fumarate HO C H
COO
CoQH2 6 Isocitrate
Succinate
I. Oxidation FAD NAD
Dehydrogenase First
complex 3
CoQ NADH, H oxidative
Isocitrate decarboxylation
Dehydrogenase CO2
COO
CH2 COO
CH2 CH2
COO CH2
Succinate
C O
COO
HS CoA α-Ketoglutarate
Substrate-level GTP (or ATP) COO HS CoA
phosphorylation 5 4
GDP (or ADP) Second
Succinyl CoA CH2 α-Ketoglutarate
Synthetase NAD oxidative
Dehydrogenase decarboxylation
CH2 NADH, H
Pi complex
C O CO2
S-CoA
Succinyl CoA
Chapter 23 The Krebs Cycle 181
Source Remark
The conceptual logic, graphics and reactions in the metabolism sections generally follow the treatment of
Horton et al, Principles of Biochemistry (4th ed.), 2006. The original material was transcribed from the facts
presented by Moran et al, Biochemistry (2nd ed.), 1994.
Stepwise Reactions
Reaction Enzyme
(1) Acetyl CoA + Oxaloacetate + H2O → Citrate + CoA-SH + H+ Citrate synthase
(2) Citrate ↔ Isocitrate Aconitase (Aconitate hydratase)
(3) Isocitrate + NAD+ → α-Ketoglutarate + NADH + CO2 Isocitrate dehydrogenase
(4) α-Ketoglutarate + CoASH + NAD+ → α-Ketoglutarate dehydrogenase
Succinyl CoA + NADH + CO2 complex
(5) Succinyl CoA + GDP (or ADP) + Pi ↔ Succinyl-CoA synthetase
Succinate + GTP (or ATP) + CoA-SH
(6) Succinate + CoQ ↔ Fumarate + CoQH2 Succinate dehydrogenase complex
(7) Fumarate + H2O ↔ L-Malate Fumarase (Fumarate hydratase)
(8) L-Malate + NAD+ ↔ Oxaloacetate + NADH + H+ Malate dehydrogenase
23.2 Reactions
Net Reaction
Acetyl CoA + 3 NAD+ + CoQ + GDP (or ADP) + Pi + 2 H2O →
CoA-SH + 3 NADH + CoQH2 + GTP (or ATP) + 2 CO2 + 2 H+
Unique and Common Characteristics
(1) The net reaction shows that each molecule of acetyl CoA produces three molecules of NADH, one
molecule of CoQH2, and one molecule of GTP or ATP. The citric acid cycle is referred to as a multistep
catalyst because during each turn, in which one acetyl CoA is oxidized to CO2, oxaloacetate is regenerated
at the end of the cycle.
(2) Succinyl CoA Synthetase catalyzes the substrate-level phosphorylation reaction (step 5). To accomplish
the reaction, a histidine in the active site is first phosphorylated then transfers the phosphate to either GDP
or ADP, forming the trinucleotide.
(3) The series of three linearly connected reactions 6, 7, and 8 accomplish the same task as three analogous
reactions that occur during the breakdown of fatty acids in the β-oxidation pathway.
23.3 Yields
Krebs Cycle Yields
Both NADH and CoQH2 are oxidized by electron transport. ATP is produced concurrently via oxidative
phosphorylation.
Krebs Cycle Reactions:
Reaction Energy- ATP
Yielding Product Equivalents
Isocitrate Dehydrogenase NADH 2.5
α-Ketoglutarate Dehydrogenase NADH 2.5
complex
Succinyl-CoA Synthetase GTP or ATP 1.0
Succinate Dehydrogenase complex CoQH2 1.5
Malate Dehydrogenase NADH 2.5
Total: 10
(1) Each molecule of NADH oxidized to NAD+ produces 2.5 molecules of ATP by oxidative
phosphorylation. Each molecule of CoQH2 oxidized to CoQ produces 1.5 molecules of ATP.
(2) Given these figures, oxidation of one acetyl CoA by the Krebs Cycle produces approximately 10
molecules of ATP.
182 Chapter 23 The Krebs Cycle
Total ATP Produced Per Glucose

Each of the three central metabolic pathways yield products that contribute to the total number of ATP
produced per glucose. Glycolysis produces a net gain of 2 ATPs, 2 NADHs and 2 pyruvates. The bridge
reaction produces 2 NADH. Combining these ATPs with those generated by two rounds of the Citric Acid
cycle, the total yield is about 32 molecules of ATP per glucose.
ATP
Glucose
equivalents
2 ATP 2 NADH 5
2 Pyruvate 2 (ATP)
2 NADH 5
2 Acetyl CoA
Oxidative
phosphorylation
2 GTP Citric
6 NADH 15
or Acid
ATP Cycle
2 CoQH2 3
Substrate-level 2 (ATP or GTP)

phosphorylation
Total: 32 ATP per glucose
Reaction ATP Equivalents
Glycolysis 2 + 5 (from 2 NADH)
Pyruvate Dehydrogenase complex 5
(Pyruvate x 2)
Krebs Cycle (Acetyl CoA x 2) 20
Total: 27 + 5 = 32
23.4 Cellular Redox Potential

The calculations above focus on ATP formation. The cell is sensitive to the ATP charge it maintains.
Disulfide Oxidation
Cells must also maintain the proper redox state. The complicated set of redox reactions in the
pyruvate dehydrogenase mechanism requires that the sulfhydryl components can form the proper oxidized
and reduced species, and that appropriately sized pools of them accumulate. If the cellular redox potential
is incorrect, these structures may not form as easily as is required for efficient metabolism. The result is a
redox imbalance, analogous to a pH imbalance during lactic acidosis.
Regulating Systems in Prokaryotes and Eukaryotes

The redox potential is regulated in prokaryotes and eukaryotes using different redox state maintenance
systems. The bacterial system involves a small protein called Thioredoxin and the accessory protein
Thioredoxin Reductase. In eukaryotes, the small tripeptide compound glutathione is adjusted by accessory
reductase and oxidase proteins to maintain the proper redox state. The redox state in E. coli encourages
disulfide formation; most eukaryotic cells favor a state in which most sulfhydryls are in the free state.
Chapter 23 The Krebs Cycle 183
23.5 Regulation
Overview
Regulation occurs at: (1) the step that feeds the cycle, the bridge reaction, and (2, 3) the two oxidative
decarboxylation steps. As expected, the energy-producing steps are regulated.
Pyruvate
Pyruvate (-) NADH, Acetyl CoA

Dehydrogenase
complex (+) NAD+, CoA-SH
Acetyl CoA
Oxaloacetate
CoA-SH
Malate Citrate
Fumarate Isocitrate
Isocitrate (-) NADH
Dehydrogenase (+) ADP, Ca 2+
Succinate α– Ketoglutarate
α-Ketoglutarate
Dehydrogenase
complex 2+
(+) Ca
Succinyl CoA
.
Isocitrate Dehydrogenase
Mammalian isocitrate dehydrogenase is allosterically activated by Ca2+ and ADP. The enzyme is inhibited
by NADH. Mammalian isocitrate dehydrogenase is not covalently modified. In contrast, E. coli isocitrate
dehydrogenase is inhibited by phosphorylation at a particular serine.
The protein containing the kinase that inhibits isocitrate dehydrogenase can also reactivate the
enzyme using its phosphatase activity. The latter activity is located on a separate domain that catalyzes
hydrolysis of phosphoserine, reactivating isocitrate dehydrogenase. The kinase and phosphatase activities
are regulated reciprocally, i.e. an inhibitor of the kinase activates the phosphatase, and vice versa.
(Kinase)
ATP ADP
Isocitrate
(-)
(ICD) (ICD-Pi) Isocitrate
Oxaloacetate
Isocitrate Bifunctional Isocitrate Pyruvate
Dehydrogenase OH kinase/phosphatase Dehydrogenase Pi 3-Phosphoglycerate
active Phosphoenolpyruvate
(+) inactive
α-Ketoglutarate Pi H2 O
(Phosphatase)
.
See the Phosphorylation-Dephosphorylation section of the Enzyme chapter for the mechanistic
consequences of phosphorylation.
α-Ketoglutarate Dehydrogenase is also activated by Ca2+.

Chapter 24
Gluconeogenesis
24.1 Reactions
The two offsetting pathways are shown below. Four reactions in gluconeogenesis (catalyzed by the
enzymes shown on the right) replace the offsetting three reactions in glycolysis (catalyzed by the enzymes
listed on the left).
Glucose
ATP Pi
Hexokinase Glucose-6-
Glycolysis Phosphatase
ADP
Glucose 6-phosphate
Fructose-6-phosphate
ATP Pi
Phosphofructokinase-1 Fructose-1, 6-Bisphosphatase

ADP
Dihydroxyacetone Glyceraldehyde
phosphate 3-phosphate
NAD + Pi
NADH + H
1, 3-Bisphosphoglycerate
ADP ADP
ATP ATP
3-Phosphoglycerate
2-Phosphoglycerate
GDP
Phosphoenolpyruvate Phosphoenolpyruvate
Carboxykinase
ADP GTP
Pyruvate Kinase Oxaloacetate
ATP ADP
Pyruvate
Pyruvate Carboxylase
ATP Gluconeogenesis
amino
Lactate acids (ala)
Chapter 24 Gluconeogenesis 185
24.2 Regulation
Fructose-2, 6-Bisphosphate
The hormone glucagon regulates the fructose-6-phosphate / fructose-1, 6-bisphosphate futile cycle as
follows. Under catabolic condition, fructose-2, 6-bisphosphate is produced and acts as a potent activator of
Phosphofructokinase-1. As a result, glycolysis predominates and gluconeogenesis is inhibited.
Glycolysis
Glucose
Phosphorylated
PFKase-2
(inactive) Fructose-6-Phosphate
PKase A
PFKase-2 PFKase-1 Fructose-1, 6-Bisphosphatase
(active)
Fructose-2, 6- (+) Fructose-1, 6-

bisphosphate bisphosphate
Protein Kinase A (PKase A)

activates Gluconeogenesis by
decreasing the concentration of Phosphoenol- PEP
fructose-2, 6-bisphosphate. pyruvate Carboxykinase
Cyclic AMP activates PKase A.
Pyruvate Oxaloacetate
Kinase
Pyruvate
Pyruvate Carboxylase
Gluconeogenesis
Lactate
Glucagon Action
In contrast, when glucagon binds its receptor, cyclic AMP (cAMP) is produced. This second messenger
then activates Protein Kinase A, which catalyzes phosphorylation of Phosphofructokinase-2. The resulting
decrease in fructose-2, 6-bisphosphate concentration removes this potent activator of Phosphofructokinase-
1 and relieves the inhibition of fructose-1, 6-bisphosphatase. The net result is that cAMP, which is
produced by the extracellular binding of circulating glucagon, activates gluconeogenesis. This example
shows how an intracellular metabolic pathway can be controlled by an extracellular hormonal stimulus.
24.3 Sources Used to Produce Glucose

The principal substrates that contribute to gluconeogenesis pathway in mammals are amino acids, most
commonly alanine and lactate. These amino acids are produced by breakdown of muscle protein. Under
particular metabolic conditions, the oxaloacetate produced by the Krebs Cycle is redirected into the
gluconeogenic pathway. Because gluconeogenesis is not saturated by the concentrations of amino acids in
the blood, an excess of free amino acids build up, causing an increased conversion of amino acids to
glucose. In the same way, the concentration of lactate in the blood does not saturate gluconeogenesis.
Pyruvate is produced from alanine by an aminotransferase reaction. The lactate that fuels
gluconeogenesis is produced in bulk from the muscle, explaining the large mass and high rate of glycolytic
activity of muscle tissue. As a result, other tissues in the body can strongly affect the level of
gluconeogenesis in the liver.
Chapter 25
Electron Transport and Oxidative Phosphorylation
25.1 Mitochondria in Red and White Muscle

A cell’s overall energy requirement depends upon the number of mitochondria present. White muscle tissue
contains a small number of mitochondria and acquires energy via anaerobic glycolysis. For example, an
alligator’s jaw is composed largely of white muscle tissue. It is able to snap open and shut swiftly but is
quickly exhausted and unable to sustain this motion for more than a few seconds. Oxygen-enriched red
muscle contains many more mitochondria. Red meat is red because it is suffuse with hemoglobins
transporting O2 into the muscle.
25.2 Overall Process

Oxidative phosphorylation is driven by a proton gradient, which is produced by the active transport of
protons from the mitochondrial matrix to the inner membrane space, through the mitochondrial inner
membrane.
CYTOPLASM
H
INNER MEMBRANE
SPACE
2e INNER MEMBRANE
ATP
synthase
H 2O MATRIX
H
1/2 O2 ADP ATP
+ + +
2H Pi H2O
H
This proton movement creates a gradient, operating as an aqueous circuit that connects the electron
transport chain to ATP Synthase. This is similar to the action of a wire in an electrochemical reaction in that
the electrons are passed from the reducing agent, NADH or CoQH2, through the electron transport chain,
and finally to the terminal oxidizing agent O2. This series of coupled redox reactions pass the electrons,
which drives the proton pumps. The transformed free energy is captured by phosphorylation of ADP to
produce ATP.
Two interlinking processes work together to drive oxidative phosphorylation:
(1) NADH and CoQH2 are oxidized by the membrane-embedded mitochondrial electron-transport chain.
This sequence of electron carriers passes the electrons from the reduced coenzymes to O2, the terminal
electron acceptor in aerobic metabolism. Coupled to some of the redox reactions in the chain, protons are
pumped across the mitochondrial membrane into the inner membrane space. The energy from the oxidation
reaction drives the buildup of protons and creating a proton concentration gradient. The inner membrane
space becomes more positively charged than the matrix.
(2) This accumulation of protons creates a potential energy gradient, called the protonmotive force, which
supplies the energy that drives the protons back across the inner membrane. The protons flow through the
integral membrane enzyme complex called ATP Synthase (F0 F1 ATPase), which phosphorylates ADP to
produce ATP.
ADP + Pi → ATP + H2O
Chapter 25 Electron Transport and Oxidative Phosphorylation 187
25.3 Chemical Potential Energies That Drive Proton Transport

Source Remark. This section is based on the treatment in Chapter 18 of Moran et al. Biochemistry (2nd ed.),
1996.
Free Energy of Proton Transport

The proton motive force is analogous to the electromotive force in electrochemistry. Similar to an electrical
circuit, the protons flow from the matrix, through the electron transport-driven proton pumps to the inner
membrane space, then back out through the ATP synthase.
Electromotive Force
(emf)
e e
X 2H 2H 1/2 O2
XH2 H2O
Protonmotive Force
(pmf)
H
H
H H
X 2e 2e 1/2 O2
MATRIX XH2 H2O
The following reaction shows the reduction of O2 by a reducing agent XH2 in an electrochemical cell.
XH2 + ½ O2 ↔ X + H2O
the reducing agent XH2 passes electrons along a wire that connects the two electrodes where the redox half-
reactions occur.
Electrons pass from the anode, where XH2 is oxidized,

XH2 ↔ X + 2 H+ + 2 e–
to the cathode, where O2 is reduced.
½ O2 + 2 H+ + 2 e- ↔ H2O
A salt bridge connects the two reaction cells, allowing electrons to flow freely between them. The
electromotive force is the potential energy difference, measured in volts, between the electrodes. The
specific reduction potentials of XH2 and O2 determine the difference in free energy between them, which in
turn determines the direction of electron flow and extent of reduction of each oxidizing agent.
Both chemical and electrical potential energy are generated by the movement of protons across the
membrane. The free energy change due to chemical potential energy is characterized by the difference in
proton concentration on each side of the membrane.
∆Gchem = n R T ln ([H+]in/[H+]out) (1)
where n is the number of protons, R is the universal gas constant (8.315 J K-1 mol-1) and T is the absolute
temperature. Equation 1 can be rewritten as:
∆Gchem = 2.303 n R T (pHin - pHout) (2)
188 Chapter 25 Electron Transport and Oxidative Phosphorylation
The free energy change due to the electric potential energy is characterized by the change in membrane
potential (∆ψ).
∆Gelec = z F ∆ψ (3)
where z represents the charge of the transported substance, and F is Faraday’s constant (96.48 kJ V-1 mol-1).
Because the charge for each proton translocated is 1.0 (n = z), the total free-energy change for proton
transport is:
∆G = n F ∆ψ + 2.303 n R T ∆pH (4)
Protonmotive Force
An expression for the potential that occurs between the two sides of the mitochondrial membrane is
obtained by dividing equation 4 by n F. The term ∆G /n F is the proton motive force (∆p).
∆p = ∆G / n F = ∆ψ + (2.303 R T ∆pH/ F) (5)
Note that ∆p is not ∆pH. At 25°C, 2.303 RT/ F = 0.059 V and
∆p = ∆ψ + (0.059 V) ∆pH (6)
The proton concentration gradient is a pH gradient. The other source of free energy in the proton
motive force is the charge gradient.
25.4 Mitochondrial Electron Transport

The electron transport is composed of four complexes which each contain protein subunits and cofactors.
Each complex undergoes cyclic reduction and oxidation via a mobile electron carrier, ubiquinone (CoQ)
and Cytochrome c, respectively, which provide a link between the subunits. The energies are calculated as
described below.
Eo ' (V) ∆Go '

(kJ mol-1)
-0.4
NADH
Complex I
NADH-ubiquinone 200
-0.2 NAD oxidoreductase Path of electrons
Succinate CoQ Complex III
0 Complex II
Ubiquinol-cytochrome c
Succinate-ubiquinone
oxidoreductase
oxidoreductase
Fumarate Cyt c
0.2
Complex IV 100
0.4 Cytochrome
oxidase
1/2 O2 + 2 H+
0.6
H 2O
0.8
0
[Cyt b]
NADH FMN Fe-S CoQ Cyt c1 Cyt c Cyt a O2
[Fe-S]
Fe-S
Succinate FAD
The standard reduction potential (V) is directly related to the standard free energy change (kJ mol-1)
by the formula:
∆G°′ = - n F ∆E°′ (7)
The energy stored during the electron transfer process occurs in discrete steps, releasing a significant
amount of energy, and is stored as a proton concentration gradient.
The following table provides standard reduction potentials of the mitochondrial redox components.
Substrate or complex E°′ (V) Substrate or complex E°′ (V)
NADH - 0.32 Complex III
Complex 1 Fe-S clusters + 0.28
FMN - 0.30 Cytochrome b560 - 0.10
Fe-S clusters - 0.25 to - 0.05 Cytochrome b566 + 0.05
Succinate + 0.03 Cytochrome c1 + 0.22
Complex II Cytochrome c + 0.23
FAD 0.0 Complex IV
Fe-S clusters - 0.26 to 0.00 Cytochrome a + 0.21
CoQH2/CoQ + 0.04 CuA + 0.24
CoQ-/CoQ - 0.16 Cytochrome a3 + 0.39
CoQH2/CoQ- + 0.28 CuB + 0.34
O2 - 0.82
(Adapted from Moran et al., Biochemistry (2nd ed.), 1996, p. 18-10; Table 18.2.)
The values are only applicable under standard conditions. The actual reduction potentials (E) can be
calculated from standard values (E°′) in a manner analogous to calculating actual free energy changes from
standard values.
E = E°′ + R T ln [red]/[ox] (8)
The difference between E and E°′ varies in proportion to the logarithm of the [reduced] to [oxidized] ratio.
The following table shows standard free energies released in the oxidation reaction catalyzed by each
reaction center.
Complex E°′reductant E°′oxidant ∆E°′ ∆G°′
(V) (V) (V) (kJ mol-1)
I (NADH/CoQ) - 0.32 + 0.04 + 0.36 - 70
II (Succinate/CoQ) + 0.03 + 0.04 + 0.01 -2
III (CoQH2/Cytochrome c) + 0.04 + 0.23 + 0.19 - 37
IV (Cytochrome c/O2) + 0.23 + 0.82 + 0.59 - 110
(Adapted from Moran et al., Biochemistry (2nd ed.), 1996, p. 18-10; Table 18.3.)
25.5 Electron Transfer and Proton Flow in Complexes I through IV

Complex I
This center contains FMN and a series of Fe-S centers transfer electrons from NADH to CoQ. Two protons
are needed for the reduction of CoQ to CoQH2. Approximately four protons are translocated per pair of
electrons transferred in electron transport.
.
INTER MEMBRANE
SPACE
2 one-electron 2 one-electron
transfers transfers CoQH2
FMNH2 FeS
e e
FMN
CoQ
MATRIX
NADH NAD 4H
H
(Adapted from Moran et al., Biochemistry (2nd ed.), 1996, pp. 18-13 to -17; Figs. 18-11, -12, -13, and -15.)
Complex II
Electrons enter this complex at the CoQH2 energy level. The proton concentration gradient is not affected
by this center.
.
INTER MEMBRANE
SPACE
2 one-electron 2 one-electron
transfers transfers CoQH2
FeS
e e
FAD b560 CoQ
2e
MATRIX
Succinate Fumarate + 2 H
2H
Recall that Succinate Dehydrogenase is also a component of the Krebs Cycle. This is that enzyme.
Reducing equivalents are transferred from FADH2 to ubiquinone (CoQ), forming the mobile electron
carrier ubiquinol (CoQH2).
Complex III
Electrons are transferred from CoQH2 to Cytochrome c. When the electrons are passed, two protons move
across the membrane from the matrix and two protons are captured by reduction of CoQH2, creating a net
gain of four protons in the inner membrane space.
2H
INTER MEMBRANE
SPACE
Cyt c
CoQ 2 one-electron
transfers
CoQH2 e
MATRIX
2H
Complex IV
Coupled electron transfer reactions occur between the iron in Cytochrome c and the copper in the first of
two forms of Cytochrome a (A), which passes them to the second copper-containing form (B). Finally, they
are passed to oxygen. Half of one molecule and two protons are reduced to form water.
.
INTER MEMBRANE
SPACE
Cyt c Two
one-electron
transfers Two
one-electron
Cyt a- Cyt a- transfers
e CuA CuB e
MATRIX
2H
1/2 O2 H2O
2H
The proton concentration gradient is affected by Complex IV in two ways: (1) As electrons flow between
the a cytochromes, protons are pumped from the matrix to the inner membrane space, and (2) Protons are
required to form water within the matrix. The net production of proton gradient by this center is nullified.
25.6 Oxidative Phosphorylation

E. coli ATP Synthase
The F0F1 ATPase in E. coli has a “knob-and-stalk” structure. In E. coli, the F1 component lies on the inside
of the plasma membrane, but in mitochondria, F1 is found on the inside of the inner mitochondrial
membrane. The F0 component forms a proton channel and spans the length of the membrane.
H+
F0
Cell exterior
Interior
ADP + Pi H+ F1
ATP
F1 is composed of three α and three β subunits, which together form the “knoblike” six-sided structure. The
“stalk” is composed of one γ, one δ, and one ε subunit, connecting the F1 and the F0 base. The F0 base piece
is composed of a complex structure of subunits and structural details that are not easily distinguishable.
ATP is produced as the complex rotates around a central axis, powered by the pumping of protons from the
inner membrane space through to the mitochondrial or cell interior. In this way, the F0F1 ATPase acts as a
molecular nanomachine.
Chloroplast ATP Synthase

The ATP synthase is made up of a four-subunit (I, II, III, IV) integral membrane protein complex (CF0)
with two parts: a proton pore which is made of 12-14 copies of subunit III and an external protein complex
(CF1) that generates ATP and is composed of five subunits (α, β, γ, δ and ε). Subunit IV (not shown) binds
to both CF0 and CF1.
Lumen
H+
CF0
Stroma
H+
CF1
ATP ADP + Pi
Chemiosmosis
The Chemiosmotic Theory proposed by Nobel Laureate Peter Mitchell holds that electron transport in
mitochondria produces a proton gradient, which drives ATP biosynthesis (i.e., the mechanism of oxidative
phosphorylation).
In 1974, Ephraim Racker and colleagues demonstrated a proof of this principle. Their proton gradient
was produced by the protein bacteriorhodopsin, which is found in the membranes of the the
archaebacterium Halobacterium salinarium. The seven membrane-spanning helices form in this protein
(shown below) form a channel that allows active transport of protons. A lysine binds one retinal, which can
absorb light and undergo the same all-trans to 13-cis photoisomerization reaction that occurs in the opsin-
bound coenzyme during visual stimulation. This isomerization releases a proton, which travels up the
channel, releasing it on the outside of the membrane.
A H+ B
C
N
G G F C
F E C
N
A E N
B D
C
A C
C N N D
C N
C B N
C
H+
The intent of Racker et al. was to couple the proton pump in bacteriorhodopsin, which can produce a
proton gradient, with a proton gradient-dependent ATP synthase, all embedded within a lipid vesicle.
+
ADP + Pi
H
ATP
+ +
Bacteriorhodopsin H H
ATP Synthase
lipid vesicle
(Adapted from Horton et al. Principles of Biochemistry (4th ed.), 2006, p. 461; Box 15.1.)
The result was that bacteriorhodopsin pumped protons into the vesicle, which formed a proton gradient
between the inside and outside of the vesicle. This gradient drove the exit of protons through the ATP
synthase, catalyzing ATP formation. The essential idea of chemiosmosis was confirmed.
In this system, light-driven isomerization of retinal drives proton gradient formation. In the
mitochondrial case, it is driven by electron transport. As described in the Photosynthesis chapter, both
modes occur in chloroplasts, electron-driven and photoelectron-driven.
Chapter 26
The Malate-Aspartate Shuttle and Proteomics
26.1 Getting NADH into the Mitochondrion: Isozymes

The purpose of this shuttle is to transfer the electron of NADH, which cannot cross the mitochondrial
membrane, into the matrix. In the first step, cytosolic NADH is used to reduce oxaloacetate to produce
malate, catalyzed by cytosolic Malate Dehydrogenase. Next, malate passes through the mitochondrial
membrane, in exchange for α-ketoglutarate, catalyzed by the antiport called Dicarboxylate Translocase,
and carrying the reducing equivalent it received from NADH. Within the mitochondrion, malate is oxidized
by the mitochondrial Malate Dehydrogenase to form oxaloacetate and regenerate NADH, which is used to
fuel electron-transport.
NADH, H
cytosolic Aspartate
NAD Transaminase
Malate Oxaloacetate Aspartate
cytosolic Malate
Dehydrogenase
a-Ketoglutarate
Glutamate-
Dicarboxylate Aspartate
Translocase Translocase
CYTOSOL Glutamate
MATRIX
Glutamate
a-Ketoglutarate
mitochondrial Malate
Dehydrogenase
Malate Oxaloacetate Aspartate
mitochondrial
NAD Aspartate
NADH, H Transaminase
Electron-Transport Chain
2e (in inner membrane)
In order to complete the shuttle cycle, oxaloacetate in the matrix must be transported back to the
cytosol. Since it cannot move through the mitochondrial membrane, it is converted into aspartate by
mitochondrial Aspartate Transaminase. Aspartate can pass through the membrane via the antiport
Glutamate-Aspartate Translocase. Oxaloacetate and glutamate react with mitochondrial aspartate
transaminase to produce aspartate and α-ketoglutarate. Glutamate enters the matrix through Glutamate-
Aspartate Translocase. The α-ketoglutarate exits the mitochondrial matrix via Dicarboxylate translocase,
allowing entry of more malate. In the last step of the cycle, cytosolic Aspartate Transaminase, converts
aspartate into oxaloacetate. Note that the cytosolic and mitochondrial versions of the two enzymes
Aspartate Transaminase and Malate Dehydrogenase operate in opposite directions.
NADH Yields: Cytosolic Versus Mitochondrial

Reduction of oxaloacetate in the cytosol uses a proton. When malate is reduced in the mitochondrial
matrix, the proton is released. Since electron transport oxidizes cytosolic NADH indirectly, it donates one
194 Chapter 26 The Malate-Aspartate Shuttle and Proteomics
less proton to the proton concentration gradient than mitochondrial NADH. As a result, the total ATP yield
for oxidation of two cytoplasmic NADHs is about 4.5, rather than 5.0 for two mitochondrial NADHs.
Regulation: Oxidation Phosphorylation Is Controlled by Cellular Energy Demand
Oxidative phosphorylation is not regulated simply by feedback inhibition or allosterism. The rate is
controlled predominantly by substrate availability and cellular energy demand. Concentrations of
intramitochondrial NADH, O2, Pi, and ADP determine the rate of respiration.
26.2 Isozymes and Proteomics
Isozymes
The Malate-Aspartate Shuttle makes use of two sets of isozymes, cytosolic and mitochondrial versions of
Malate Dehydrogenase and Aspartate Transaminase. The cytosolic and mitochondrial Aspartate
Transaminase and Malate Dehydrogenase operate in opposite directions. They are tuned to catalyze the
opposite reaction in the two compartments.
Proteins are often made in several isozyme forms in cells. For example, we encountered the adult and
fetal versions of hemoglobin. Both are hemoglobin, but each is endowed with specific capabilities that
allow the cell to adapt to particular circumstances, e.g., adult and fetal O2 availability.
In 1984, the front lobby of the developmental biology building at Argonne National Labs contained a
20’ by 12’ picture of a beautifully resolved summary version of a two-dimensional gel of human serum
proteins. It was a fascinating set of groups of isozymes in different patterns, with many examples of three or
more different resolved isozymes! These isozymes are discussed below.
Motivation for Proteomics
The Malate-Aspartate Shuttle demonstrates the essential coupling that occurs between different cellular
compartments. These couplings are essential. They can be inhibited, enhanced, or disconnected by cellular
malfunctions, and so forth.
A typical life science problem might address the following sorts of questions:
(1) How does a particular toxin induce cancer?
(2) How does a plastic additive affect the level of a particular hormone?
To answer such inquiries, one must look at how complex protein mixtures change as the toxin is
administered during the time frame when it induces its effect(s). In such scenarios, researchers find that a
set of proteins typically changes, sometimes dozens are affected.
26.3 Characterization by Two-dimensional Gel Electrophoresis
Two-dimensional (2D) Gel Electrophoresis is used to analyze complex mixtures of proteins (Google
“O’Farrell gels”).
(1) Proteins are separated in the first dimension by isoelectric focusing, in which the proteins migrate to the
position of their respective isoelectric points (pI) on a pre-constructed stabilized pH gradient material.
(2) The second dimension involves separating the gels based in different molecular weights using sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
The advantage of this 2D approach over one-dimensional electrophoresis gel is that one can separate
and characterize proteins even when they are nearly the same molecular weight, and cannot be resolved on
the SDS-PAGE gel.
.
Molecular Weight (Mass, kiloDaltons)
.
400 200 100 50 10
12
Second Dimension
Isoelectric Point (pI)
(SDS PAGE)
10
8
First Isozymes
Dimension Same enzyme,
(Isoelectric 6 slightly different
Focusing) sequences
4
.
Chapter 26 The Malate-Aspartate Shuttle and Proteomics 195
This method allows one to separate large mixtures of proteins and thereby analyze the gene products of that
particular genome. 2D Gels have been used extensively to characterize human plasma protein populations.
Many of the proteins really consist of two or several isozymes. Such techniques are used in many fields,
e.g., medicine, criminology, genealogy, archaeology, migration and habitat mapping, and many others.
26.4 Mass Spectrometry and Proteomics

Proteomics is the study of protein populations and comparisons of these populations after isolation from
one or more sources, for example, healthy and specifically infected tissues.
Mass Spectrometry (MS) can be used to determine the individual masses of a set of proteins then
separate them into single species, which can then be analyzed for amino acid content, and types and
locations of modifications. A single gel spot, corresponding to a selected protein, is taken from the 2D gel,
chemically modified, as required for the MS experiment, then injected into the machine. Capillary
Electrophoresis is also used as the first separation step, replacing the 2D gel.
Chapter 27
Degradation and Synthesis of Lipids
27.1 Beta Oxidation of Saturated Fatty Acids

The β Oxidation process forms a spiral metabolic pathway, with each rotation consisting of four enzyme-
catalyzed reactions. The products of each round are a single molecule of CoQH2, NADH, acetyl CoA, and a
fatty acyl CoA molecule that is two carbon atoms shorter than the molecule that entered that cycle.
O Electron Transport/
CH2 CH2 C S CoA Oxidative Phosphorylation
R CH2
Fatty acyl CoA
3+ CoQH2
O FAD FADH2 Fe S
O
CH3 C S CoA Acyl CoA
R CH2 C S CoA ETF: ubiquinone
dehydrogenase ETF
reductase
Thiolase Fatty acyl CoA
2+
shortened by two carbons FADH2 FAD Fe S CoQ
HS CoA
(4) thiolysis (1) oxidation
ETF = Electron-transferring flavoprotein
H O
O O
R CH2 C C C S CoA
R CH2 C CH2 C S CoA H
3-Ketoacyl CoA
trans-∆2-enoyl CoA
(3) oxidation (2) hydration

H2O
NADH + H
H O
Enoyl-CoA
L-3-Hydroxyacyl-CoA R CH2 C CH2 C S CoA Hydratase
Dehydrogenase NAD OH (Redox regenerating system)
L-3-Hydroxyacyl CoA
(1) In the first step of β oxidation, enoyl CoA combines with water to form the L isomer of 3-hydroxylacyl
CoA.
(2) Using NAD+, L-3-hydroxyacyl CoA is then oxidized to 3-ketoacyl CoA.
(3) Next, the nucleophilic sulfhydryl group of Coenzyme A attacks carbonyl C3 of the 3-ketoacyl CoA
group, catalyzed by Thiolase. This reaction cleaves the methylene-carbonyl C3 bond, releasing acetyl CoA,
and producing a fatty acyl CoA molecule two carbons shorter than the initial substrate.
(4) This shortened acyl CoA molecule becomes the substrate for the next round of β oxidation, continuing
the cycle until the fatty acyl CoA molecule is reduced to acetyl CoA.
As the fatty acyl chain shortens, the reactions are catalyzed by particular isozymes of acyl-CoA
dehydrogenase that are specific for short, medium, long or very long chains.
Note that steps 1 through 3 of β oxidation are the same chemical reactions as in the last three steps of the
Krebs Cycle, which convert succinate to oxaloacetate. In addition, these three steps are reversed to
accomplish the reverse effect during fatty acid biosynthesis.
Chapter 27 Degradation and Synthesis of Lipids 197
27.2 Biosynthesis of Fatty Acids.

Mechanism
The biosynthesis of fatty acids in E. coli begins with acetyl CoA and malonyl CoA. (1) The loading stage
consists of the esterification of both acetyl CoA and malonyl CoA to ACP. (See the Coenzyme A section for
the definition of ACP.) (2) The condensation stage is characterized by action of Ketoacyl-ACP Synthase,
also known as the “condensing enzyme,” to accept an acetyl group from acetyl-ACP, thereby releasing ACP-
SH. An acetyl group is then transferred to malonyl-ACP by ketoacyl-ACP synthase, forming acetoacetyl-
ACP and CO2. Loss of CO2, a good leaving group, drives the reaction to completion. The odd point is that
each added two-carbon unit in fatty acid biosynthesis is contributed by a three-carbon compound.
O
Carboxylated
O
acetyl CoA
H3C C S CoA OOC CH2 C S CoA
Acetyl CoA (C2) Malonyl CoA (C3)
HS ACP Malonyl CoA:ACP HS ACP

Loading Acetyl CoA:ACP
Transacylase Transacylase HS CoA
HS CoA
O O
O
H3C C S ACP C CH2 C S ACP
O
Acetyl ACP Malonyl-ACP
HS ACP
O
H3C C S Ketoacyl-ACP
Synthase
HS Ketoacyl-ACP
Condensation Synthase CO2
O O
H3C C CH2 C S ACP
Acetoacetyl-ACP
Ketoacyl-ACP NADPH H
Reductase
NADP
Reduction OH O
H3C C CH2 C S ACP
D-β-Hydroxybutryl-ACP
Subsequent β-Hydroxybutryl-ACP
rounds Dehydrase H2O
(Repeat from the
Dehydration O
condensation step) H
H3C C C C S ACP
H
trans-Butenoyl-ACP
NADPH H
Enoyl-ACP
Reductase NADP
Reduction
O
H3C CH2 CH2 C S ACP
Butryl-ACP
198 Chapter 27 Degradation and Synthesis of Lipids
In the first reduction, NADPH-dependent Ketoacyl-ACP Reductase catalyzes the conversion of

acetoacetyl-ACP to D- β-hydroxybutyrl-ACP. Dehydration removes a water molecule from D-β-
hydroxybutanoyl-ACP, forming a double bond and producing trans-butenoyl-ACP. In the second
reduction, trans-butenoyl-ACP is reduced to butyryl-ACP.
The last four stages of this reaction are repeated in the next turn except that butanoyl-ACP undergoes
the condensation stage with another malonyl-ACP instead of acetyl-ACP. No loading is necessary. The
third turn uses hexanyl-ACP, the fourth uses octanyl-ACP, and so forth. Trimming the mechanism down to
the essential details:
1. Reduction
2. Dehydration
3. Reduction
3-Ketoacyl ACP (Cn) Acyl ACP (Cn)
3-Ketoacyl ACP (Cn + 2)

(Cn + 4 )
CO2 Malonyl ACP (C3)
...
(1) Initiation involves condensation of the acetyl and malonyl groups to produce the four-carbon butyryl
skeleton.
(2) In the elongation stage, three reactions modify the carbonyl-containing precursor, two reduction
reactions and a dehydration reaction.
(3) These steps produce acyl ACP, which becomes the substrate for the new condensation reaction with
malonyl ACP, beginning the next turn of the spiral.
Biological Occurrence. Fatty acid synthesis always occurs in the cytosol. In humans, fatty acids are mostly
produced in liver cells, lactating mammary glands, and to a lesser degree, in adipose tissue. The sequence
of reactions is catalyzed by a multifunctional enzyme in animals.
Synthesis of Malonyl ACP and Acetyl ACP

Malonyl ACP is the primary substrate for chain elongation in fatty acid biosynthesis. It contributes the two-
carbon units in two steps.
CO2
Acetyl CoA (C2) Malonyl CoA (C3)
Prokaryotes Eukaryotes both
Acetyl ACP (C2) Malonyl ACP (C3)
CO2
Acetoacetyl ACP (C4)
In the first, acetyl CoA is carboxylated to form malonyl CoA, catalyzed by Acetyl-CoA Carboxylase. This
carboxylation reaction is similar to the mechanism catalyzed by pyruvate carboxylase. The bicarbonate
anion (HCO3-) is activated by ATP, then transferred to biotin to form carboxybiotin. This activated CO2 is
then transferred to acetyl CoA to form malonyl CoA.
O
OOC CH2 C S CoA
HCO3 + ATP E Biotin Malonyl CoA
O
ADP + Pi E Biotin COO H3C C S CoA
Acetyl CoA
Bacterial acetyl-CoA carboxylase is a heterotrimeric complex composed of Biotin Carboxylase and a
heterodimeric transcarboxylase. In eukaryotes, acetyl CoA is converted to acetyl ACP by Acetyl CoA-ACP
Transacylase. In both types of cells, Malonyl CoA-ACP Transacylase catalyzes transfer of the malonyl
fragment from CoA to ACP.
Regulation. Acetyl-CoA Carboxylase is metabolically irreversible and the focus of metabolic regulation.
27.3 Length Determination of Fatty Acids

The enzymes that catalyze fatty acid biosynthesis are specialized with respect to preferred chain length.
Different isozymes prefer the following size classes: short (< C6), medium (C6 to C12), long (> C12), or very
long (> C16).
An elongase reaction occurs in the synthesis of arachidonate, the fatty acid precursor to the
eicosanoids (step 2; see Sect 12.8). Long-chain fatty acids of length C20 and C22 are common; longer chains
such as C24 and C26 are less so.
O
12 9
C S CoA
Linoleoyl CoA (18:2 ∆9, 12)
O2 NADH + H+
6
∆ -Desaturase
2 H2O NAD+
O
18
C S CoA
12 9 6
γ-Linolenoyl CoA (18:3 ∆6, 9, 12)
O Malonyl CoA
OOC CH2 C S CoA
Elongase
added
CO2 + HS CoA fragment
20 O
CH2 C S CoA
14 11 8
Eicosatrienoyl CoA (20:3 ∆8, 11, 14)
O2 NADH + H+
∆5-Desaturase
2 H2O NAD+
14 11 8 5 O
CH2 C S CoA
20
Arachidonoyl CoA (20:4 ∆5, 8, 11, 14)
(Adapted from Moran et al., Principles of Biochemistry (5th ed.), 2012, p.480; Fig. 16.8.)
27.4 Synthesis of Acidic Phospholipids.

Carbohydrate-carbohydrate joining reactions, such as lactose biosynthesis, require the nucleotide UDP as a
cofactor. The UDP is a good leaving group, similar to the loss of pyrophosphate in nucleoside biosynthesis
using phosphoribosylpyrophosphate (PRPP). See the Uridylyl Carbohydrates and ATP sections of the
Coenzymes chapter for details.
CTP donates a cytidylyl group to phosphatidate to create CDP-diacylglycerol. In the next reaction,
CMP can be displaced by the hydroxyl oxygen of either (1) serine to form phosphatidylserine, or (2)
inositol to form phosphatidylinositol. The other modified phosphatidyl compounds described in the Lipids
chapter are made by analogous reactions.
O
O H2 C O C R1
R2 C O CH O Phosphatidate
-
H2C O P O
O-
CTP
PPi
O
NH2
O H2C O C R1 H
R2 C O CH O O N
H
H2C O P O CH2 O
P ON
O
O- O-H H
H H
+ OH OH
NH3 CDP-diacylglycerol
HO CH2 CH COO - inositol
Serine
some bacteria and
CMP bacteria CMP eukaryotes
O O
O H2C O C R1 O H2C O C R1
R2 C O CH + R2 C O CH O O
O NH3
H OH
H2C P O CH2 - H2C O P O P O H
O CH COO
OH H
O- O- O- OH HO
H OH
H H
Phosphatidylserine Phosphatidylinositol
(Adapted from Moran et al., Principles of Biochemistry (5th ed.), 2012, p.482; Fig. 16.11.)
27.5 Cholesterol Biosynthesis

Stage 1
The first stage in cholesterol synthesis is making isopentenyl diphosphate. The steps are:
(1) In the first process, three acetyl CoA molecules undergo condensation to produce HMG-CoA.
(2) Next HMG-CoA is reduced to form mevalonate. The enzyme that catalyzes this reaction, HMG-CoA
Reductase, is the primary control point in cholesterol biosynthesis. The use of statins to control cholesterol
metabolism through this reaction in humans is described below.
(3) Next, mevalonate is phosphorylated, in two sequential reactions, using ATP.
(4) Finally, an ATP-dependent decarboxylation reaction produces isopentenyl diphosphate.

O O
H3C C S CoA H3C C S CoA
Acetyl CoA Acetyl CoA
Acetoacetyl CoA
thiolase
O O O
ATP
H3C C S CoA H3C C CH2 C S CoA Phosphomevalonate
Acetyl CoA Acetoacetyl CoA kinase
ADP + Pi + HCO3
H2C O O
HMG CoA H2O
C CH2 CH2 O P O P O
synthase H3C
H+ + HS - CoA O O
Isopentenyl diphosphate
OH O
OOC CH2 C CH2 C S CoA
Isopentenyl
H3C diphosphate
isomerase
3-Hydroxymethyl-3-methylglutaryl CoA (HMG CoA)
2 NADPH + H+ O O
HMG CoA H3C
reductase 2 NADP+ + HS - CoA C CH CH2 O P O P O
H3C
O O
OH Dimethylallyl diphosphate
OOC CH2 C CH2 CH2 OH
H3C Mevalonate
ATP PPi
Mevalonate O O
kinase
ADP H3C CH2 O P O P O
C C
H O O
OH O H2C H
+
OOC CH2 C CH2 CH2 O P O CH2 Isopentenyl diphosphate
H3C
H3C O C CH
Mevalonate-5-phosphate H3C
Prenyl transferase
ATP Dimethylallyl
Phosphomevalonate carbocation
kinase
H+
ADP
OH O O
O O
OOC CH2 C CH2 CH2 O P O P O
O P O P O
H3 C O O
O O
Mevalonate-5-diphosphate
Geranyl diphosphate
(C10, BOOM!)
(Adapted from Moran et al., Principles of Biochemistry (5th ed.). 2012, p. 490; Fig. 16.17.)
Stage 2
The second stage of cholesterol synthesis is marked by isoprene-to-isoprene condensation reactions.
O O
O O H2 C CH2 O P O P O
O P O P O C C
H O O
H3 C H
O O
Geranyl diphosphate
(C10) Isopentenyl diphosphate
Prenyl
transferase
PPi
O O
O P O P O
Farnesyl diphosphate O O
(C15)
NADPH + H+
Squalene +
synthase Second
2 PPi + NADP+ Farnesyl
diphosphate
Squalene
(Adapted from Moran et al. Principles of Biochemistry (5th ed.), 2012, p. 491; Fig. 16.18.)
Stage 3
The concluding stage of cholesterol synthesis converts squalene to cholesterol. Converting lanosterol to
cholesterol is an intensive process, requiring up to 20 steps.
Squalene
HO
Lanosterol
HO
Cholesterol
(Adapted from Moran et al. Principles of Biochemistry (5th ed.), 2012, p. 491; Fig. 16.19.)
27.6 Regulating Cholesterol Levels

HMG-CoA Reductase is one of the most highly regulated enzymes known because it catalyzes the rate-
limiting step in cholesterol biosynthesis. It is controlled by three primary regulatory mechanisms: covalent
modification, repression of transcription and control of degradation.
Cholesterol levels in cells are highly regulated because it can suppress transcription. Both intracellular
cholesterol in plasma lipoproteins and cholesterol in dietary chylomicrons contribute to transcriptional
activation.
By decreasing the serum cholesterol levels in the body, one can decrease their risk of coronary heart
disease. A group of drugs known as statins are often used to treat hypercholesterolemia. They work by
competitively inhibiting HMG-CoA reductase. The structures of HMG-CoA and two common statins are
shown below.
HO
O
HO O
H3C C
O C
C O O
HO O
F
S O C
N CH3 O
C
H3C O
NH CH3 H3C
O C O
N H3C
NH H
O
HMG CoA
H3C
Atorvastin Lovastin
H3C
(Lipitor R ) (Mevacor R )
O
3'-ADP
(Adapted from Moran et al. Principles of Biochemistry (5th ed.), 2012, p. 492; Box 16.3.)
However, using statins to control cholesterol through inhibition of HMG-CoA reductase can
potentially affect other processes. For example it also inhibits mevalonate synthesis, which is necessary for
make a number of important biomolecules, such as ubiquinone.
Chapter 28
Photosynthesis
28.1 Light and Dark Reactions
From childhood, we associate photosynthesis with the green color of plants. Plants use the green molecule
Chlorophyll, as well as a number of other “accessory pigments,” as antennae to capture the energy of light.
This energy is converted into NADPH, which is used in the Ribulose Bisphosphate Carboxylase
(RUBISCO) reaction to “fix carbon,” in the form CO2, to produce the metabolite 3-phosphoglycerate.
28.2 Photo-Gathering Pigments

Molecules. Chlorophylls (Chl) are the primary membrane-embedded light receiving molecules.
H3C R1
I
H N
H3C R2 Saturated in
2+
IV N Mg N II BChl a and
H2 BChl b
C R3
Phytol side chain - made from H
three isoprene units CH2
N
III
O C H V
O C
CH3 CH3 O O
O
H CH2 CH CH2 CH2 CH2 C CH CH2
3 CH3
Chlorophyll
species R1 R2 R3
Ch1 a CH CH2 CH3 CH2 CH3

O
Ch1 b CH CH2 C H CH2 CH3
O
BCh1 a C CH3 CH3 CH2 CH3
O
BCh1 b C CH3 CH3 CH CH2
(1) β-Carotene - orange and red pigments in carrots and tomatoes

CH3 CH3 H3 C
H3C CH3
CH3 CH3 H3C CH3

CH3
(2) Neoxanthin - contains cyclohexane and cyclohexane epoxide ring

systems
CH3 CH3
O
CH3 CH3 OH
HO OH
(3) Phycoerythrin - linearized heme ring, pyrrazoles
OOC
CH3 CH2 H2C COO CH2 Ethyl group in
CH3 CH CH3 CH2 CH2 CH3 CH3 CH phycocyanin
O N N N N O
H H H
Unsaturated in phycocyanin
Chapter 28 Photosynthesis 205
Absorption Spectra
The following set of spectra show that the photosynthetic antenna pigments span the visible region,
assuring that all wavelengths are captured.
let ndigo w e
Blu
e een llo ang d
Colors: Vio I Gr Ye Or Re
Chl b
Carotenoids
Chl a
Phycocyanin Phycoerythrin
Absorbance
Chl b Chlorophyll a
0
400 500 600 700
Wavelength (nm)
.
Note. The plant pigments appear as to us visually as the colors that are not absorbed. For example, the
chlorophyll a and b molecules absorb in the blue and red but have a lack of absorbance in the green, so they
appear to be green to our eyes.
28.3 Photosynthetic Electron Transport Pathway (Z scheme)

Electrons are transferred between membrane-associated photosynthetic electron carriers depending upon
the difference in reduction potential.
Photosystem I
-1.5
P700*
Photosystem II A0
-1.0 A1
P680* FX
FB
Reduction Potential (V)
Ph α Fd
FA
-0.5
NADP
PQA
Fd -NADP
0
PQpool Oxidoreductase
PQB
Cytochrome bf Plastocyanin
Oxygen- complex
evolving P700
+0.5 complex
proton
gradient Light
2 H 2O
Z
+1.0
P680
O2
+
+1.5 proton gradient Light
206 Chapter 28 Photosynthesis
As in mitochondrial electron transport, electrons flow naturally from lower reduction potential
carriers (stronger reductants) to higher reduction potential carriers (stronger oxidants).
When light energy is absorbed in photosynthesis, the reduction potential of the primary donor is
forced to decrease dramatically. The photo-excited complexes in the Z-scheme called P680* and P700*
have significantly lower reduction potentials than their ground-state equivalents P680 and P700. This
indicates that they are stronger reductants (electron donors). The electron is activated to a higher energy
state. The two connected consequences of photosynthetic electron transport are:
(1) Light drives the production of a proton gradient.
(2) Excess protons are transported from the granal lumen to the stroma, which drives ATP production via
oxidative phosphorylation.
More stacked grana, which also contain

embedded photosynthetic components
Stroma ATP
Granal Synthase
Lumen Ferredoxin
(Fd) Fd-NADP
Oxidoreductase H
hν
Cytochrome bf ADP + Pi ATP
complex
hν
Mobile NADP
hν 4H hν -
2H LHC NADPH Cl CF0
+H
CF1
P680 -
2e P700 LHC
4e-
Stromal
Mg2+ H lamella
LHC 2 H2O 4 H + O2
8H
Oxygen-evolving Fd-NADP
PS II PS II Plastocyanin Oxidoreductase
antenna complex
reaction Plastoquinone
complex center pool PS I
reaction Granal Lumen
center
The Chlorophyll Special Pair. The light energy is received by chlorophyll antenna pigments and
focused into the pathway shown by the dashed lines. When it reaches the “special pair” of chlorophylls, an
electron is contributed to the electron transfer pathway. All of these chlorophylls are held tightly by
membrane-bound proteins.
light
e-
.
(Adapted from Moran et al., Principles of Biochemistry (5th ed.), 2012, p. 446, Fig. 15.3.)
Chapter 28 Photosynthesis 207
Photosystem I
Photosystem I electron transfer is initiated with the special pair of chlorophylls (P700). The energy is
directed through the branch to phylloquinone, then on to the Fe-S clusters and finally to ferredoxin. P700*
is reduced by either cytochrome c or plastocyanin.
light
Cytochrome c or
P700 Plastocyanin
e
e
Pyhlloquinone e
FA
e
A-branch
FA
FB
e
Ferredoxin or
e Flavododxin
(Adapted from Moran et al., Principles of Biochemistry (5th ed.), 2012, p. 451, Fig. 15.9.)
Chapter 29
Carbon Fixation: The Calvin Cycle and C4/CAM

Pathways
This pathway was worked out using the unusual radioisotope Carbon-11. The ‘usual’ tracer isotope used
now is C-14. The key effect is capture of gaseous carbon dioxide (CO2) to form the common metabolic
intermediate 3-phosphoglycerate.
29.1 The Dark Reactions: Carbon Fixation

The enzyme complex Ribulose Bisphosphate Carboxylase (RuBisCO) catalyzes ‘carbon fixation.’ Ribulose
1,5-bisphosphate (C5) and three CO2 molecules form six 3-phosphoglycerates.
Reduction Reactions
Carbon Fixation Step
3 CO2 6 NADPH + 6 H 6 NADP
3 H 2O 6H 6 ATP 6 ADP (1) G3P DHAP
O OPO3 2- + 6 Pi
(6) COO
(6) C
H C OH H C OH
RuBisCO Phosphoglycerate
CH2OPO3 2- CH2OPO3 2- Glyceraldehyde-3-phosphate
O
3-Phosphoglycerate Kinase 1,3-Bisphosphoglycerate Dehydrogenase (5) C
H
CH2OPO3 2-
(3) H C OH
C O
CH2OPO3 2-
H C OH
CH2OH CH2OPO3 2- Glyceraldehyde
H C OH
C O Pi H2O 3-phosphate (G3P)
CH2OPO3 2- C O
Ribulose Aldolase
CH2OH HO C H HO C H
Ribulose 5-phosphate Triose
1, 5-bisphosphate O H C OH Fructose- H C OH
3-Epimerase C
1, 6-bisphos- CH2OH Phosphate CH2OH
H C OH
CH2OH HO C H phatase H C OH
C
Isomerase O
O C
C O H C OH CH2OPO3 2- CH2OPO3 2-
CH2OPO3 2- CH2OPO3 2-
H C OH CH2OPO3 2- Fructose- Fructose-
6-phosphate 1, 6-bisphosphate (2) Dihydroxyacetone
3 ADP H C OH Xylulose-
5-phosphate O Phosphates (DHAP)
CH2OPO3 2- H
Phospho- C
ribulokinase Ribulose- Aldolase
5-phosphate H C OH
3 ATP Transketolase H C OH
CH2OPO3 2-
CH2OH Erythrose-
CH2OH Ribulose 4-phosphate
C O CH2OPO3 2- CH2OH
C O
5-phosphate
3-Epimerase HO C H C O H 2O Pi C O
H C OH HO C H HO C H
H C OH
CH2OPO3 2- Sedoheptulose-
CH2OPO3 2- H C OH H C OH
Xylulose- 1, 7-bisphosphase
Ribulose- 5-phosphate H C OH H C OH
5-phosphate
O H
Sedoheptulose- Sedoheptulose-
CH2OH C
1, 7-bisphosphate 7-phosphate
C O Ribulose-5-phosphate H C OH
H C OH
Isomerase Transketolase
H C OH
H C OH H C OH
Ribulose- Ribose-
5-phosphate 5-phosphate
Regeneration Reactions
29.2 Biosynthesis of Ribose-5-phosphate

Two Transketolases catalyze carbon skeletal rearrangement steps in the Calvin cycle. Transketolases use
Vitamin B12 (Cobalamin) to rearrange the skeleton (see the Coenzyme section). These rearrangements
provide a pool of differently sized skeleton fragments for a variety of biosynthetic reactions.
Chapter 29 Carbon Fixation: The Calvin Cycle and C4/CAM Pathways 209
In one reaction, a Transketolase converts glyceraldehyde-3-phosphate (C3) to xylulose-5-phosphate (C6)

and ribose-5-phosphate (C5), the sugar subcomponent in RNA, DNA, NADH, and so on.
In another reaction, fructose-6-phosphate (C6) is converted to xylose-5-phosphate (C5) and erythrose-4-
phosphate (C4).
The Calvin Cycle can be summarized as follows:
CO2
Ribulose 1, 5-
bisphosphate
1. Carbon fixation
3-Phosphoglycerate
3. Regeneration ATP
Carbohydrates
ADP
2. Reduction
Glyceraldehyde
3-phosphate 1, 3-Bisphosphoglycerate
Pi NADP+ NADPH
+ H+
29.3 RuBisCO Mechanism

RuBisCO in plants, algae and cyanobacteria are composed of 8 small and 8 large subunits. The “carbon
fixation” mechanism consists of five steps:
(1) Enolization: keto-enol tautomerism
(2) Carboxylation (carbon fixation): the “ene” electrons attack the carbon of CO2.
(3) Hydration: OH- from H2O attacks the central carbonyl carbon; the carbonyl oxygen picks up the H+.
(4) Cleavage: the “3-gem diol” intermediate cleaves to produce 3-phosphoglycerate (3PGA).
(5) Protonation: the remaining carbanion abstracts H+ from an active site base to form a second 3PGA.
CH2OPO3 2- CH2OPO3 2- O CH2OPO3 2-

C O H C OH C HO C COO
B enolization B carboxylation O H
H C OH H C O H O B
H C O
H
CO2
CH2OPO3 2- CH2OPO3 2- CH2OPO3 2-
Ribulose- 2, 3-Enediol
intermediate 2-Carboxy-3-ketoarabinitol
1, 5-bisphosphate
1, 5-bisphosphate
hydration
CH2OPO3 2-
protonation HO C cleavage HO C COO
HO C H B COO B
H H HO C O H
COO H C OH
Carbanion
3-phosphoglycerate
CH2OPO3 2-
COO
3-Gemdiol
H C OH
intermediate
CH2OPO3 2-
3-phosphoglycerate
210 Chapter 29 Carbon Fixation: The Calvin Cycle and C4/CAM Pathways
(1) Carbon is fixed to produce 3-phosphoglycerate, which can interconvert with dihydroxyacetone
phosphate, catalyzed by Triose Phosphate Isomerase.
(2) Under suitable conditions, these compounds fuel Gluconeogenesis. In woody plants, this provides
glucose for the biosynthesis of cellulose, fruit, leaves, wood, and other essential tissues. Plants are our
ultimate source of many, if not most, of our essential nutrients and metabolites. Approximately 70% of the
atmospheric O2 is produced by photosynthesis in land plants, especially in tropical forests. Nearly all of our
food comes directly or indirectly from plants, energized by sunlight.
(3) Reductive conversion of CO2 to carbohydrate compounds is driven by ATP and NADPH—as a result of
the “light reactions.” Carbon fixation and carbohydrate synthesis occur in in the cytoplasm of cyanobacteria
and the stroma of the chloroplast in algae and plants.
Melvin Calvin: Parking at Berkeley and the Nobel Prize:

As a postdoc at Berkeley, I recall walking past Melvin Calvin’s unique plant-green Saab parked in his
exclusive spot—right next to the Laboratory of Chemical Biodynamics building. Parking at Berkeley was a
hassle. If I drove my car, it usually meant a five-block walk, which is no big deal except in the miserable
late November rain. I even bought a motorcycle because I could park it under the building I worked in.
One October day, it was announced that Professor Yuan Lee had won the Nobel prize—as had
Professor Calvin before him for his Cycle. The student newspaper ran a two-inch headline that read: “Yuan
Lee Gets Parking Space!”… At Berkeley, one receives a lifelong parking space upon winning the Nobel
prize!
29.4 The C4 and CAM Pathways

Plants that grow in high light intensities and temperatures, e.g., corn, sorghum and maize, have evolved a
way to avoid wasteful photorespiration (light-dependent water loss) by using a second carbon fixation
method called the C4 pathway. The net effect is to increase the ratio of CO2 to O2, thereby increasing the
activity of RuBisCo and facilitating delivery of CO2 to the interior of the leaf, where the Calvin cycle is
active.
A similar pathway called Crassulacean Acid Metabolism (CAM) occurs in succulent plants such as
cactus, which grow in an arid climate and must carefully avoid water loss. The solution is that they
assimilate CO2 at night, thereby limiting the water loss that would occur if CO2 was absorbed during the
day. The key is that the plant is covered with a waxy coating and CO2 exchange occurs through bicellular
port structure called stomata.
Details of both pathways are described in most standard biochemistry textbooks.
Chapter 30
The Urea Cycle
30.1 Purpose and Reactions

The purpose of the Urea Cycle is to convert ammonium (NH4+) to the less toxic urea. The overall reaction
is:
CO2 + NH4+ + aspartate + 3 ATP + 2 H2O
urea + fumarate + 2 ADP + 2 Pi + AMP + PPi + 5 H+
The enzymes that catalyze the individual transformations only occur in liver cells and are distributed in
both the cytoplasm and mitochondria, analogous to the reactions in the Malate-Aspartate Shuttle.
(1) The initial steps occur in the mitochondrial matrix and involve the synthesis of carbamoyl phosphate
from carbonate and ammonium, the sources of the carbonyl group and one of the nitrogen atoms in the
final urea product. These reactions, which require two ATPs, are shown below.
O O O O NH3 Pi O
HO C O + O P O ADP HO C O P O H2N C O
O O ammonia carbamate
ADP
carbonate ATP
O H H H H H
ATP
1
O C C C C C N H ADP
NH2
urea +H N H H H
C NH2 3
O O
O ornithine
H2N C O P O
H2O
5 O
carbamoyl phosphate
Mito-
NH2 2 chondrial
Pi matrix
C NH2+
Cytoplasm
H N
arginine
H O H H H H H O
C H
H O C C C C C N C NH2
C H
argininosuccinate 3
+H N H H H
H C H 3
4 ATP citrulline
H C NH3+ O
ADP
NH2+ C O
O C
O
O C N C H
O C O
H N H H C H +H N C
C O 3 H
H H C H C O aspartate
C H C H
fumarate H C H O
C C O
H H C H
O C
H C NH3+ O
O
O C
O
212 Chapter 30 The Urea Cycle
(2) Next, carbamoyl phosphate reacts with ornithine to produce citrulline, which is transported out of the
mitochondria in exchange for ornithine. This step is driven to completion by loss of phosphate from
carbamoyl phosphate. Note that ornithine is regenerated by the cycle, a role analogous to that of
oxaloacetate in the Krebs Cycle.
(3) In the third reaction, aspartate, which is produced by transamination of oxaloacetate, contributes the
second nitrogen atom that ends up in urea.
(4) Reaction 4 produces fumarate and arginine, which is cleaved in reaction 5 to yield urea and regenerate
ornithine.
The enzymes, which are indicated by the circled numbers, are summarized in the following table.
Number Enzyme Reaction

1 Carbamoyl Phosphate Synthase HCO3- + NH4+ + 2 ATP → carbamoyl phosphate
+ 2 ADP + 3 H+
2 Ornithine Transcarbamoylase carbamoyl phosphate + ornithine → citrulline + Pi
3 Argininosuccinate Synthase citrulline + aspartate + ATP → argininosuccinate
+
ADP + PPi
4 Argininosuccinate Lyase Argininosuccinate → fumarate + arginine
5 Arginase Arginine + H2O → ornithine + urea
Ornithine is transported in exchange for either H+ or citrulline. The fumarate is is transported back
into the mitochondrial matrix, where it is converted to malate (see the Malate-Aspartate Shuttle chapter).
30.2 Regulation
The Urea Cycle is regulated by several interconnected mechanisms.
(1) Dietary protein consumption changes the levels of all five of the Urea Cycle enzymes.
(2) Glucagon and the glucocorticoid hormones activate their transcription, while insulin represses their
production.
(3) Carbamoyl Phosphate Synthetase I is allosterically activated by N-acetyl glutamate, which is made
from glutamate and acetyl CoA. Glutamate is a major source of excreted NH4+. This reaction, which is
catalyzed by N-Acetylglutamate Synthase, is a good indicator of the cellular glutamate nitrogen load.
(4) N-Acetylglutamate Synthase is allosterically activated by arginine, so higher arginine levels activate the
Urea Cycle at the first committed step.
Chapter 30 The Urea Cycle 213
30.3 Comparative Nitrogen Excretion

Nitrogen is enriched in the nucleic acid bases, especially the purines adenine and guanine. Adenine is
converted to hypoxanthine by deamination. The following reactions show how the purines are degraded
prior to excretion. Note that different end products exist depending on the type of organism. Marine
organisms produce ammonia on surfaces such as gills, where it is washed away before accumulating to
toxic levels.
O H2O + O2 H2O2 O O
C 1 C NH3 H2O C
HN C N HN C N HN C N
CH or CH CH
HC C N C C N Guanase C C N
N 2 O N H2 N N
H H H
H2O + NAD+ NADH + H+ H
Hypoxanthine Xanthine Guanine
1 = Xanthine oxidase H2O + NAD+ H2O + O2

2 = Xanthine dehydrogenase 2 or 1
NADH + H+ H2O2
O
H H2O + O2 H2O2 + CO2 O H
C
HN C N H2N C N
C O C O
C C N Urate oxidase C C N
O N O N H H
H H
H
Uric acid Allantoin
H2 O
(birds, some (most mammals,
reptiles, Allantoinase turtles, some
primates) insects, gastropods)
Allantoicase
O O-
H2O 2 H2O
Urease H2N H2N C H2N
2 CO2
+ C C
C C
4 NH3 O NH2 O N H N O
2 H2O H H
O O-
Urea C Allantoate
(most fish, amphibians, Glyoxylate (some bony fish)
freshwater mollusks) CH
O
30.4 Protein Degradation and Programmed Cell Death

Eukaryotic cells contain a set of enzymes in both the cytosol and nucleus that link a protein called
ubiquitin, which is connected via the C-terminal end to the lysine of the target protein. As a result the
protein is degraded by a large multi-protein complex called the proteosome. ATP is required for the
proteosome to degrade the ubiquinated protein.
Programmed cell death is called apoptosis. The affected cell decreases in volume, the chromatin
becomes fragmented, the plasma membrane becomes damaged and the mitochondria swell. The enzymes
214 Chapter 30 The Urea Cycle
that are responsible for cell death are called caspases, which generally contain cysteine and react with
aspartate residues on the substrate target proteins. Cells can die due to normal developmental turnover, in
order to control antibody production and as a result of disease, for example, neurodegenerative disease.
It is interesting to note that another targeting protein analogous to ubiquitin exists. The SUMO-
mediated pathway targets SUMO-linked proteins for import into the nucleus. The SUMO-protein cross-
linkage reaction is called sumoylation.
STUDY GUIDE
Review Session for Chapters 1—3

Organelle Functions
Review the organelles and concepts in the Cell Biology Review. Example first quiz questions, in our typical
format, are shown below. The quiz will be a combination consisting of one of the first three questions, or
some variant that addresses the properties and functions of organelles. We typically give some variation of
the fourth question, which requires drawing a relatively simple organic molecular structure and describing
some typical inherent property.
1. (a) Name and explain the functional process that occurs on the rough endoplasmic reticulum (RER).
(b) What is the name of the “machines” that are present on the RER surface and what do they make?
2. (a) Explain what a chloroplast does.

(b) How are chloroplasts special in a genetic sense?
3. (a) Explain what a mitochondrion does.

(b) How are mitochondria special in a genetic sense?
4. Draw the ethanol molecule. Draw every atom and bond clearly. Circle then label the two parts that differ
with respect to solution properties and provide a suitable label for each. (Hint. Think about charges and
solubility/insolubility.)
Organic Chemical Functional Groups and Their pKas.

Look up the following items in the textbook, Wikipedia, etc. Draw an example of each. Differentiate the
class of the molecule from a specific functional group. What would the pKa values of each be? Why?
alcohols (pKa = 15–16 for aliphatic); aromatic (pKa = 10)
carbonyls, aldehydes, ketones
carboxylic acids (pKa = 5)
amines (pKa = 9)
thiols, sulfhydryls (pKa = 8)
esters
amides
ethers
anhydrides
hemiacetals (acetals)
phosphoryl (pKa = 2, 6, & 12)
2-mercaptoethanol
urea
guanidinium (and guanidine)
pyrophosphate (pKa values: 1.52; 2.3; 6.60; 9.25) How much is ionized at pH 7?
acetylphosphate
216 Study Guide
Chemical Principles
Review the following concepts. Write a carefully worded definition, using proper grammar, punctuation
and usage. Include any useful illustration material that will enhance the clarity of your explanation, for
example, graph, equation, chemical detail, and/or mechanism.
Oxidation, Reduction
Review the concept associated with the acronym “oil rig.” Oxidation is loss of one or more electron(s).
Reduction is a gain of electrons. These reactions always involve electron exchange.
Reaction Equilibrium and Thermodynamics

The relation between the equilibrium constant of a reaction and its Gibbs free energy is:
ΔG = - R T ln K (1)
Two contributions lead to the net ΔG are:
ΔG = ΔH – T ΔS - the Gibbs Equation- (2)
where ΔH is the enthalpy change, and -TΔS is the negative product of temperature times the entropy
change, that is, the entropic contribution to ΔG.
Prediction of Reaction Spontaneity.

When ΔH < 0 and ΔS > 0, the reaction is always spontaneous.
When ΔH > 0 and ΔS < 0, the reaction is never spontaneous.
When ΔH < 0 and ΔS < 0, the reaction is spontaneous at low temperature.
When ΔH > 0 and ΔS > 0, the reaction is spontaneous at high temperature.
Physical Chemistry Concepts

(1) Bond polarity (i.e., the carbonyl C=O bond has a δ+ / δ- charge distribution)
(2) Hydride (H•): A hydride is a hydrogen atom with an attached electron. It is not H+. This is an electron-
free hydrogen nucleus, that is, a proton.
Water-Solute Interactions
(1) Hydrophobic effect
(2) Hydrogen bonds
(3) Van der Waals interactions (London dispersion forces, induced dipole:induced dipole); dipole:induced
dipole; dipole:dipole; dipole:monopole (ion)
(4) Ionic bonds (salt bridges); (monopole:monopole)
(5) Hydrogen bonding: Plot the relation between interaction energy and distance between the two hydrogen
bonded atoms.
pH and pKas
(1) A functional group is in the basic form when the pH is greater than the pKa of the group. The group is
in the acidic form when the pH < pKa. To accomplish this transformation, H+ dissociates from its conjugate
base.
(2) Practice drawing the pH titration curves for each amino acid.
Study Guide 217
Review Session for Chapter 4
The following examples of Quiz 2 questions constitute a review of the structures of the amino acids.
The name and score information are included as part of our typical quiz format.
Quiz 2A
1. Draw the structures of the following amino acids at pH 7. Show all bonds and atoms.
(a) L-methionine (b) L-serine
2. Name (below) all of the functional groups in the two molecules drawn in #1, including those on the R
group. Be precise with your nomenclature.
Quiz 2B
(a) L-histidine (b) L-glutamate
(a) L-histidine (b) L-glutamate

218 Study Guide
Quiz 2C
(a) L-leucine (b) L-asparagine
(a) L-leucine (b) L-asparagine
Quiz 2D
(a) L-proline (b) L-aspartate
(a) L-proline (b) L-aspartate

Study Guide 219
Quiz 2E
(a) L-lysine (b) L-valine
(a) L-lysine (b) L-valine
Quiz 2F
(a) L-isoleucine (b) L-cysteine
(a) L-isoleucine (b) L-cysteine

220 Study Guide
Review Session for Chapters 5 and 6
Look up and define each of the following concepts. These topics are incorporated into example quiz
format on the following pages. In subsequent sections, the quizzes will not be shown explicitly.
(1) Van der Waals interactions.
(2) Hydrogen bonds.
(3) Edman degradation. Describe the two different purposes.
(4) Disulfide bonds.
(5) α-helix. Provide an illustration of the hydrogen bonding pattern as part of your definition.
(6) β-sheet. Also provide an illustration of the hydrogen bonding pattern.
(7) Hydrophobic effect.
(8) Ramachandran plot.

Study Guide 221
Quiz 3A
1. Define a Van der Waals interaction. Include a description of the relation between interatomic distances
and free energies.
2. Define the following terms as completely as you can:

(a) Protein primary structure:
(b) Protein quaternary structure:
Quiz 3B
1. Define a hydrogen bond interaction. Include a description of the relation between interatomic distances
and free energies.
2. Define a protein β-sheet; include a drawing of the key hydrogen bonds.

222 Study Guide
Quiz 3C
1. Define the following terms as completely as you can:

(a) Protein secondary structure:
(b) Protein tertiary structure:
2. Describe Edman degradation. Draw the structure of the key reagent.
Quiz 3D
1. Define a chaotropic agent. Give two specific examples used with proteins.
2. Define a protein α-helix; include a drawing of the key hydrogen bonds.

Study Guide 223
Quiz 3E
1. Define a disulfide bond. Draw one between two fully drawn amino acids.
2. Explain the purpose of a Ramachandran plot. What are φ and Ψ?
Quiz 3F
1. Define a zwitterion. Draw a generic amino acid zwitterion.
2. Define and describe the energetic origins of the hydrophobic effect with respect to protein stability.
224 Study Guide
Review Session for Chapters 7—8.6
Note. From here on, the quiz questions are presented as lists. Any combination of two or three questions
might appear on a given quiz. In order to provide practice at the process of composing answers, none are
provided for Review Sessions 4 through 7. To help accelerate the pace in the latter sessions, briefly
sketched versions of answers are provided.
1. Define an “initial rate” in the context of enzyme kinetics. Give the relevant equation and name the terms
in it.
2. Does an act of catalysis change an enzyme? Explain and give the expression for the generic enzymatic
reaction.
3. Define the term “maximum velocity” in the context of enzymatic catalysis. Give the relevant equation
and name the terms.
4. Write out the Michaelis-Menten equation and name each term.
5. (a) What are the two definitions of KM?
(b) Define the Michaelis complex.
6. Define the parameter kcat and provide the key defining equation.
7. Write out the linked chemical equilibrium reactions involving E, S, and other terms that describe simple
Michaelis-Menten catalysis.
8. Show how the reactions involving E, S and the inhibitor I differ for competitive, noncompetitive, and
uncompetitive inhibition of enzyme catalysis.
9. List the three requirements for enzyme catalysis.
10. How do the initial rate and steady state ideas coincide in the description of Michaelis-Menten kinetics?
Why must they coincide for Michaelis-Menten analysis to work.
Study Guide 225
Review Sessions for Chapters 8.7—8.8
1. Which external factors limit the rate/velocity of an enzyme-catalyzed reaction? Give three examples and
explain how each modulates the catalytic rate.
2. (a) Draw acetylcholine. See part b before you start.
(b) Which atom, on which amino acid in the esterase site of Acetylcholine Esterase, reacts with the
substrate? Draw a simple depiction of the enzyme surface beneath your acetylcholine drawing in part
(a). Show how the recognition and catalytic entities of the enzyme interact with the substrate. Include an
arrow to show how the electrons of the key enzymatic atom attack the substrate.
3. (a) Explain in general how the nerve gas antidote pyridine aldoximine methiodide (PAM) reactivates
acetylcholine esterase.
(b) What is the key functional group on the antidote?
(c) What kind of reaction produces the reactivated enzyme?
4. Draw the generic reaction for the bisubstrate-enzyme “ping-pong” reaction. Define the letters used.
5. What is meant by an enzyme cascade. Include a drawing and indicate how amplification of the initial
signal is achieved.
6. Blood clotting is initiated by very few enzymes. Explain how a clot composed of many, many proteins is
produced. Include a reaction or reactions and emphasize how such a large number of products is achieved.
Name this phenomenon.
7. Define the term zymogen and give one specific example important for food digestion.
8. Describe feedback inhibition. Why is it a logical way to regulate a metabolic pathway?
9. Define allosterism. Describe how it is initiated using the appropriate name.
10. Describe one example of kinase and phosphatase reactions that regulate either glycolysis or the Krebs
Cycle. Name the regulated enzyme and explain which form is active and which is inactive.
11. (a) Which global cellular process does Cyclin Kinase regulate? Which two amino acids can be
phosphorylated?
(b) Describe the composition of the Mitosis Promoting Factor complex phosphoryl-
ation/dephosphorylation state of the activated form.
226 Study Guide
1. Give two examples of reversible factors that control the catalytic capability of an enzyme. Explain each
briefly.
2. Give two examples of irreversible factors that control the catalytic capability of an enzyme. Explain each
briefly.
3. List the two chemical modes of catalysis. Define each briefly.
4. List the two binding modes of catalysis. Define each briefly.
5. Explain how uncatalyzed and catalyzed reaction coordinate diagrams differ. Explain each change you
show.
6. Define a nucleophile and explain what happens in a nucleophilic substitution (SN2) reaction. Provide a
mechanism diagram.
7. (a) Define an electrophile. (Think about how it reacts in contrast to a nucleophile.)
(b) Explain how to create an electrophile via acid-base catalysis from histidine. Explain why it is an
electrophile.
8. (a) Why is histidine used so often by enzymes to carry out acid-base catalysis?
(b) Explain why enzymes typically have an optimal pH.
9. Draw and label the participants in the catalytic triad of amino acids typically present in the active site of
Serine Proteases.
(b) Explain how the three amino acids collaborate to accomplish acid-base catalysis.
10. (a) What parameters appear in the Arrhenius equation? Which variable changes and what causes it to
change? What are the constants and/or energies?
(b) Is it a thermodynamic equation or a kinetic equation? Explain. (Hint. The changing variable allows
one to distinguish between an equilibrium- or rate-dependent equation.
11. Draw the structure of ATP. Include all atoms and bonds.
12. Why is Mg2+ typically required to achieve optimal activity with ATP cosubstrate in enzyme-catalyzed
reactions?
Study Guide 227

1. Define the term coenzyme. Give one example and describe how the coenzyme is used in a typical
biochemical application.
2. Briefly describe one example in which a heavy (transition) metal is used to facilitate a biochemical
reaction.
3. What is ATP used for in most biochemical applications? Give two different examples that involve
different parts of the molecule.
4. (a) Draw NAD+ (NADH) in both the oxidized and reduced forms. Use the contraction “R” to signify the
common parts of the structure in the latter drawing (i.e., only show the part that changes, connected to “R”).
(b) Describe how this coenzyme is used in a typical biochemical application.
5. Draw FAD/FADH2 in both oxidized and reduced forms.
6. (a) Draw pyridoxal phosphate prior to and after forming a Schiff base with the ε-amino group of a lysine
residue from an enzyme E.
7. (a) What chemical group does coenzyme A typically carry in the course of its biochemical function?
Draw the covalent complex in an abbreviated manner that emphasizes the ligand-carrying functional group
of the coenzyme.
(b) Why is this function such a crucial link in intermediary metabolism?
8. Explain how the noncovalent complex between biotin and avidin is used in biotechnology to capture
ligand-binding entities, such as DNA-binding proteins. Provide a step-by-step procedure and diagram that
illustrates your explanation.
9. (a) What is the crucial function of N5, N10-methylenetetrahydrofolate in the production of DNA?
(b) Explain how our understanding of this function can be used in a strategy for anticancer
chemotherapy.
10. (a) How does a ketose differ from an aldose? Give one example of each.
(b) How does a pyranose differs from a furanose? Give one example of each.
11. (a) What is UDP-galactose and how is it used to make lactose?
(b) Draw the structure of lactose.
12. How does cis-retinal function in transducing the signal of a photon of light into a chemically
recognizable form?
228 Study Guide
Review Session for Chapters 10—11.4: Key
Note Regarding the Key Material. The Key information provided in the remaining sections of the
Review material contain rudimentary answers, which should not be misconstrued as sufficiently detailed
for use on formal quizzes and tests. Fully developed explanations should be written (and revised) as a
means to practice fleshing out informative definitions at the level expected on a formal quiz or test.
Students ask me regularly “Do I need to include (some given piece of an explanation or definition)?” My
answer is “Will it make it more complete and clear? If so, include it.” Our goal is to get you to write
excellent answers, which will in turn earn an excellent grade. More importantly, since you wrote it out,
you’ll be better prepared to remember and use the information in the future.
Structure Functional Part Purpose

1. e.g., NAD+, FAD, PLP, CoA, ATP (cosubstrate) Organic cofactor
coenzyme (vitamin) that aids in
the catalysis of an
enzymatic reaction.
2. heavy e.g., Molybdenum—Xanthine Oxidase (purine catabolism) Metallic atom
metal Zinc—Carbonic Anhydrase; Zinc Finger proteins (transition metal – 3rd
Cobalt—vitamin B12 (one carbon transfers) period) used as the
Iron—FexSy clusters – redox reactions focus of an enzymatic
reaction
The following metals are not transition metals: Mg2+, Ca2+, K+,
Na+.
3. ATP cosubstrate; activated monophosphate , pyrophosphate ATP + glucose →
Pi O ADP + glucose-6-Pi
Pi O O
O
OH O PPi
ATP + ribose-5-Pi →
HO OH OH OH AMP + phosphoribosyl
OH pyrophosphate
glucose-6-phosphate phosphoribosyl pyrophosphate (PRPP)
+
4. NAD acceptor/donor of
H O
H H H O reducing equivalent
H C NH2 H C NH2 (redox partner)
(glycolysis, Krebs) →
H H H H electron transfer
N N
H
5' ribose R
Adenine
Pi Pi 5'
ribose
NAD+ NADH
Sreduced + NAD+oxidized Poxidized + NADHreduced + H+
5. acceptor/donor of
FAD/FAD reducing equivalent
FAD H FADH2
H2 (redox partner)
N
(glycolysis, Krebs) →
R2 N R2
C C electron transfer
R1 R1
C R4 C R4
R3 N R3 N
X X H
Study Guide 229
E E substrate
binding
H O NH2 H N
6. PLP C C
H O R1, R2 from substrate
Pi CH2 OH Pi CH2
O
R1 C R2 removed; weakens
bonds α – to the Schiff
H N CH3 H N CH3 (e.g., Acetoacetate base with PLP
Decarboxylase)
H H
7. pyruvate takes acetyl group

O derived from glycolysis
pyruvate
and uses it as “fuel” for
CoA S C CH3
the Krebs Cycle
CoA SH CO2 (e.g., the Pyruvate Dehydrogenase
"Bridge Reaction")
O
8. Biotin chemically C
ligand 1. Capture a ligand
your bait, e.g., a DNA
attached
biotin
HN NH whose binding proteins binding protein
HC CH you're interested in
O
isolating (usually) and 2. Capture
H2C CH (CH2)4 C
S the complex and purify
+
it from everything else
by avidin-biotin affinity
cell or nuclear extract,
e.g., a mixture that target chromatography
contains the DNA- protein
binding protein of
interest to isolate
O
biotin C
HN NH
HC CH O
H2C CH (CH2)4 C ligand
S
target
protein
bead Avidin
affinity chromatography column
containing beads bound to the
biotin-capture protein Avidin.
O
C
HN NH
HC CH O
bead Avidin H2 C CH (CH2)4 C
S
9. Tetra- (a) Contributes a methyl to group form dTMP in biosynthesis

hydrofolate from dUMP.
(THF)
The THF analog methotrexate inhibits Dihydrofolate
Reductase, which makes the N5,N10-THF for Thymidylate
Synthetase.
(b) This allows preferential killing of cells that are rapidly

incorporating dT into DNA. Cancer grows more rapidly than
most “normal” cells, results in preferred cancer cell destruction.
230 Study Guide
10. (a) (a) ketose aldose

ketose H O
versus CH2OH C
aldose C O H C OH
(b) CH2OH CH2OH

pyranose dihydroxyacetone D-glyceraldehyde
(5 C’s)
(b)
versus pyranose 5 C's & 1- O in ring furanose 4 C's & 1-O in ring
furanose (4 HO CH2
C’s) HO CH2 OH
O O
OH
HO OH
OH OH
OH
α -D-glucose β-D-ribose
11. UDP- cosubstrate; activates galactose for di- or oligosaccharide

galactose synthesis
& lactose HOCH2

O
CH2OH glucose β
HOCH2 OH
HO O OH
O O O
OH H HO
OH
O PP OH
i
HO Uridine
OH
UDP-galactose lactose
12. Retinal visual

photon
H H
R1 R1 R2
H chemical signal
absorbance,
R2 photoisomerization H
trans-retinal
cis-retinal
receptor linked
to optic nerve
visual sensation
Study Guide 231
Review Session for Chapters 11.5—12.5
1. (a) Draw the chair and boat conformations of the β-D-glucopyranose structure.
(b) Explain why a polysaccharide chain would turn directions much more in one case than in the other
and which conformational isomer would be expected to produce the most radical change in chain
direction.
2. (a) Why is the hexa-atomic ring of inositol more stable than that of galactose?
(b) Given what you know about the biosynthesis of lactose from UDP-galactose and glucose, why
would you expect inositol to be less likely to polymerize than glucose or galactose? (Hint: What are
their relative capabilities in serving as nucleophilic centers?)
3. (a) What does NAG-α (1→6)-gal-α (1→4)-glc-β (1→4)-gal mean?
(b) Draw the molecule with emphasis on the meaning of α and β.
4. (a) How does glycogen differ from the amylopectin in starch?
(b) How would this facilitate the function of glycogen?
5. How does penicillin work selectively on bacteria but not harm us significantly? (What does it do?)
6. (a) How do extra-cellular surface carbohydrates regulate osmotic pressure around cells?
(b) What advantage does this impart to cells?
7. Explain how the terms “export” and “clearance” apply to the functional status of carbohydrate-bearing
proteins.
8. (a) How does Phospholipase C produce two different second messengers in the corresponding signal
transduction pathway?
(b) Draw an appropriate reactant.
9. What function does chondroitin sulfate serve in cartilage and skeletal joints?
10. (a) Assuming equal length, do saturated or unsaturated fatty acids have a lower melting temperature
(Tm)?
(b) What is the nature of the reaction/equilibrium one measures? What molecular property causes the
differences in Tm values?
11. How do lipid bilayers and micelles differ? Include a simple drawing.
12. (a) Draw the general structure of a phosphatidyl choline molecule.
(b) Name the functional groups.

232 Study Guide
Review Session for Chapters 11.5—12.5: Key

1. (a) Sugar pucker conformations:
H OH
HO OH OH
CH2 H
HO CH2 H
HO
HO O
H HO HO
OH H OH
H
H
chair boat
conformation conformation
(b) The boat C1 and C4 hydroxyls produce a kink in the polysaccharide that is more pronounced than in
the chair polymer.
extended
O chain O
O
O O O
O
O turn
O
O
axis
O O
O
O
chair boat
conformation conformation
2 (a) Galactose forms the pyranose via acetal – H+ catalysis; a reversible reaction. As a result the ring is
easily hydrolyzed to form the linear form. In contrast, the cyclohexane ring of inositol must be oxidized by
a more powerful oxidant than 10–7M OH-/H+ solution. The six OHs on inositol provide plenty of routes to
ring opening, but it’s harder to do than with galactose.
CH2OH OH OH no ring oxygen
O
HO OH OH
OH OH
HO
OH OH
β-D-galactose inositol
(b) Eliminating UDP by nucleophilic attack at C1 is more likely for galactose than with inositol because
there is more electron withdrawl by the ring oxygen, which is present in galactose but not in inositol.
Much weaker
nucleophilic center
H
HO glucose
H H HO OH
O O 2- relatively
- Pi
HO H Pi unreactive
H H H H O
δ+
OH δδ+ Urd
H H OH
O - HO H H
Pi 2-
Pi H
H HO much less O
leaves H HO reactive
Urd glucose
galactose inositol
(protected phosphate)
Study Guide 233
3 (a) α (1→6) refers to the linkage between C1 of the first residue and C6 of the second residue, with the
linked O pointing down from the 1st to second residue. β (1→4) refers to pointing up at C1.
(b) The molecule
. NAG - α(1 6) - gal - α(1 4) - glc - β (1 4) - gal is:

Key:
NAG N-acetylglucosamine
H gal galactose
HO H glc glucose
O
H H H The α and β linkages are shown.
OH H αO H H
HO HO H
H H HO
H
N-acetyl- H NH O O β O
HO H H O H OH
glucosamine C H H H
H3C O OH H α or β
H α O OH H
HH OH H not specified
OH H
galactose H HO H HO
glucose H HO
galactose
4 (a) Glycogen contains more α (1→6) linkages and on a percentage basis fewer α (1→4) linkages than
amylopectin. It is more branched.
(b) Glycogen functions to store glucose units in the liver that can be mobilized during glycolysis as fuel
when needed. A higher branching number results in a higher storage density, resulting in more efficient
storage, allowing simultaneous liberation of terminal glucoses from more branches.
5. Penicillin inhibits linkage of the second peptide in the peptidyl-glycan cell coat cross-linkage of the
bacterial cell wall. Human cell membranes are constructed of cytoskeletal elements that differ significantly
from those of bacteria. As a result, the bacteria are killed while human cells remain relatively unaffected.
6 (a) The osmotic pressure of the concentrated poly-glucose polymers drives them to absorb water from the
bulk bathing extra-cellular fluids. Water tries to dilute the carbohydrates, which cannot be diluted because
they are interconnected and connected to the extracellular surface. These carbohydrates thereby both wet
the cell and inflate it. This helps cell-cell adhesion and fluid behavior, making them communal and
wanderers with the same forces: hydrogen bonding and osmotic pressure.
(b) Protection, polyfunctionality, recognition of “self” and “non-self” entities, sequestration, and so on.
7. CHOs function to tag and chaperone proteins in cells, between cells and in circulating cells. This
amounts to export. For example, antibodies are CHO tagged. The CHO also determines the “age” of a
protein. When the CHO is shortened (but still present), it targets certain proteins for destruction and
clearance.
8. (a) Phospholipase C (PLC) functions in the phospholipids – IP3 signal transduction pathway by
hydrolyzing the IP3-DAG connection. Each component is a second messenger, IP3 activates Ca2+ influx
(indirectly activating Ca2+ sensitive protein kinase DAG directly activates PKC).
234 Study Guide
8 (b)
O
14 11 8 5 C O CH2 H2O
C O CH IP3
DAG + IP3
DAG O H2C Pi2- 5
H
Pi1- Phospholipase C
OH H
HO HO 4
P 2- (two biologically
H i
H H active products)
phospatidyl inositol triphosphate

(reactant)
9. Chondroitin sulfate is one of several glycosaminoglycans that forms a core of the cushion material in
cartilage, bone and cornea. These materials absorb water and buffer against shocks, bumps, and so on.
10 (a) Unsaturated fatty acids; double bonds disorder the interaction stacking/aggregation tendencies and
thereby reduce their stabilities in liquid crystal formation.
(b) One follows “melting” from the liquid crystal to the disordered (dissolved) emulsion (solution). At
lower temperatures, chains form ordered lattices; at higher temperatures, the hydrophobic effect is
overcome (water is released from the surrounding solved) and these lattices melt.
11.
head
groups
+/- +/- +/-
+/-
+/-
hydrocarbon
chains
micelle bilayer vesicle
A bilayer has two layers, back to back, of hydrocarbon chains in the center of a sandwich with outer layers
composed of the polar headgroups of the phosphatidyl-X lipid molecules.
The micelle is a single droplet of phospholipid molecules with the solvent-exposed surface composed of the
polar headgroups.
12 (a) and (b)

O
C O CH2
C O CH O CH3
+
O H2C O P O CH2 CH2 N CH3
O CH3
fatty acids
phosphate choline
glycerol
Study Guide 235
1. Explain five details that describe the fluid mosaic membrane model.
2. (a) Draw and label the structures of the four nucleic acid bases in DNA.
(b) Label the bases drawn in part (a).
3. (a) Draw and label a RNA trinucleotide using three different bases.
(b) Label the bases and other structural subunits drawn in part a.
4. Draw the structures of the two Watson-Crick base pairs. Scientists typically use the symbol ••• to
designate hydrogen bonds.
5. Define a glycosidic bond in a nucleoside.

236 Study Guide
Review Session for Chapters 13—14.7: Key
1. The “fluid mosaic” model includes the following details:

(a) It is supported by and assembled within/around the lipid bilayer. The mixed composition “liquid
crystal” is interrupted by integral membrane proteins, cholesterol, lipids, phospholipids, terpenes,
glycosphingolipids, and so forth.
(b) Peripheral membrane proteins are not covalently bound, they are often “anchored” to phospholipid
anchor
fragments.
(c) Integral membrane proteins – embedded in the lipid bilayer with one or both faces exposed (2
different exposures, inside & out). for example, 7-helix transmembrane motif.
(d) The lipid-anchored proteins are connected by covalent “tails” of
(i) fatty acids (myristoyl, etc.),
(ii) polyprenes (farnesyl, squalyl), or
(iii) glycosphingolipids.
(e) Oligosaccharide tree-like structures are connected to proteins, glycosphingolipids, proteoglycans,
and so forth.
2. The Watson-Crick base structures are:
NH2 NH2
4 6
H Cytosine 5 N7
N
3 5
(Cyt) 1N H
Adenine
1 8 (Ade)
O 2 N 6 H H 2 N 4 N9
3
H H
O O
H 4 H 6 5 N
Uracil (in RNA) 7 Guanine
H 1N
3N 5 (Ura) H
8 (Gua)
1
6 H H2N 2 N 4 N9
O 2 N 3
H
H
O
H 4 CH3
3N Thymine (in DNA)
5
1 (Thy)
O 2 N 6 H
Adenine (Ade) base pairs with Thymine (Thy) in DNA and Uracil (Ura) in RNA.
Cytosine (Cyt) base pairs with Guanine (Gua).

Study Guide 237
3. The following figure shows the details of an RNA trinucleotide:
5' terminus
H adenosine-3', 5'-diphosphate
H
O H N N H
O P O H
H N N
O
O H H N
H H
H H H cytosine-3', 5'-diphosphate
O OH N
H H
O P O H
H N
O N
O
H H O
H H
O OH
O P O
O
H N guanosine-5'-monophosphate
phosphodiester O H
H
phosphate O N N H
H H N
H H N H
ribose O OH
H
H
3' terminus
4. The Watson-Crick base pairs are:
A U (or T)
δ -
(a)
H O H CH3 in DNA A U
N H+
H N δ or
δ+
δ- H N dA dT
H
N N
R1 O N
H N δ- O O
R2
H
H H R3
H R4
OH glycosidic HO
bonds
H in DNA
G C
H
(b)
H +N H G C
O
H N δ
δ- or
N H dG dC
N N H+
R1 O δ δ- N
N O
R2 O R3
H N H+ H
δ δ- R4
OH H H
HO
glycosidic
H in DNA bonds
238 Study Guide
Review Session for Chapters 16.2—17.6
1. Explain how and why the absorbance at 260 nm (A260) can be used to determine if a double helix forms
from 2 single strands of DNA or RNA.
2. (a) Define “base stacking.”
(b) Describe the three predominant types of forces that contribute to stabilization of stacked bases in a
double helix.
3. (a) What are counterions and why do they bind all nucleic acids?
(b) How do histones serve this function in the case of most chromosomal DNAs?
4. Give two reasons why G•C base pairs are more stable than A•T (or A•U) base pairs. Rank these
contributions according to importance in inducing stability.
5. Describe four differences between A and B forms of DNA.
6. (a) Draw the mechanism of alkaline hydrolysis of RNA.
(b) Why is DNA not subject to this mechanism?
7. Why and how does an antisense oligonucleotide functionally inactivate a mRNA for use in translation by
a ribosome?
8. Name the four classes of RNA and briefly explain their functional significance. In what reaction(s) do
they participate?
9. List four distinctive features of most eukaryotic mRNAs.
10. What is the primary use of a DNA probe and how is this process accomplished?
11. Why are restriction endonucleases required to produce, manipulate and clone specific pieces of DNA?
12. What are the two functional ends of transfer RNA, what are their functions and how do they accomplish
them?
Study Guide 239
Review Session for Chapters 16.2—17.6: Key
1. When two strands of DNA anneal to form a double helix, their absorbance at 260 nm decreases relative
to the sum of A260 of the individual strands. This hypochromicity is used to determine the extent of duplex
formation.
2. (a) Bases “stack” on each other, face to face, like coins in a roll. Base-pair stacking is the primary
stabilizing force maintaining the double helical structure.
(b) Forces listed according to relative contributions to stabilization are:

(1) the hydrophobic effect – in which water encages the bases at low T.
(2) London dispersion forces – transient dipole-dipole attractions that involve the π electrons.
(3) hydrogen bond formation – which helps reinforce the stacked geometry.
3. (a) Nucleic acids have one electronegative phosphodiester phosphate, of which 0.1 unit of negative
charge is present per nucleotide. Ninety percent of the negative charge is neutralized by the cation charges,
which achieves near electroneutrality, and thereby maintain thermodynamic stability.
(b) Histones are rich in the electropositive (basic) amino acid residues lysine and arginine. These
positive charges neutralize the negative charge of chromosomal DNA, which allows the strands to
become condensed and packaged within the chromosome.
4. The bases are ranked according to hydrophobicity as follows: G > A > C > T (or U), so G-C is more
hydrophobic than A-T (or A-U). The hydrophobic effect is the predominant stabilizing force in base
pairing. In addition, G-C base pairs have 3 hydrogen bonds while A-T (or A-U) only have 2. Due to more
extensive electronic overlap, London dispersion forces and stacking are also more stabilizing for G-C
versus A-T or A-U.
5. A-DNA versus B-DNA:
A-DNA B-DNA
(a) C3’ endo sugar conformation (a) C2’ endo sugar conformation
(b) base pairs tilted relative to a plane located (b) base pairs are approximately perpendicular to
perpendicular to the helical axis the helix axis
(c) base pairs are splayed away from center of (c) base pairs cross the center of the helix since no
helix, leaving a central axial cavity down the central axial cavity is present
center
(d) slightly shorter and wider (squatter) helix than (d) slightly longer and narrower than A-form
B-form
6.
Base 1 R1 Base 1
R1 O O
OH
OH
H 2O
O O O O
H
δ + P
O P O
O O
O Base 2
O H
HO Base 2
H O
O OH
O OH
R2
R2
Hydroxide abstracts the 2’ hydroxyl hydrogen, producing a 2’ hydroxylate nucleophile, which then attacks
the adjacent 3’ phosphate to produce the 2’, 3’ cyclic phosphodiester structure. As a result, the attached 5’
hydroxyl oxygen of the attached 3’-terminal nucleotide is eliminated, resulting in strand cleavage.
240 Study Guide
(b) DNA does not have a 2’ hydroxyl group, so it cannot form the interal nucleophile that attacks the 3’
phosphate group.
7. An antisense oligonucleotide forms a double helix with the mRNA, preventing it from being usable (in
single-stranded form) in the decoding process of translation (protein synthesis).
8. RNA class Functional Importance

(a) ribosomal RNA (rRNA) rRNA is the predominant structural and catalytic
component of ribosomes
(b) transfer RNA (tRNA) tRNA plays a key role in decoding the mRNA by
encoding the placement a specific amino acid in a
defined location in a synthesized protein.
(c) messenger RNA (mRNA) mRNA is produced by transcribing the permanently
encoded chromosomal DNA. It carries the
trinucleotide-based message to the ribosome, where
it is decoded during translation, resulting in
insertion of a defined amino acid into a protein.
(d) “small” RNA (snRNA, snoRNA, gRNA, siRNA, These species serve a number of roles, usually
miRNA) involved in catalysis and guiding processes required
for RNA maturation.
9. The 5’-terminal m7G+ cap is linked by a 5’ to 5’ bond to the 5’end of the mRNA. The poly (A) “tail” is at
the 3’ end. These mRNAs often contain introns, which are not usually intended to encode amino acid
sequences, and are removed by splicing. If only one protein product results from the mRNA, it is said to be
monocistronic (1 gene/message per mRNA).
Eukaryotic mRNA structure:

m7G
intervening sequence " 3'-NTR "
O CH3 (non-coding)
H 5' to 5' triphosphate
N + linkage
N 3'
H
N 5' exon (coding) exon OH
H2N N
H
O
H
O
P
O
O
5'
O
Base 2 intron(s) ... AAA(A)
20-200
O
3
O O CH3 O O (H, CH3)
H H Coding-"reading frame"
base 3
The 5' end is really a

3' end. This prevents
5' exonuclease action. poly(A)n tail
(not universal, e.g., histone
mRNAs contain a WC
hairpin.)
" 5'-NTR "
(non-translated region)
cap (a) 5’-terminal m7G cap structure 5’ to 5’ linked to 2nd nucleoside
1 gene per mRNA (b) generally monocistronic. Some intervening sequences contain a separate encoded
gene.
introns (c) genes in parts. Introns are removed from mRNAs by the splicing process,
exons catalyzed by the spliceosome, a snRNAs/protein complex.
poly (A)n (d) The poly (A) “tail” is added enzymatically and helps determine the lifetime of
a given mRNA. This process is analogous to that of punching a train ticket.
Study Guide 241
10. A labeled probe DNA is used to detect the presence of a specific complementary nucleic acid sequence
by annealing/hybridization to form a (radioactively or otherwise) labeled duplex.
11. Restriction enzymes allow one to cut out and classify DNA fragments with specific terminal sequences,
then splice them specifically into plasmids/vectors for designed uses.
12. The anticodon forms base pairs with the triplet codon of a mRNA during the decoding process of
translation.
The acceptor end is composed of the 2’ or 3’ hydroxyl of the 3’ terminal ribose of the tRNA. The amino
acid is esterified to the carboxyl group of a (cognate) amino acid, which is then transferred into a growing
protein chain during translation.
242 Study Guide
1. (a) Name and briefly describe the purpose (point) of the three most central catabolic pathways of
intermediary metabolism. (The third has two coupled parts. Name both of them.)
(b) What is (are) the product(s) of each of these pathways? (list according to pathway)
2. Describe the two major ways energy is captured in a chemically usable form by metabolic pathways.
3. The following imaginary reaction from wizard Merlin’s notebooks occurred with a standard free energy
(ΔG°′) of -8.5 kJ per mol:
carborandum + gold ↔ essence of life
(a) Assume that the poise of the equilibrium does not change (which is often referred to as being “at
equilibrium”). A sample contains 44 mM carborandum, 0.1 mM gold and 45 µM essence of life. What is
the actual free energy (ΔG) of the reaction under these equilibrium conditions at room temperature (298
K)? Show your work.
(b) A different sample contained 0.9 M carborandum, 0.1 mM gold and 45 µM essence of life. What is ΔG?
(c) How did adding more carborandum affect the equilibrium poise of the reaction? What was the mass
action ratio (Q) in each case?
4. (a) Why do only three steps in glycolysis control most of the flux through the pathway under actual
cellular conditions?
(b) What are the three steps, the three enzymes that catalyze the reactions? What do the reactions have in
common?
5. (a) Define the concept ‘metabolically irreversible.’
(b) Define concept ‘near equilibrium.’
6. Explain why the kinetics of an enzyme reaction are most easily controlled when KM is approximately
equal to the concentration of the reactant.
7. Consider the following data (where E is the standard state reduction potential):
Reaction E°′
Acetyl CoA + CO2 + H + 2 e-
+
→ Pyruvate + CoA-SH -0.48
+
NAD + 2 H+ + 2 e - → NADH + H+ -0.32
+ -
FAD +2H +2e → FADH2 -0.22
(a) Under standard state conditions (T = 298 K; all component concentrations = 1 M), will it require
more energy if the breakdown of pyruvate to acetyl CoA is coupled to FAD formation or to NAD+
formation? Show work.
(b) Does the answer change if pyruvate is present at 1 mM, while the other species are still present at 1 M?
Study Guide 243
8. (a) What are the reactant and products of the reaction catalyzed by Triose Phosphate Isomerase?
(b) What do the products get converted to next? Explain the reason this occurs.
9. (a) Why should citrate negatively regulate (discourage) the Phosphofructokinase-1 reaction? What is the
general name for this phenomenon?
(b) Why should fructose-1, 6-bisphosphate stimulate the pyruvate kinase reaction? What is the general
name for this phenomenon?
244 Study Guide
Review Session for Chapters 19—20: Key
1. (a) i. Glycolysis: conversion of glucose (“glc”) to 2. pyruvate, then to two acetyl CoA
ii. Krebs Cycle: fixing OAA to acetate, makes citrate, which fuels the Krebs Cycle
iii. electron transport/oxidative phosphorylation – conversion of “reducing power” (NADH, FADH2,
CoQH2) to water, via the electron transport chain (cytochromes, CoQ) and using O2 as the terminal
electron acceptor.
(b) i. Glycolysis and the Bridge Reaction: 2 ATP per glc, 4 NADH (+2 acetyl CoA) = 12 ATP
equivalents
ii. Krebs Cycle: CoA-SH, 3 NADH + CoQH2 + GTP + 2 CO2 + 2 H+ = 20 ATP equivalents
iii. oxidative phosphorylation: NAD+, fumarate, H2O [½ (XH2 → O2)], proton gradient
2. Nucleoside triphosphates: X + Pi ↔ X-Pi (e.g., X + ATP ↔ X-Pi + ADP)

Energy is used in the course of the bond formation.
Phosphorylation of a substrate energizes it in preparation for subsequent reactions, in which

dephosphorylation of that substrate drives product formation.
Reducing power of reduced coenzymes is used to reduce a compound, creating the energized species ½
YH2. That substrate is subsequently oxidized, which drives that reaction, analogous to the role played by
dephosphorylation above.
Also, energy is accrued as the reduced material ½ YH2, that is, NADH or CoQH2. They are used in electron
transport to produce the proton gradient between the mitochondrial inner membrane space and the matrix.
This gradient drives the protonmotive force, which powers ATP formation by the F0F1 ATPase complex.
2 H+, 2 e
Y YH2
2 H+, 2 e
3. (a) Using the equation from Chapter 20, ∆Greaction = ∆Go’ + RT ln Q
= ∆Go’ + RT ln {[essence of life]/[carborandum][gold]}
Using RT = 2477.9 J/mol:
∆Greaction = -8.5 kJ/mol + (8.315 J mol-1 K-1 )(298 K) ln [(45 x 10–6 M)/(44 x 10–3 M)(0.1 x 10–3 M)]
= -2.74 kJ/mol ↨
The value of Q = 10.22
(b) ∆Greaction = -8.5 kJ/mol + RT ln [(45 x 10–6 M)/(0.9 M)(0.1 x 10–3 M)]
= -10.22 kJ/mol ↨
The value of Q = 0.5
(c) Adding carborandum decreases the partition coefficient (Q), meaning the reaction has a much higher
likelihood of occurring; this is reflected in the much more negative ∆G value, (-10.2 << -2.7) kJ/mole.
4. (a) Steps catalyzed by hexokinase, phosphofructokinase-1 and pyruvate kinase are “metabolically
irreversible,” with ∆G values of -33, -22 and -17 kJ/mol, while the other 7 reactions are “near equilibrium,”
with ∆Gs of ~0.
(b) The 3 steps are:
Reactions Coupled Reactions
(i) glucose → G6P (enzyme: Hexokinase) ATP → ADP, H+
(ii) fructose-6-Pi → fructose-1, 6 (Pi )2 (enzyme: PFK-1) ATP → ADP, H+
(iii) PEP → pyruvate (enzyme: Pyruvate Kinase) ADP, H+ → ATP
All three enzymes are kinases; note that reaction (iii) is phosphorylation of ADP by PEP, while the
other two are sugar phosphorylation reactions in which the phosphate contributor is ATP.
Study Guide 245
5. (a) Having a large negative ∆G; highly spontaneous (thermodynamically) (and therefore kinetically
predisposed to product formation, too).
(b) Having a ∆G approximately equal to zero; flux is approximately the same in both directions (i.e., if the
[reactant] is equal to the [product], the Keq is close to or equal to 1).
6. When KM is approximately equal to substrate concentration, the reaction is occurring at a rate of ½ Vmax.
The rate is not maxed out, yet is occurring at a rate >> 0. As a result both negative and positive regulation
are possible within a relatively small range of [S] changes.
Consider what happens if the concentration is either 100 times KM or 1/100 of KM.
7. (a) The net reactions, constructed by combining the pyruvate (pyr) oxidation reaction with either NAD+,
H+, 2 electron reduction or FAD, 2 H+, 2 electron reduction are:
(i) pyr + CoA-SH + NAD+ + 2 H+ + 2 e- ↔ Acetyl CoA + CO2 + H2 + 2 e- + NADH
net: pyr + CoA-SH + NAD+ ↔ Acetyl CoA + CO2 + NADH

(Ared) (Box) (Aox) (Bred)
∆Gº′ = -n F ∆Eº′ = -n F [(-0.32) – (-0.48)]V = -n F (0.48–0.32) V

= -(2)(96.48 kJ mol-1 V-1)(0.16 V)
= -30.87 kJ/mol (Q = 1) ; (n = 2)
(ii) pyr + CoA-SH + FAD + 2 H+ + 2 e- ↔ Acetyl CoA + CO2 + H2 + 2 e- + FADH2
net: pyr + CoA-SH + FAD + 2 H+ ↔ Acetyl CoA + CO2 + FADH2

(Ared) (Box) (Aox) (Bred)
∆Gº’ = -n F ∆Eº′ = -n F [(-0.22) V – (-0.48) V] = -(192.96)(0.26V) = -50.17 kJ/mol (Q = 1); (n = 2)

This situation is more spontaneous than (a) (i).
(b) (i) ∆E = ∆Eº′ – (0.026/n) ln { [Aox][Bred]/[Ared][Box] } (Q = 1000); (n = 2)

= 0.07 V = {(0.16 V) – [0.09 V]} = [(1 M)(1 M)/(0.001 M)(1 M)]
∆Gº’ = -n F ∆Eº′ = -13.50 kJ/mol (Q = 1000)
(ii) ∆E = ∆Eº′ – (0.026/n) ln {[Aox][Bred]/[Ared][Box]} (Q = 1000); (n = 2)

= 0.17 V = {(0.26 V) – [0.09 V]} = [(1 M)(1 M)/(0.001 M)(1 M)]
∆Gº’ = -n F ∆Eº′ = -32.80 kJ/mol (Q = 1000) This situation is more spontaneous than (b) (i).
Summary of the results:

Calculation Reaction Q ∆Eº’ ∆E ∆Gº’ ∆G
(a) (i) pyr, CoA, NAD+, 2 H+, 1 0.16 V -30.87 kJ/mol
2 e- passed
(b) (i) pyr, CoA, NAD+, 2 H+, 1000 0.07 V Compare ↓↑ -13.50
2 e- passed kJ/mol
(a) (ii) Pyr, CoA, FAD, 2 H+, 1 0.26 V -50.17 kJ/mol Compare ↓↑
2 e- passed
(b)( ii) Pyr, CoA, FAD, 2 H+, 1000 0.17 V -32.80
2 e- passed kJ/mol
The two conclusions are:

(1) The FAD reaction remained more spontaneous than the NAD+ reaction under both [pyruvate]
conditions.
(2) Lowering the [pyruvate] from 1 M to 1 mM made the reaction less spontaneous with both NAD+ and
FAD.
246 Study Guide
8. Fructose-1, 6-bisphosphate is dissociated to form dihydroxyacetone phosphate (DHAP) and

glyceraldehyde-3-phosphate (G3P) by aldolase (rxn 4). DHAP is converted to G3P by triose phosphate
isomerase (rxn 5). Finally, G3P is converted to 1, 3-bisphosphoglycerate by G3P dehydrogenase (rxn 6).
2
O H O O Pi
2
CH2 O Pi C [Rxn 6] C
2 [Rxn 5]
H
H OH OH
PiO C O H C H C OH
O
2 2
H HO 2 CH2OH CH2 Pi
O CH2 O Pi
H CH2 O Pi
OH H Dihydroxyacetone Glyceraldehyde-3- 1, 3-bisphosphoglycerate
[Rxn 4] phosphate phosphate
Fructose-1, 6-
bisphosphate
9. (a) The negative regulation of PFK-1 by citrate is an example of (negative) feedback inhibition of
glycolysis by a key Krebs Cycle intermediate. Glycolysis is purposefully turned off when plenty of citrate
is already present.
(b) Stimulation of pyruvate kinase by fructose-1, 6-bisphosphate is an example of feed-forward stimulation.

The presence of too much fructose-1, 6 (Pi)2 indicates that it is accumulating, so more of the later reaction
should occur in order to use the accumulated intermediate.
Study Guide 247
1. We discussed three branching catabolic fates of pyruvate. Draw the three reaction (reaction sequences),
including cofactors and enzymes.
2. (a) Why does the absence of alcohol dehydrogenase produce even more scurrilous behavior than if the
person has the enzyme?
(b) How is the blocked catabolic intermediate related to the typical role of pyridoxal phosphate in
enzymatic catalysis?
3. (a) Why does “carbonation” accompany ethanol production?
(b) How does the staff of life of the western world benefit (and us as a consequence)? [Bread, not beer!]
4. Dihydrolipoamide Acetyl Transferase uses a coenzyme not previously described. (a) What is it and how
does it function in fueling the Krebs Cycle?
(b) What other coenzyme is also involved in this process? How?
5. (a) What “symport” reaction accompanies import of pyruvate into the mitochondrion and what enzyme
catalyzes the reaction?
(b) Does the pH of the cytoplasm increase or decrease as a result?
6. List the reactions, coenzyme(s), cofactor(s) and enzymes involved in the two “oxidative
decarboxylation” reactions of the Krebs Cycle.
7. List the reactions, coenzyme(s), cofactor(s) and enzymes involved in the “substrate-level
phosphorylation” reaction of the Krebs Cycle.
8. List (only once each) all of the “energy conserving” compounds formed by the Krebs Cycle
accompanied by the number of “ATP equivalents” finally accrued after oxidative phosphorylation of 1
molecule of each of these compounds.
9. (a) How do fumarase and malate dehydrogenase “fix” a carbonyl group on succinate in the production of
oxaloacetate (OAA).
(b) What crucial 2 carbon compound is then “fixed” to OAA?
(c) How is the product used in food flavoring?
10. What amino acid and what product of pyruvate metabolism are the principle substrates for
gluconeogenesis in mammals?
11. What is protonmotive force and what enzyme complex uses this phenomenon as the driving energy for
ATP synthesis in oxidative phosphorylation? (Contrast it with electromotive force, i.e., emf, voltage.)
248 Study Guide
12. How does electron transport drive production of the protonmotive force?
Study Guide 249
Review Session for Chapters 21—25: Key
1. Three fates of pyruvate:
I
CoA SH CO2
O O NAD+ NADH, H+ CoA
C S
C O C O
Pyruvate
CH3 Dehydrogenase
complex CH3
pyruvate acetyl CoA Krebs cycle,
Fatty Acid
NADH, H+ synthesis
Lactate
CO2
Dehydrogenase
NAD+ (see # 3)
NADH, H+
NAD+ II
III
H O
O C H2 C OH
O
C
Pyruvate Alcohol CH3
CH3
Decarboxylase Dehydrogenase
HO C H ethanol
acetaldehyde
CH3
(baking,
lactate brewing)
(cheese, yogurt) (preservation, brewing!!)
(hallucinations, violence?)
2. (a) The intermediate between pyruvate and ethanol, acetaldehyde, is not broken down effectively by
people who are ADH-deficient. As a result, they form acetaldehyde as the ethanol accumulates via the
reversible ADH reaction. Accumulated aldehyde-containing acetaldehyde reacts to form Schiff base
compound with amine-containing compounds. These compounds have the problematical pharmacological
properties that can induce drunken behavior. As a result, when ADH-deficient people consume ethanol they
are much more vulnerable to the effect.
(b) Recall the way in which a pyridoxal phosphate in Aspartate Transaminase is neutralized by Schiff base
formation with an active site lysine.
3. In ethanol fermentation, CO2 is evolved by pyruvate decarboxylase as acetaldehyde is made (see Answer
1). The released CO2 produced by the activated yeast culture is captured by the moistened wheat (etc.)
fibers of the dough.
4. (a) Dihydrolipoamide is a lipid-based hydrocarbon that is covalently attached to the pyruvate

dehydrogenase E2 subunit (PD-E2). It accepts ethanol from HETPP to form a thioacetate
H H
H SH
H
S C CH3
E2 H
O
then transfers the acetyl group to CoA-SH. In pyruvate dehydrogenase (PD), CO2 is evolved as HETPP
(hydroxyethylthiamine pyrophosphate) is formed from (1) pyruvate and TPP (thiamine PPi). This is one of
three cycles that couples to the dihydrolipoamide acetyltransferase (DLAAT) cycle.
The next steps involve transfer of HETPP-bound (2) acetate to (3) lipoic acid then transfer to CoA, making
(4) acetyl CoA. The cofactors, co substrates, H+, and so forth are omitted.
250 Study Guide
Glycolysis
[PD-E2] 3 CoA SH
(DLAAT) E2-lip- acetate
4
1 TPP
pyr S C CH3 acetyl CoA
[PD-E1] E2-lip
S O
H
HETPP SH
S E2-lip
CO2 (g) TPP E2-lip SH Kreb's
2 S Cycle
(evolved as gas) HO C CH3
One
H step
Indigestion, a medically significant problem and money-maker for pharmaceutical companies.
(b) Thiamine pyrophosphate (TPP) (on PD-E1) accepts ethanol and transfers it to the lipoic acid (on PD-
E2). Five coenzymes are used: lipoamide (amide), thiamine pyrophosphate, CoA-SH, FAD and NAD+.
5. (a) Pyruvate translocase imports 1 H+ and pyruvate simultaneous (in the same direction). This coordinate
translocation mechanisms defines a “symport”.
A
B
An “antiport” exchanges two species coordinately in opposite directions:
A
B
(b) Since 1 H+ leaves the cytoplasm for each translocase reaction, the cytoplasm becomes less acidic and
the pH increases;–log [H+] is larger.
Study Guide 251
6 and 7.
CoA - SH,
NADH, H+ NADH, H+
COO NAD+ NAD+ COO
COO
CO2 CO2
CH2 CH2 CH2
H C CO2 is lost
COO CH2 CH2 from C3 and
HO CH α-KG C O C4 in two
isocitrate C O
dehydrogenase successive
COO dehydrogenase S CoA
COO steps.
isocitrate succinyl CoA
(C6) α-ketoglutarate (C4)
(αKG)
(C5)
COO GDP (or ADP)

COO
Pi
CH2 CH2 CoA SH is lost from
CH2 succinyl CoA, accompanied
CH2 by GTP (or ATP) synthesis.
C O succinyl CoA COO
S CoA synthetase CoA SH succinate
succinyl CoA
GTP or ATP
CO2 is lost from C3 and C4 in two successive steps.
8.
Energy source ATP equivalents/molecule
NADH 2.5
GTP (or ATP) 1
CoQH2 1.5
9.
NADH,
CoQ CoQH2 COO H+ COO
COO COO H2O NAD+
CH2 HO CH C O
H C
CH2 FAD CH2 CH2
C H
COO Succinate COO Fumarase COO Malate COO
Dehydrogenase malate Dehydrogenase oxaloacetate
succinate fumarate
The O is derived from water which is fixed after C=C formation and oxidized from R-OH. For the role of
FAD in the CoQ → CoQH2 process, see the Review Session for Chapter 27, question 3.
(b) Acetate is “fixed” to OAA, producing citrate.
(c) Citrate is a common flavoring as a result of its acidic tartness. Its acidity and redox properties lead to
food processing events that change the consistency and preservation of foods.
10. Alanine and lactate are the principle gluconeogenic substrates. Compare pyruvate and alanine to
determine how one is made from the other. (Hint. How are oxaloacetate and glutamate interconverted?)
252 Study Guide
11. Oxidative loss of reducing equivalents due to a proton gradient across a membrane (of the
mitochondrion).
XH2 + ½ O2 + 2 e- + 2 H+ ↔ H2O + 2 e- + 2 H+ + X
The electrons are the connecting (driving) “wire.”
Oxidative phosphorylation is driven by a proton gradient, which is produced by the active transport of
protons from the mitochondrial matrix to the inner membrane space, through the mitochondrial inner
membrane.
CYTOPLASM
H
INNER MEMBRANE
SPACE
2e INNER MEMBRANE
ATP
synthase
H2O MATRIX
H
1/2 O2 ADP ATP
+ + +
2H Pi H2O
H
This proton movement creates a gradient, operating as an aqueous circuit that connects the electron
transport chain to ATP Synthase. This is similar to the action of a wire in an electrochemical reaction in that
the electrons are passed from the reducing agent, NADH or CoQH2, through the electron transport chain,
and finally to the terminal oxidizing agent O2. This series of coupled redox reactions pass the electrons,
which drives the proton pumps. The transformed free energy is captured by phosphorylation of ADP to
produce ATP.
Viewed another way: (membrane)

The enzyme is
F0F1 ATP Synthase.
X Δ pH 1/2 O2
2 H+
XH2 H 2O
2e 2e driven by e transport
Δψ
(outside) (inside)
Mathematically, there are two contributions to the protonmotive force (∆p), due to the difference in [H+]
inside versus outside, and the change in membrane potential (∆ψ). (F is the Faraday constant)
∆p = ∆G/n F = ∆ψ + (0.059V) ∆pHin-out
12. The electron transport reactions pump H+ out of the mitochondrion matrix and build up the [H+] in the
inner membrane space, which drives ATP synthesis by F0F1 ATPase. Four H+ are pumped per ATP
generated.
Study Guide 253
1. (a) How many reactions does each round of β-oxidation of a fatty acid require?
(b) What are the products of one round of β-oxidation and what’s the tally in terms of ATP equivalents of
energy conserving products?
2. (a) Draw the coupled cofactor regeneration cycles that siphon off reducing equivalents then fix them into
coenzyme Q in reactions that are coupled to the first oxidative step of fatty acid β-oxidation.
(b) Write down the names and a short definition for the cofactors involved in this siphon.
3. Which three steps of the Krebs Cycle do the first three steps of the fatty acid β-oxidation cycle resemble?
Draw (horizontally) the three analogous reactions from (i) β-oxidation, (ii) the Krebs Cycle, and (iii) the
three generic chemical transformations that occur beneath the four analogous substrates.
4. Draw the analogous (but backward) [reduction-dehydration-reduction] series of reactions of fatty acid
synthesis starting from reactants and going to products beneath your three reaction series from problem 3.
Point the arrows in the opposite direction drawn for problem 3.
5. Draw the reactions for the condensation sub-step in Acyl-Carrier Protein (ACP)-mediated fatty acid
synthesis.
Note. In the absence of proper exercise and portion control, this pathway can lead to obesity, a modern
human calamity.
254 Study Guide
Review Session for Chapter 27: Key
1. (a) Four reactions occur during each β-oxidation cycle: (1) oxidation #1, (2) hydration,
(3) oxidation #2, and (4) thiolysis.
(b) Each cycle yields 1 CoQH2, 1 NADH, H+, 1 acetyl CoA and 1 fatty acyl CoA shortened by 2 carbons.
In ATP equivalents: 1 CoQH2 = 1.5 ATP eq.; 1 NADH = 2.5 ATP eq. and 1 acetyl CoA = 10 ATP (i.e.,
because it feeds one round of the Krebs cycle).
2. (a)
fatty acyl CoA Oxidation (Redox regenerating system)
#1
H H H O
R C C C C S CoA 3+ CoQH2
FAD FADH2 Fe S
H H H
Acyl CoA Electron transport
dehydrogenase ETF ETF: ubiquinone
reductase
H H O
R C C C C S CoA 2+ Oxidative Phosphorylation
FADH2 FAD Fe S CoQ
H H
ETF = Electron-transferring flavoprotein
trans- Δ2 enoyl CoA
(b) (i). FAD/FADH2 – oxidized/reduced in the Acyl CoA Dehydrogenase reaction.

(ii). Fe-S 3+/2+ – oxidized/reduced iron-sulfur center in ETF-Ubiquinone Reductase
(iii). CoQ/CoQH2 – oxidized/reduced CoQ; ubiquinone (dione) and ubiquinol (diol).
3.
(FAD)
(i) Krebs COO CoQ CoQH2 COO COO COO
Cycle : H2O NAD+ NADH, H+
CH2 H C HO C H C O
CH2 Succinate C H Fumarase CH2 Malate CH2
Dehydrogenase Dehydrogenase
COO COO COO COO
succinate fumarate malate oxaloacetate
CoQ CoQH2
2+ 3+
Fe S Fe S
(ii) β-oxidation R R R R
(Fatty acid ETF
degradation) CH2 ETF FADH2 H C HO C H +
FAD H2O NAD + NADH, H C O
CH2 C H CH2 CH2
C O Acyl CoA C O Enoyl CoA C O L-3-Hydroxyacyl C O
dehydrogenase hydratase dehydrogenase
S CoA S CoA S CoA S CoA
Study Guide 255
4.
(iv) Fatty acid

biosynthesis : reduction dehydration reduction
CH3 CH3 CH3 CH3

CH2 NADP+ NADPH, H C H2 O HO C H + C
NADP NADPH, H+ O
H+
CH2 C H CH2 CH2
C O Enoyl-ACP C O β HA-ACP C O Ketoacyl ACP C O
Reductase dehydrase Reductase
S ACP S ACP S ACP S ACP
butryl-ACP butenoyl-ACP β-hydroxybutryl-ACP acetoacetyl-ACP
Note : Not a CoQ / CoQH2 reaction.
5. Initial stage of fatty acid biosynthesis:
O
-
O O
O CH2 C S ACP
C
CH3 C S ACP O
acetyl-ACP
(n)
ACP SH
O
KAS S C CH3
δ+
H+
CO2
HS-ketoacyl-ACP
synthase
("KAS")
O O
CH3 C CH2 C S ACP
acetoacetyl-ACP
(n + C2)
256 Study Guide
Practice Exam 1
1. Draw the predominant structures of the following molecules at the pH indicated. Be sure to include all
carbon and hydrogen atoms.
a. (1) urea at pH 12 b. (1) L-aspartate at pH 3.9
Also affected
O O O- O O
-
H C H C
C pKa = 3.9
H N N H H N C H H N C H
H H H H H
C H H C H
(no pKa) C
-O H C
O O O
50% 50%
c. (5) histidyl-argininyl-glycyl-tryptophanyl-methioninyl-tyrosine at pH 7.
H H O H O H O H O H O H
O
H N C C N C C N C C N C C N C C N C C
H HH C H H HH C H H H C H HH C H O
H C H H H
C H C H H C H C H H C H
H C C C
C C
C N H H C H hydrogen C H
S
no pKa C C N C C H
N C H C H C
H H N H H C H
H
C H H O
H H H
Methyl imidazole N N
methyl indole
pKa = 6 ethyl, methyl
H H no pKa thioether methyl phenol
10% protonated n-propyl guanidinium pKa = 10.5
90% deprotonated no pKa
pKa = 12.5
(Note that the upper carboxylate in 1b will also adopt two forms: ~ 95% deprotonated and ~5%
protonated.)
2. (0.25 each) List and name the entire functional group (R) and pKa (if appropriate) under each amino acid
side chain you drew in #1a, 1b and 1c.
(answers shown above)
3. a. (1) What is the charge of the fully protonated molecule in problem #1c? +3
b. (1) Name each ionizable functional group in the deprotonated form.
histidyl: – imidazole
argininyl: – guanidine
cysteinyl: – thiolate
tyrosine: – phenolate
Study Guide 257
c. (4) Draw a titration curve for the oligopeptide in problem #1c. Assume the pKa of the C-terminal
carboxylate is the same as that on a typical protein C-terminus. Label your axes and include a scale and units.
# of H+ 3
removed
2
0
0 2 4 6 8 10 12 14
pH
pKa,1 pKa,3 pKa,4 pKa,5
pKa,2
4. Explain the following terms and explain their use in protein work:
a. (0.75) Gel filtration chromatography:
It is a protein purification technique in which molecules flow through a column containing porous beads,
which separates the proteins due to size differences. Smaller molecules are included (enter) in the beads
and take longer to reemerge (and finally elute from the column) than larger molecules, which don’t enter
the beads (as often).
b. (0.75) Sodium dodecyl sulfate (SDS):
SDS is an amphipathic molecule with a hydrophilic sulfate and hydrophobic C12 hydrocarbon tail. Used in
gel electrophoresis to coat and denature proteins so they electrophorese as rod-shaped particles. Separation
is generally predominantly based on differences in protein chain length because the SDS charges
overwhelm those of the protein.
c. (0.75) BCA assay:
A method derived from the Biuret reaction in which Cu2+ is reduced to Cu1+ in proportion to the amount of
peptide bonds present (protein concentration). A dye is added to the protein that turns deep purple in
proportion to Cu+1 produced, thereby amplifying the signal which is indirectly due to [protein].
d. (0.75) A280:
Absorbance at 280 nm, which is the result of trp, tyr (and to a lesser extent, phe) in proteins. If the number
of these amino acids in the protein is known, one can calculate the molar concentration (c) of protein using
Beer’s law (A= ε c l), where ε is the molar extinction coefficient (which can be looked up) and l is the
cuvette pathlength.
e. (0.75) Chymotrypsin:
A protease that cleaves the chain, at peptide bonds adjacent to aromatic side chains, producing free
carboxyl-termini.
258 Study Guide
f. (0.75) Ramachandran plot:
A plot of the two peptide torsion angles of φ (phi, pronounced “fy”) and Ψ (psi, pronounced “sigh”) as the
two axes. A point on the plot corresponds to 1 type of conformation since the peptide bond is constrained to
planarity. Each of the 2º structures falls in a unique part of the plot. This characterizes the structure of the
molecule.
5. (3) Explain why a peptide bond has partial double and partial single bond character. Give the structures
of the two contributing forms.
The carboxyl group and peptide bond undergo enol ↔ keto tautomerism. The enol contribution produces
the “partial double bond” character, the keto produces the partial single bond character.
H
O O
R1 CH C N CH R4 R1 CH C N CH R4
R2 H R3 R2 R3
keto enol
6. (10) Multiple choice: One point maximum each. Circle the single number that corresponds to the best
answer. Read every answer carefully and completely. Answers can receive either positive or negative
partial credit. (Note. Plus signs indicate correct answers.)
A. The following functional groups can form hydrogen bonds:

i. methyl and amide
ii. sec-butyl and thiol
iii. amide and hydroxyl (+)
iv. benzyl and phenol
v. alcohol and phenylisothiocyanate
B. The torsion angle of a bond defines

i. the rise over the run
ii. the rotational conformation (+)
iii. the distance
iv. the juxtaposition of bound ligands
v. the temperature
C. Thermolysin cuts
i. lys and arg leaving a free C-terminus
ii. ile, leu, val (+)
iii. ile, leu, tyr (partial credit)
iv. phe, tyr, trp
v. lys and arg leaving a free N-terminus
D. Methionines are identified using

i. DTT and β-mercaptoethanol
ii. urea and H2O
iii. CNBr and carboxypeptidase B
iv. PITC and guanidinium
v. DNFB and CNBr (+)
Study Guide 259
E. The hydrophobic effect involves

i. hydrophobic bonds
ii. H2O and peptide bonds (partial credit)
iii. aliphatic residues (partial credit)
iv. conjugate bases
v. nonpolar residues and H2O (+)
F. The ratio of conjugate bases and acids is calculated using the Henderson-Hasselbalch equation and ___.
i. pO2 and pKa
ii. pKa
iii. pO2
iv. pH and pKa (+)
v. pH and log ([A-][HA])
G. Zwitterions formed by amino acids

i. are present when pH = pI
ii. denature easily
iii. are present at neutral pH
iv. are called dipoles
v. contain both positive and negative charges (+)
H. Chaotropic molecules include

i. ether, H2O and sulfates
ii. thiols and ammonium
iii. urea and ethanol
iv. guanidinium and urea (+)
v. SDS and ammonium
I. Hemoglobin binds
i. H+
ii. O2
iii. thiols
iv. i. and ii.
v. all of the above
J. What controls the hydrophobic effect

i. enthalpy
ii. entropy
iii. free energy
iv. pH
v. all of the above (+)
7. (PUZZLER) (3) Why do proteins typically denature at more acidic and alkaline pHs. Illustrate your
answer.
Increasing the pH results in deprotonation of side chains. Decreasing the pH results in hyperprotonation of
side chains.
In either case, salt bridges or hydrogen bonds that require both types of charge or protonation state to form
cannot form. The result of these disruptions is loss of secondary and tertiary structure, and therefore
denaturation.
260 Study Guide
Practice Exam 2
(Note. The letters Ω, Г, and Ψ are used to indicate different versions of the test.)
1. Draw the predominant structures of the following molecules (corresponding to your Greek letter) at the
pH indicated. Be sure to include all carbon and hydrogen atoms.
a. (1) b. (1)
Ω: 5-phosphoribosyl-1-pyrophosphate at pH 7.4 Ω: carbamoyl aspartate at pH 7.4
Г: NAD+ at pH 7.4 Г: α-D-glucose-6-phosphate at pH 7.4
Ψ: α-D-glucose at pH 7.4 Ψ: NADP+ at pH 7.4
O
Ω
O
P
O H
O H
H
Ω O O
O
C
H H
NH2 CH2
O
H O
OH HO H
P C C
O
O O N C
O
O
P H O
O
O
H
O
H
Γ C NH2 Γ O
O
H H P
H N H O
O O
O H H CH2
H H H
O P O H
O OH OH OH H
O HO OH
NH2
H OH
O P O N
H N
O HH
O N
N H
H H
H H
OH OH
Study Guide 261
Practice Exam 2 (key 2 of 2)
pH indicated. Be sure to include all carbon and hydrogen atoms.
Ω: 5-phosphoribose-3-pyrophosphate at pH 7.4 Ω: carbamoyl aspartate at pH 7.4

Г: NAD+ at pH 7.4 Г: α-D-glucose-6-phosphate at pH 7.4
Ψ: α-D-glucose at pH 7.4 Ψ: NADP+ at pH 7.4
H
O
H
C NH2
Γ H Γ
O
O
H N H P
H O
O O
O H H CH2
H H H O H
O P
O OH OH H
OH
NH2 HO OH
O H OH
N
O P O N
H
O H N H
H N
O
H H
H H
OH OH
H O
H C NH2
Ψ Ψ
H H
N
HO H
H
CH2 O
O H O H H
H H H
O P
OH H O OH OH
HO OH NH2
H OH O
N
O P O N
H
O H N H
H N
O
H H
H H
OH O
O P O
O
262 Study Guide
c. (5) Draw the catalytic triad in a generic serine protease. Order the residues from left to right; label the
residues from left to right 1, 2, and 3 for question 2. Leave room for the substrate.
his 2 ser 3
N
CH2 CH2 H N
asp1 H
H
H O C O
CH2
O
- H N N
C N
H
O R2
peptide/protein
substrate
2. a. (1) Draw a generic substrate juxtaposed next to the key catalytic atom in your drawing in 1c.
b. (0.5) Explain which atom on which residue in 1c attacks the substrate.
The hydroxyl oxygen atom of serine 3 attacks the carbonyl carbon in nucleophilic reaction.
c. (0.5) Explain which atom on the substrate is attacked and why.
The carbonyl carbon is attacked by the hydroxyl oxygen of serine 3 since it has a partial positive charge.
d. (3) Explain how the residues in the “catalytic triad” in question 1c collaborate to induce the first step in
the catalytic mechanism. Use the letter 3 residue nomenclature and numbers assigned in your drawing in
your explanation. Name the type of chemical reaction with the appropriate term.
(Useful terms: “charge relay” and “acid-base catalysis”)
The electronegatively charged oxygen of aspartate 3 hydrogen bonds with the secondary amino hydrogen
of histidine 2. This enhances the electronegitivity of the lone pair electrons on the other ring nitrogen on
histidine 2, thereby strengthening the hydrogen bond with the hydroxyl hydrogen of serine 3 and eventually
removing it. The resulting negatively charged serine oxygen is then activated to attack the substrate in a
nucleophilic attack.
4. Explain the following terms and explain their use in the study of enzyme catalysis. Use one or a few
complete sentences.
a. (1) Ω, Г: Acid-base catalysis (show the generic enzymatic reaction):
catalytic transfer of a proton (e.g., between E-H and B); E-H + B → E- + B-H+
Ψ: Covalent catalysis (shown the generic enzymatic reaction):
Catalytic transfer of a group X between enzyme and substrate; substrate 1, enzyme & substrate 2; etc.
e.g., E-X + S → E + S-X

S1-X + E → S1 + E-X; EX + S2 → S2-X + E
Study Guide 263
b. (1) Ω, Г: Michaelis-Menten constant (show the generic enzymatic reaction):

k1 kcat
E + S ↔ [ES] → E + P; KM = (k-1 + kcat)/k1
k-1
Ψ: Catalytic constant (show the full reaction):
kcat
E + S ↔ [ES] → E + P
kcat is the number of catalytic events per second.
c. (1) Ω, Г: Cyclin kinase:
Kinase activity of cyclin heterodimer that phosphorylates substrates and controls cell cycle “checkpoints”.
Ψ: Allosterism:
Allosteric effector binds, often to a regulatory subunit, and changes the binding constant or catalytic rate of
a linked reaction. Can involve feedback, but may also be induced by an apparently unlinked effector (not
synthetically related).
d. (1) Ω, Г: a zymogen (include a reaction scheme):
Typically described as an inactive enzymes precursor (usually prior to proteolysis) that is covalently
modified (typically proteolyzed) to become activated.
Ψ: an enzymatic cascade (a picture would help): A series of linked catalytic reactions, in which the first
stage releases a population of activated enzymes, each of which go out and create a population of activated
enzymes of a second type. This stage-by-stage amplification leads to a very large set of activated species in
later stages.
realized net activity
(amplification factor)
activation
nA n A* (n A* A* n)
Stage 1
mB (n x m) B* n x m B* B* (n x m)
Stage 2
... ...
(further
...
stages)
e. (1) Ω, Г: an uncompetitive enzyme inhibitor (Draw the relevant enzymatic reactions):
E + S ↔ [ES] → E + P
[ESI]
Uncompetitive – bind to the [ES] complex only and block product formation.
264 Study Guide
Ψ: a competitive enzyme inhibitor (Draw the relevant enzymatic reactions).
E + S ↔ [ES] → E + P
[EI]
Competitive inhibitors bind E. No [ES] or P form.
f. (2) Ω, Г: a Schiff base. Explain its utility. (Draw a generic chemical structure, include as much detail as
you can. Include all chemical bonds and atoms.
Ω, Γ E
+
O H N H
C
O P O CH2
O
O
N CH3
H
PLP forms Schiff bases with enzymes and substrates. This weakens bonds to the alpha carbon, leading to
bond breakage, electron shifts and isomerizations.
Ψ: the redox center of FAD and FADH2. Explain its utility. (Draw the key parts of both chemical structures
and include the number of rings. Include all chemical bonds and atoms.)
Ψ proton
hydride
O H, H O
H H
H3C C C H
N
riboflavin C C C N H H3C
C
C
C
N
C
C
NH
vitamin B2 C C C C
N N C C C C
H3C C O H3C N N O
C H
H CH2 H R
the redox
(CH OH)3 center
FAD FADH2
CH2
5' ADP
Used in redox reactions in intermediary metabolisms. Transports electrons (reducing equivalents) to the
electron transport apparatus in the mitochondria.
5. (3) What chemical does Coenzyme A carry in its typical biochemical function? Draw the abbreviated
structure as you’ve been taught. What biochemical pathway or cycle is this covalent compound used to
fuel?
CoA-SH carries an acetyl group, derived by decarboxylation of pyruvate, which is then transferred to
oxaloacetate from the Krebs Cycle to form citrate, thereby fueling it. This reaction links liver glycogen
stores, through Glycolysis to the Krebs Cycle (and many other pathways).
Study Guide 265
answer.
A. Lactose synthesis requires:
i. UDP-glucose and fructose
ii. UDP-glucose and galactose
iii. UDP-galactose and glucose (+)
iv. ATP, glucose and galactose
v. ATP, glucose and fructose
B. The following coenzyme and protein are required for our eyes to absorb visual photons:
i. FADH2 and isocitrate dehydrogenase
ii. ubiquinone and rhodopsin
iii. trans-retinal and opsin
iv. cis-retinal and opsin (+)
v. trans-retinal and rhodopsin
C. The following are examples of ketoses:

i. D-ribose and dihydroxyacetone
ii. D-glucose and D-galactose
iii. D-fructose and D-ribose
iv. D-fructose and D-lactose
v. dihydroxyacetone and D-fructose (+)
D. Fructose ribopyranose formation involves:

i. an enamine and acid-base catalysis
ii. an intermolecular Haworth projection
iii. a hemiacetal and acid catalysis (+)
iv. reducing sugar and RNA
v. sucrose and glucose
E. The following are examples of second messengers in signal transduction pathways discussed in class:
i. cyclic AMP and glucose
ii. inositol and vitamin E
iii. inositol diphosphate and steroids
iv. cyclic AMP and phospholipase C
v. inositol triphosphate and diacylglycerol (+)
F. Acetylcholine esterase:
i. contains an active site serine and is inactivated by the nerve gas (+)
ii. contains an active site serine and is activated by the nerve gas antidote CoA
iii. DEP contains an active site threonine and is inactivated by cAMP
iv. contains a “catalytic triad” and is inactivated by the nerve gas DFP
v. is embedded in the finger nails of frogs
G. The proximity effect and transition state stabilization

i. are confusing and hard to separate
ii. enhance the rate of an enzymatic reaction (+)
iii. involve catalytic transfer of H+
iv. i. and ii. above
v. i. through iii. above
H. The following metals occur naturally in biological cofactors

i. calcium, molybdenum and cesium
ii. sodium, iron, phosphorus and cobalt
iii. potassium, molybdenum and zinc (+)
iv. all of the above
v. ii. and iii. above
266 Study Guide
I. Coenzyme Q
i. can transform between diol and dione forms
ii. carries reducing equivalents in glycolysis
iii. can be reduced by NADH or FADH2
iv. i. and ii
v. i. and iii. (+)
J. D-ribose is not
i. illustrated as a Haworth projection
ii. a pyranose in solution
iii. a furanose in solution
iv. present as the sugar acid in chewing gum (+)
v. in RNA
7. PUZZLER (5 total) Trypsin catalyzes proteolysis, The ‘specificity site’ has a high affinity for arginine so
the protease cuts specifically adjacent to this amino acid.
C
H N + N H
H H
a. (2) Explain why the compound benzamide inhibits the reaction.
It is an active site-directed mimic of arginine (e.g., see Horton et al.)
b. (1.5) Draw out the likely reaction scheme, including the effect of benzamide.
Study Guide 267
Practice Exam 3
1. Draw the predominant structure of the molecule corresponding to your Greek letter at the pH indicated.
Be sure to include all carbon and hydrogen atoms (or “get it right” if you use abbreviated structural
nomenclature).
a. (1) b. (1)
Ω: lauric acid at pH 7.4 Ω: 2’-O-methyl guanosine-5’-monophosphate at pH 7.4
Г: palmitate acid at pH 7.4 Г: ribothymidine-3’-monophosphate at pH 7.4
Ψ: arachidonic acid at pH 7.4 Ψ: cytidine-2’, 3’-diphosphate at pH 7.4
Ω: O
C12 Ψ:
C O O
O C
14
O
Γ: 8
C 5
O 11
b. (1)
Ω: syn 2’-O-methyl guanosine-5’-monophosphate at pH 7.4
Г: ribothymidine-3’-monophosphate at pH 7.4
Ψ: cytidine-2’, 3’-diphosphate at pH 7.4
Γ: O Ψ: NH2 Ω:
H H O
CH3 N
N H N
N
N H H
N H O
O H2N N N
O
H H
O H
O OH H H H O P O O
H H H H
H H O H H O
O HO O H
HO H O O CH3
H O P OH
O P O O H
O P O
O
O
268 Study Guide
c. (5) Draw the structure of a generic “fluid mosaic” membrane. Label each of the five typical
components with appropriate name. Number them 1 to 5.
[extracellular space]
5 oligosaccharide of
glycoprotein
CHO, proteoglycan
4 lipid anchored
protein
2 peripheral
membrane lipid
protein anchor
1
lipid bilayer
[cytosol]
3 integral membrane protein
2. (2) Briefly describe 4 of the 5 components of the fluid mosaic pointed out in your drawing in Question 1.
Make the numbers match.
(i). lipid bilayer (1)
This refers to a mixed composition liquid-crystal, interrupted by integral membrane proteins, lipid
derivatives, anchors, glycosphingolipids, and so forth. The head groups are composed of ethanolamine,
inositol, choline, etc. The “tails” are composed of even-numbered carbon-containing hydrocarbon chains.
(ii). peripheral membrane proteins (2)
These are non-covalently membrane-bound proteins. They adhere to the membrane at the H2O interface,
predominantly by electrostatic interactions (salt bridges, H-bonds).
(iii). integral membrane proteins (3)
These proteins are embedded in the lipid bilayer with one or both faces exposed to the external solution.
They often contain attached tree-like carbohydrate chains.
(iv). lipid anchored proteins (4)
These proteins are connected to lipid tail, for example, the farnesyl group, which is embedded within the
lipid bilayer.
(v). oligosaccharide trees (5)
These structures are connected to proteins, proteoglycans, and glycosphingolipids.

Study Guide 269
3. Explain the following terms with respect to their purpose in lipid or nucleic acid biochemical pursuits.
a. (1) Ω, Г: Sodium deoxycholate (What it’s used for and why does it work?):
It is a detergent that is commonly used to solubilize membrane proteins. It’s also produced naturally by the
gall bladder to facilitate bile excretion, which is used to facilitate lipid absorption by the gut.
Ψ: a seven-helix transmembrane protein (Explain why it embeds and orients within the membrane bilayer
as it does.):
A seven-helix transmembrane protein contains seven alpha helices that span the bilayer region of a
membrane. The connecting loops extend out into the extracellular space or into the cytoplasm. The alpha
helices are hydrophobic, so they remain embedded with the hydrocarbon-rich interior of the membrane.
b. (1) Ω, Г: H2O-octanol partition coefficients (explain the practical significance):
This coefficient characterizes the ratio of a molecule (e.g., an amino acid) that resides in the aqueous phase
divided by the amount of the same molecule that enters the octanol phase in an equilibrated two-phase
partitioning sample. This is related to the free energy for a partitioning into water versus octanol, a generic
mimic for the hydrophobic interior of a protein (or membrane). The results are used to predict whether
amino acids in a protein will lodge within a membrane, or inside the protein, versus surface exposure to
solvent.
Ψ: farnesyl anchor (explain why it works):
Lipid anchored membrane-bound proteins can be linked to an isoprenoid molecule called farnesene (which
contains 3 isoprene units). The farnesyl anchor is hydrophobic and therefore prone to becoming embedded
in the lipid bilayer on the extracellular surface of a cell. This anchors the protein to the cell.
(Note: Two farnesyl pyrophosphates react to form squalene, which, through a series of reactions, zips up to
form cholesterol.)
O O
P O P O
O
O O
Squalene
2 farnesyl pyrophosphate (C15) (C30)
HO HO
Cholesterol Lanosterol
270 Study Guide
c. (1) Ω, Г: hypochroism (Explain the practical significance.)

Hypochroism is a decrease in absorbance (at 260 nm) that occurs when two single strands form a double
helix. This effect has been used to determine relative stabilities of different sequences and lengths of double
helix constructs. By measuring equilibrium constants, one can calculate ∆G values. By doing this at
different temperatures, one can calculate the ∆H and -T∆S values at any given temperature. Thus, one can
calculate ∆G, ∆H, and -T∆S for duplex formation. Doing so with the right set of molecules has produced a
database which is used extensively to predict structures from sequences.
(Note. One important application is used when one selects optimal PCR primers. Primers predicted to form
unwanted secondary structures are avoided.)
Ψ: phosphodiester versus carboxymonoester (also draw the generic structures):
The backbones of DNA and RNA are composed of phosphodiester bonds, in which two different R groups
are connected to the phosphoryl group. A carboxymonoester occurs in the linkage that connects a fatty acid
to glycerol. Only one R group is connected to the single carboxyl oxygen.
O O
R1 O P O R2 R1 C O R2
O
carbon monoester
phosphodiester
d. (1) Ω, Г: counterions (draw a picture):
The phosphodiester backbone carries about 1 negative charge per nucleotide so electropositive counterions
such as Na+, K+, Mg2+, polyaminen+ must bind to the external surface of DNA or RNA in order to achieve
(almost) electroneutrality.
R1
O
O
P H
O O H
base
O
H H
Mg2+ H H
O (H or OH)
O
P
O
O
R2
Ψ: major and minor grooves (Explain how these structures are decoded by proteins.):
The double helical A- and B- form structures have 2 “sides,” of which one is larger than the other, so the
two are referred to as major and minor grooves. Different functional groups are presented to the two
grooves, and as such they act as specific protein recognition elements. This is the nature of the structural
code that allows regulated use of nucleic acids in their biochemical functions.
e. (2) Ω, Г: Z-DNA repeating unit (also explain why the unit is as your state; illustrate your point(s) with a
simplified structure):
Z-DNA has a zig-zagged phosphodiester backbone because the duplex has a dinucleotide repeating unit.
The typical sequence, alternating CG has: (1) two different phosphodiester backbone conformations, (2)
anti C, glycosidic torsion angle and a syn G, and (3) both C2’ endo and C3’ endo deoxyribose
conformations.
Study Guide 271
Ψ: Duplex melt analysis (Draw the relevant reaction. Draw a simple graph that illustrates the results.):
One can determine the ∆G, ∆H, and T∆S for duplex dissociation to form 2 single strands by raising the
sample temperature and following the increase in A260 (and varying the nucleic acid concentration). This is
used to understand how different sequences affect the stabilities of the corresponding duplex structures.
duplex 2 complementary
single strands (ss)
ss
1.00
typical
numbers A260 Tm
(50% of the duplex
population is "melted"
to form single strand)
duplex
0.85
20 40 60 80 100
T (oC)
f. (2) Ω, Г: antisense mRNA. (Explain the functional utility and draw the functioning structure.):
Antisense mRNA is the Watson-Crick complementary sequence corresponding to a “sense” mRNA. The
former binds the latter to form a duplex. Since mRNA must be single stranded to be used in translation,
antisense binding prevents translation of the bound mRNA.
mRNA + antisense mRNA ↔ mRNA•antisense mRNA
active inactive
Ψ: Hoogsteen base pairing. [Illustrate the relevant functional groups and explain how the bases interact
with each other.]
Hoogsteen base pair formation involves the top/back side of one of the purines, specifically the N7 atom on
the imidazole ring. This sort of base-base interaction also occurs in triple- and quadruple-strand complexes.
H H O
N
N H N
N N
H and H
H N H N N N
N
R H R
272 Study Guide
4. (2) Explain and illustrate the difference between anti and syn glycosidic torsion angles using guanosine.
Anti and syn conformations interconvert by rotation of the base relative to the (deoxy) ribose. The anti
conformation involves a base swung away from the “top” of the sugar; syn nucleosides have a base above
the sugar.
O
O
H N H
N N
N
H H
H2N N H
N H N N NH2
HO O
O H H H
H H H H
H
H H
HO OH H
H OH
syn G anti G
answer. Read every answer carefully and completely.
A. Eukaryotic mRNAs contain the following:

i. 5’-O-methyl
ii. m’G cap and exons
iii. introns and codons (+)
iv. introns and histones
v. 5’-terminal poly(A)
B. Restriction endonucleases do not

i. cleave specific DNA duplex sites
ii. create “sticky ends”
iii. produce cloned plasmids (+)
iv. define DNA map locations
v. protect bacteria
C. Transfer RNA
i. is typically in the cruciform secondary structure
ii. of pivotal importance in decoding nucleic acid information into protein (+)
iii. contains a 5’-terminal amino acid
iv. has a dinucleotide anticodon
v. is a cloverleaf
D. The hydrophobic effect is not the primary stabilizing force of

i. protein primary structure (+)
ii. lipid micelles
iii. the double helix
iv. stacking
v. transmembrane cation gradients
E. The following molecules are not listed correctly from largest to smallest (from highest to lowest Mr):
i. tryptophan, lysine, leucine, glycine, CO2
ii. cAMP, cytidine, N1-methyl uracil, methylphosphate, ammonium
iii. arachidonate, stearate, oleate, palmitate, myristate, laurate
iv. 1-stearoyl-2-oleoyl-phosphatidyl inositol, glucuronate, glycerol
v. [3H-2] erythrose, [(2H-6)2] lactose, [14C-1] glycerol
Study Guide 273
F. The ratio of absorbances of samples measured at lower and higher temperatures (Aλ, low / Aλ, high)
i. is proportional to the ratio of liquid crystal to dispersed fatty acids in re-equilibrated samples.
ii. is proportional to the ratio of duplexes to single strands in re-equilibrated samples. (+)
iii. will produce faster yields at higher temperatures if the reaction proceeds with Boltzmann kinetics.
iv. i. and ii.
v. i. and iii.
G. Phospholipase C
i. is involved in the tyrosine kinase signal transduction pathway.
ii. catalyzes hydrolysis of phosphatidyl inositide diphosphate to form diacylglycerol and inositol
triphosphate in the cAMP-dependent signal transduction pathway.
iii. is a hydrolase and esterase. (+)
iv. is missing in the Eskimo genotype.
v. co-starred with Paul Newman and Grace Kelly in a Elia Kazan-directed off-Broadway psychodrama.
H. The following molecules are commonly incorporated into phosphatidylglycerides

i. serine and cholinesterase.
ii. choline and stearate. (+)
iii. oleate and myrtylistate.
v. octanolamine and inositol.
I. DNA probes
i. cannot be labeled with biotin.
ii. have been used as convincing evidence in many celebrated murder trials. (+)
iii. hybridize with RNA to form a reverse transcript
iv. will all plasmose in the thermonuclear heat of a distant white dwarf Sun.
v. react with arachidonate to form nucleolipids.
J. The following molecules fall within the realm of lipids and derivatives thereof.
i. cerebrosides, steroids and endorphins
ii. isoprenoids, glyceraldehydes and sphingomyelins
iii. phosphatidyl inositides, insect juvenile hormone and lipid-anchored proteoglycans (+)
iv. waxes, pyridoxal phosphate, vitamin E
v. sn-(1-oleoyl, 2-myristoyl, 3-palmitoyl) triglyceride, zest of lemon, mannose
274 Study Guide
6. (PUZZLER #1) (2) A duplex DNA fragment formed by two annealed 13 nucleotide oligonucleotides
was obtained from the chloroplast genome upon digestion with the enzyme EcoRI. This duplex fragment
was treated with the enzymes HpaII and BglII. Two fragments were obtained.
Specificities of Restrictions Endonuclease Recognition Sites.

Enzyme Recognition sequence
5’
BglII A V GATCT 3’
5’
EcoRI G V AATTC 3’
5’
HpaII C V CGG 3’
(a) What is the sequence and structure of the original chloroplast EcoRI duplex DNA fragment? Explain
the logic that led you to your result.
1 2 3 4 5 6 7 8 9 10 11 12 13
5' 3'
AATTCCGGAATTC
. . . . . . . . . . . .
3'
C T T A A G G C C T T A A 5'
EcoRI HpaII EcoRI
The two ends were created by the EcoRI reaction. The center of the molecule can only be composed of a
single HpaII site which overlaps the two EcoRI site fragments BglII “treatment” did not have any effect on
the fragment since it has not BglII site.
(b) What are the sequence and structures of the two fragments produced by HpaII and BglII treatments?
Explain the logic that led you to your result.
5' 5'
A A T T C 3' CGGAATTC
. . . . .
3'
. . . . . +
3'
C T T A A G G C 5' 3' C T T A A 5'
"sticky
ends"
HpaII cut the initially isolated EcoRI fragment, which already had a 3’C extension, to produce two
fragments with both 3’C and 5’CG overhangs. The fragments are the same, so they’d appear to be 1
fragment on a gel.
7. (PUZZLER #2) (2) Draw structures of the following and name the functional groups:
(a) a nucleotide second messenger (Cyclic adenosine monophosphate, cAMP)
glycosidic NH2
bond amino
N N
H H
5' H
O N N H
O
H H adenine
cyclic 3', 5' O
P H H
phosphodiester O
O 3' OH
hydroxyl
Study Guide 275
(b) a covalent lipid-carbohydrate complex that forms two different second messengers
8 14
O C 11
Hydrolyzed by 5
Phospholipase C H2C O
O C
H C
O
CH2 Phospholipase C
O O
O O DAG + IP3
P P
O H O O
O H
OH H
HO HO
H O O
H H
P
O
O
8. (PUZZLER #3) (4) Similar functional roles are accomplished by cyclic AMP in the Adenylate Cyclase
Signaling Pathway and diacylglycerol in the Inositol-Phospholipid Signaling Pathway”. Briefly explain
their respective functions (Illustrate your answer). Explain how they are analogous.
Cyclic AMP (cAMP) activates Protein Kinase A, which produces cellular responses by phosphorylating
regulatory, sensory, and growth-controlling proteins.
Phosphatidylinositol triphosphate is lysed by the enzyme phospholipase C to produce diacylglycerol

(DAG) and inositol triphosphate (IP3), which are both second messengers. DAG and Ca2+ activate Protein
Kinase C (PKC); IP3 activates a Ca2+ channel which imports Ca2+ from the extracellular lumen. PKC is
shifted from inactive to active (when DAG is present) by an increase in [Ca2+] from 0.5 to 1 mM.
PKC produces a number of cellular responses by phosphorylating regulatory, sensory, and growth-
controlling proteins.
Both mechanisms regulate kinases, which phosphorylate analogous cellular functions in target cells.
276 Study Guide
Practice Exam 4
pH indicated. Be sure to include all carbon and hydrogen atoms (or “get it right” if you use abbreviated
structural nomenclature).
a. (1) b. (1)
Ω: glucose-6-phosphate at pH 7.4 Ω: phosphoenolpyruvate at pH 7.4
Г: fructose-6-phosphate at pH 7.4 Г: dihydroxyacetone phosphate at pH 7.4
Ψ: fructose-1, 6-bisphosphate at pH 7.4 Ψ: pyruvate at pH 7.4
O
Ω: Γ: P
O O
O O
O O
2-
Ω: Γ: O P
O
P O CH2 O CH2
O 2-
O
O CH2 O OH C O
OH C O
C O
HO OH HO H2C OH
O
OH CH2OH H2C O P
2-
O
HO
O Ψ: O O
O
Ψ: O
P
O
2-
C
C O
CH2 O OH
CH3
O
HO
CH2 O P O
2-
HO O
c. (1) d. (1)
Ω: oxaloacetate at pH 7.4 Ω: succinate at pH 7.4
Г: citrate at pH 7.4 Г: fumarate at pH 7.4
Ψ: isocitrate at pH 7.4 Ψ: malate at pH 7.4
O O
Ω:
O
C
O
Γ: C Ψ:
O
C
O
Ω: O
C
O Γ: O
C
O Ψ: O
C
O
CH2 CH2
C O O H C HO C H
O CH2
HO C C H C C
CH2 C H CH2
O CH2
CH2 O
C HO C H C C
C
O O C O O O O O
C O
O O
O O
Study Guide 277
2. a. (4) Name and briefly describe the purpose (point) of the three most central catabolic pathways of
intermediary metabolism. (The third has two coupled parts. Name both of them.) Leave room for the
answer to part b following each of your definitions.
b. (2) Also list the product(s) of each of these pathways. (See Review Session 11, problem 1.)
(i) Glycolysis + the Bridge Reaction
This involves conversion of glucose to pyruvate (two per glucose) then to 2 acetyl CoA. Products are 2
ATP per glucose, 2 acetyl CoA, 4 NADH, yielding 10 ATP in oxidative phosphorylation.
(ii) Krebs Cycle
Fixing oxaloacetate to acetyl CoA (produced by glycolysis), producing the C6 substrate citrate, which is
broken down to oxidatively to oxaloacetate in 1 turn of the cycle. Products include (quantitatively) CoA-
SH, 3 NADH (x2 per glucose, yielding 15 ATP), 1 CoQH2 (x2 per glucose, yielding 3 ATP), 1 GTP (x2 per
glucose), 2 CO2, and 2 H+.
(iii) oxidative phosphorylation/electron transport
conversion of reducing equivalents to H2O (NADH, FADH2, and CoQH2) via the electron transport chain
and used O2 as the ultimate electron acceptor. The F0F1 ATPase converts the protonmotive force generated
by electron transport into phosphorylation of ADP to produce ATP. Products are NAD+, FAD, coenzyme
Q (all oxidized) and ATP; H2O is produced by the reduction of ½ O2.
3. (2) Describe how the following concept applies to the regulation of glycolysis. Be as detailed as possible.
Ω, Г: Define “metabolically irreversible.”
Having a large negative ∆G; highly spontaneous thermodynamically and predisposed almost completely to
product formation.
Ψ: Define “near equilibrium.”
Having a ∆G close to or near zero; flux is approximately equal in both directions.
Three reactions in glycolysis are metabolically irreversible; those catalyzed by phosphofructokinase 1,

hexokinase and pyruvate kinase. The others are near equilibrium. As a result, they constitute the key
control points for metabolic regulation, specifically by allosteric effectors.
(See Review Session 11, problem 5.)
4. (3) Explain why the kinetics of an enzyme reaction are most easily controlled when KM is approximately
equal to the actual concentration of the reactant. Include the appropriate chemical reaction and defining
equation in your discussion.
When KM is approximately equal to [S], the reaction is occurring at a rate of ½ Vmax; at lower [S], the
reaction is not occurring to much of an extent (rate); at higher [S], the reaction is occurring with a rate
approaching Vmax.
278 Study Guide
(Continued)
Vo only changes a lot when [S] is equal or close to KM, so only then can the rate be regulated upward or
downward with small changes in [S]. The relevant equation and reaction are:
k1 kcat
E + S ↔ [ES] → E + P (‘↔’ indicates an equilibrium)
k -1
Vo = Vmax [S]/(KM + [S])
5. (everyone) Explain the following terms and explain their use to study or purpose in metabolism or
regulation.
a. (1.5) Why should citrate negatively regulate the phosphofructokinase-1 reaction? What is the general
term for this phenomenon? (Review Session 11, problem 9a)
The negative regulation of PFK-1 by citrate is an example of negative feedback inhibition of glycolysis by
a key Krebs Cycle intermediate. If plenty of citrate is present, sufficient acetyl CoA is being produced by
glycolysis, and the pathway should be inhibited.
b. (1.5) Why should fructose-1, 6-bisphosphate stimulate the pyruvate kinase reaction? What is the
general term for this phenomenon? (Review Session 11, problem 9b)
Stimulation of pyruvate kinase by fructose-1, 6-bisphosphate is an example of feed-forward stimulation.

The idea is that accumulation of fructose-1, 6-bisphosphate stimulates the later reaction, in order to use the
accumulated intermediate.
c. (2) triose phosphate isomerase, including the definition, reactant (s), reaction and product(s):
(Review Session 11, problem 8.)
Fructose-1, 6-bisphosphate dissociates to form two C3 compounds, dihydroxyacetone phosphate (DHAP)
and glyceraldehyde-3-phosphate (G3P) in the Aldolase reaction. Since only G3P can be used in subsequent
Glycolytic reactions, DHAP must be converted to G3P. This reaction is catalyzed by Triose Phosphate
Isomerase.
O
O H
CH2 O P O C
2-
C O O H C OH
O
CH2OH CH2 O P O
O 2-
DHAP G3P
d. (2) Aldolase, including the definition, reactant(s), reaction and product(s):

Enzyme that catalyzes aldol condensation in the Gluconeogenic direction, and cleaves fructose-1, 6-
bisphosphate to form dihydroxyacetone phosphate and glyceraldehyde-3-phosphate in the Glycolytic
direction:
2-
O H
2- H CH2 O Pi C
H Glycolysis
Pi O H
O C O + H C OH
2-
H HO
H O Pi Gluconeogenesis CH2OH 2-
H H Pi O CH2
OH H
(See Review Session, problem 8.)

Study Guide 279
6. (2) (everyone): Name the coenzyme used by dihydrolipoamide acetyl transferase and explain how it
functions fueling the Krebs Cycle. (A picture would help.)

Lipoic acid is a lipid-based hydrocarbon that acts as a covalently attached tether between the pyruvate
dehydrogenase E2 subunit and the acetyl group. It functions by accepting the ethanol group from the
thiamine pyrophosphate group (on the E1 subunit), by forming a thioacetate, and finally by transferring the
acetate to CoA-SH.
TPP
S C CH3 CoA SH
E2 lip
TPP ethanol SH O
acetyl CoA Krebs
S Cycle
E2 lip
S
SH
E2 lip
SH
7. (everyone) a. (3) Define “protonmotive force”. Explain what enzyme complex uses this phenomenon as
the driving energy for ATP synthesis in oxidative phosphorylation. Give a diagram and the relevant
equation.

Oxidative loss of reducing equivalents due to a proton gradient across a membrane (or the mitochondrion).
Diagrammatically:
X 1/2 O2
2 H+
XH2 H2O
2 e- 2 e-
(outside the cell) (inside the cell)
ΔpH across
the membrane
Mathematically, there are two contributions to the protonmotive force (∆p) due to (1) the difference
between inside and outside [H+], and (2) the change in membrane potential (∆Ψ).
∆p = ∆G / n F = ∆ψ + ∆pH in-out (0.059V)
ATP Synthase catalyzes H+ → back in.
b. (1) How does electron transport drive production of the protonmotive force?
(Review Session 12, problem 11):

The electron transport reactions “pump” H+ out of the mitochondrial matrix and build up the [H+] in the
inner membrane space, which drives ATP synthesis by the FoF1 ATPase.
280 Study Guide
answer.
A. Coenzyme/substrates in the pyruvate dehydrogenase complex shuttles C2 fragments in the following order:
i. pyridoxal phosphate-E1, lipoyl-E2, CoA-SH, oxaloacetate
ii. thiamine pyrophosphate-E1, lipoyl-E2, CoA-SH, citrate (+)
iii. thiamine pyrophosphate-E1, lipoyl-E2, CoA-SH, oxaloacetate
iv. thiamine pyrophosphate-E1, lipoyl-E2, ACP-SH, oxaloacetate
v. thiamine pyrophosphate-E1, lipoyl-E2, ACP-SH, citrate
B. Pyruvate translocase
i. is a pyruvate: malate antiport
ii. is a pyruvate: OH- symport
iii. is a pyruvate: H+ antiport
iv. exits the mitochondrion
v. fuels the Krebs Cycle (+)
C. The correct order of substrate production in the Krebs Cycle is:

i. succinate, fumarate, malate, oxaloacetate, coenzyme A
ii. glucose-6-phosphate, fructose-6-phosphate, fructose-1, 6-bisphosphate
iii. fumarate, succinate, dihydroxyacetone phosphate, oxaloacetate
iv. succinate, fumarate, malate, oxaloacetate, citrate (+)
v. succinate, succinyl CoA, fumarate, malate, oxaloacetate
D. β-oxidation yields the following:

i. 1 FADH2, 1 CoQH2 (indirectly), 1 NADH, H+, 1 acetyl CoA and a 2 C unit shorter fatty acid (+)
ii. 1 GTP, 1 CoQH2 (indirectly), 1 NADH, H+, 1 acetyl CoA and a 2 C unit shorter fatty aldehyde
iii. 1 FADH2, 1 CoQH2 (indirectly), 2 NADH, H+, 2 acetyl CoA and a 2 C unit shorter fatty acid
iv. 1 FADH2, 1 CoQH2 (indirectly), 2 NADH, 2 H+, 1 acetyl CoA and a 2 C unit shorter fatty acid
v. fire and water, sufficient supplies for 1 cell for 1 second
9. (PUZZLER) a. (2) Draw the mechanism for acetoacetyl-Acyl Carrier Protein (ACP) formation from
acetyl-ACP. Include both key proteins with substrates bound and unbound, reactants and products.
O
O C
O CH2 O
ACP-SH
CH3 C S ACP O C
S
(n) acetyl-ACP Ketacyl ACP Synthase S C CH3 ACP
CO2
(HS-) Ketoacyl ACP
Synthase
O O
CH3 C CH3 CH3 C S ACP
(n + 2) acetylacyl ACP
b. (2) Which product do fatty acid synthesis and ethanol fermentation of pyruvate have in common?
Explain.
Both processes produce 1 carbon dioxide molecule per single C2 unit added (fatty acid synthesis) or
fermented (pyruvate decarboxylation in ethanol production).
Study Guide 281
Final Test Review
1. Draw the structure of the following amino acids at pH 7. Show all bonds and atoms.
(c) L-histidine (d) L-glutamate
(e) L-leucine (f) L-asparagine
(g) L-proline (h) L-aspartate
(i) L-lysine (j) L-valine
(k) L-isoleucine (l) L-cysteine
3. Define a van der Waals interaction. Include a description of the relation between interatomic distances
and free energies.
4. Define the following terms:

(a) Protein primary structure
(b) Protein secondary structure.
(c) Protein tertiary structure.
(d) Protein quaternary structure
5. Define a hydrogen bond interaction. Include a description of the relation between interatomic distances
and free energies.
6. Define a protein β-sheet; include a drawing of the key hydrogen bonds.
7. Describe Edman degradation. Draw the structure of the key reagent.
8. Define a chaotropic agent. Give two specific examples used with proteins.
9. Define a protein α-helix; include a drawing of the key hydrogen bonds.
10. Define a disulfide bond. Draw one between two fully drawn “residues.”
282 Study Guide
11. Define the purpose of a Ramachandran plot. What are φ and ψ?
12. Define a zwitterion. Draw a generic amino acid zwitterion.
13. Define and describe the energetic origins of the hydrophobic effect with respect to protein stability.
14. Define an “initial” rate” in the context of enzyme kinetics. Give the relevant equation and name the
terms in it.
15. Does an act of catalysis change an enzyme. Explain and give the enzymatic reaction.
16. Define the term “maximum velocity” in the context of enzymatic catalysis. Give the relevant equation
and name the terms.
17. List two requirements for enzymatic catalysis.
18. Write down the Michaelis-Menten equation and name all of the terms in it.
19. (a) What is the definition of KM?
(b) What is a Michaelis complex?
20. What is the definition of kcat?
21. Write out the linked chemical equilibrium reactions involving E, S and other relevant parameters that
describe simple Michaelis-Menten catalysis.
22. Show how the reactions for E, S and I differ for competitive and uncompetitive inhibition of enzyme
catalysis.
23. List the three (3) requirements for enzyme catalysis described in class.
24. How do the initial rate and steady state postulates differ in how they describe enzyme kinetics?
25. If an enzyme catalyzes a reaction extremely quickly, what external factor still limits the velocity of the
reaction? Explain.
26. (a) Draw acetylcholine. See part (b) before you start.
(b) What atom on what amino acid in the esterase site of acetylcholine esterase reacts with the substrate?
Include a cartoon depiction of the enzyme surface (drawn under your acetylcholine drawing in part a)
Study Guide 283
showing how it interacts with the substrate. Include an arrow to show how the electrons of the key enzyme
atom attack the substrate.
27. (a) Explain in general how the nerve gas antidote pyridine aldoximine methiodide (PAM) reactivates
acetylcholine esterase.
(b) What is the key functional group on the antidote?
(c) What kind of reaction produces the reactivated enzyme?
28. Draw the generic reaction for the bisubstrate-enzyme ping-pong reaction. Define the letters used.
29. Explain how an enzyme cascade works. Include a drawing and emphasize how amplification of the
initial signal is achieved.
30. Blood clotting is initiated by very few enzymes. Explain how a clot composed of many, many, many
proteins is produced. Include a reaction or reactions and emphasize how such a large number of products is
made. Name this phenomenon.
31. Define a zymogen and give one specific example that is important for digesting food.
32. Describe how feedback inhibition works. Why is this a logical way to regulate a metabolic pathway.
33. Define allosterism. Describe how it is initiated using the appropriate name.
34. Describe one specific example of how kinase and phosphatase reactions regulate either glycolysis or the
Krebs Cycle. Name the regulated enzyme and explain which form is active and which is inactive.
35. What does cyclin kinase regulate? What two amino acids are phosphorylated?
36. Give two examples of reversible factors that control the catalytic capability of an enzyme. Explain each
briefly.
37. Give two examples of irreversible factors that control the catalytic capability of an enzyme. Explain
each briefly.
38. (a) The Arrhenius equation describes the relation between which physical parameters and constants?
(b) Is it a thermodynamic equation or a thermodynamic equation? Explain.
39. List the two “chemical modes of catalysis.” Define each briefly.
40. List the two “binding modes of catalysis.” Define each briefly.
284 Study Guide
41. Explain, providing the appropriate diagrams, how uncatalyzed and catalyzed reaction coordinate versus
energy trends differ. Explain each change you show.
42. Define a nucleophilic substitution (SN2) reaction. Provide a mechanism diagram. Define a nucleophile
in your explanation.
43. (a) Define an electrophile.
(b) Explain how to create one via acid-base catalysis.
44. (a) Why is histidine used so often by enzymes to carry out acid-base catalysis?
(b) Explain why enzymes typically have an optimal pH.
45. (a) Draw the catalytic triad of amino acids typically present in serine proteases. Label them.
(b) Explain how they collaborate to accomplish acid-base catalysis.
46. Draw the structure of ATP. Include all atoms and bonds.
47. Why is Mg2+ typically required to achieve optimal activity with ATP cosubstrate enzyme reactions?
48. Define a coenzyme. Give one example and describe how the coenzyme is used in a typical biochemical
application.
49. Briefly describe one example of a scenario in which a transition metal is used to facilitate a biochemical
reaction.
50. What is ATP used to do in most biochemical applications? Give two different examples that involve
different parts of the molecule.
51. (a) Draw NAD+ (NADH) in both the oxidized and reduced form. Use the contraction R to signify the
common parts of the structure in the latter drawing (i.e., only show the part that changes, connected to R).
(b) Describe how the coenzyme is used in a typical biochemical application.
52. (a) Draw the business end of FAD (FADH2) in both the oxidized and reduced form.

Study Guide 285
53. (a) Draw pyridoxal phosphate prior to and after forming a Schiff base with the ε-amino group of a
lysine residue from an enzyme E.
54. (a) What chemical group does coenzyme A typically carry in biochemistry? Draw the covalent complex
as you’ve been instructed in class (i.e., not the entire thing).
(b) Why is this function such a crucial link in intermediary metabolism?
55. Explain how the biotin-avidin noncovalent binding interaction is used to capture ligand-binding entities.
Provide a diagram that illustrates your explanation.
56. (a) What is the crucial function of N5, N10 tetrahydrofolate in the production of DNA?
(b) Explain why our understanding of this function can be used in a strategy for anti-cancer chemotherapy.
57. (a) What is UDP-galactose and how is it used to make lactose?
(b) Draw the structure of lactose as given in your notes.
58. How does cis-retinal function in transducing the signal of a photon of light into a chemically
recognizable form?
59. (a) Explain how a ketose differs from an aldose and give one example of each.
(b) Explain how a pyranose differs from a furanose and give one example of each.
60. (a) Draw the chair and boat conformations of the β-D-glucopyranose structure.
(b) Explain why a polysaccharide chain would turn directions much more in one case than in the other and
which conformational isomer would be expected to produce the most radical change in chain direction.
61. (a) Why is the hexa-atomic ring of inositol more stable than that of galactose?
(b) Given what you know about the biosynthesis of lactose from UDP-galactose and glucose, why would
you expect inositol to be less likely to polymerize than glucose or galactose? (Hint. What are their relative
capabilities in serving as nucleophilic centers?)
286 Study Guide
62. (a) What does NAG-α(1→6)-NAM-α(1→4)-glc-β(1→4)-gal mean?
(b) Draw a simplified diagram with specific emphasis on the meaning of α and β.
63. (a) How does glycogen differ from the amylopectin in starch?
(b) How would this facilitate the function of glycogen?
64. How does penicillin work selectively on bacteria but not harm us significantly? (What does it do?)
65. (a) How do extra-cellular surface carbohydrates regulate osmotic pressure around cells?
(b) What advantage does this impart to them?
66. Explain how the terms export and clearance apply to the functional status of carbohydrate-bearing
proteins.
67. (a) How does Phospholipase C produce two different second messengers in a signal transduction
pathway we discussed in class?
(b) Draw the appropriate reactant.
68. What function does chondroitin sulfate serve in cartilage and skeletal joints?
69. (a) Do saturated or unsaturated fatty acids of the same length have a lower melting temperatures (Tm)?
(b) What reactions are followed? What do differences in the Tm values monitor?
70. How do lipid bilayers and micelles differ? Include a simple drawing.
71. (a) Draw the structure of a general phosphatidyl choline molecule.
(b) Name the functional groups on your drawing.
72. Explain five details that describe the “fluid mosaic” membrane model.
73. (a) Draw the structures of the four nucleic acid bases in DNA.
Study Guide 287
(b) Label the bases drawn in part (a).
74. (a) Draw a RNA trinucleotide using three different bases.
(b) Label the bases and other structural subunits drawn in part (a).
75. (a) Draw the structures of the two Watson-Crick base pairs. Use “ | | | | | ” to designate hydrogen bonds.
76. Define a glycosidic bond in a nucleoside.
77. Explain how and why the absorbance at 260 nm (A260) can be used to determine if a double helix forms
from 2 single strands of DNA or RNA.
78. (a) Define “base stacking.”
(b) Describe the three predominant types of “forces” that contribute to stabilization of “stacked” bases in a
double helix.
79. (a) What are counterions and why do they bind all nucleic acids?
(b) How do histones serve this function in the case of most chromosomal DNAs?
80. Give two reasons why G•C base pairs are more stable than A•T (or A•U) base pairs. Rank these
contributions according to importance in inducing stability.
81. Describe four differences between A and B forms of DNA.
82. (a) Draw the mechanism of alkaline hydrolysis of RNA.
(b) Why is DNA not subject to this mechanism?
83. Why and how does an antisense oligonucleotide functionally inactivate a mRNA for use in translation
by a ribosome?
84. Name the four classes of RNA and briefly explain their functional significance. In what reaction(s) do
they participate?
85. List four distinctive features of most eukaryotic mRNAs.
86. What is the primary use of a DNA probe and how is this process accomplished?
288 Study Guide
87. Why are restriction endonucleases required to produce, manipulate and clone specific pieces of DNA?
88. What are the two functional ends of transfer RNA and how do they work to accomplish these
functions?
89. (a) Name and briefly describe the purpose (point) of the three most central catabolic pathways of
intermediary metabolism. (The third has two coupled parts. Name both of them.)
(b) What is (are) the product(s) of each of these pathways? (list according to pathway)
90. Describe the two major ways that energy is captured in a chemically usable form by metabolic reaction
pathways.
91. The following reaction from Merlin’s notebooks occurred with a standard free energy (ΔG°′) of -8.5
kcal per mol:
carborandum + gold ↔ essence of life
(a) At equilibrium, a real sample of these three solutes contained 44 mM carborandum, 0.1 mM gold and 45
µM essence of life. What is the actual free energy (ΔG) of the reaction under these equilibrium conditions
at room temperature (298 K)? Show your setup and work.
(b) A different sample contained 0.9 M carborandum, 0.1 mM gold and 45 µM essence of life. What is ΔG?
(c) How did adding more carborandum affect the equilibrium poise of the reaction? What was the mass
action ratio (Q) in each case?
92. (a) Why do only three steps in glycolysis control most of the flux through the pathway under actual
cellular conditions?
(b) What are the three steps, the three enzymes that catalyze the reactions and what do the reactions have in
common?
93. (a) Define metabolically irreversible
(b) Define near equilibrium.
94. Explain why the kinetics of an enzyme reaction are most easily controlled when KM is approximately
equal to the actual concentration of the reactant.
95. Consider the following data:

Reaction E° ′
Acetyl CoA + CO2 + H+ + 2 e- --> Pyruvate + CoA-SH -0.48 V
NAD+ + 2 H+ + 2 e - --> NADH + H+ -0.32 V
FAD + 2 H+ + 2 e- --> FADH2 -0.22 V
Study Guide 289
(a) Under standard state conditions (T = 298 K), will it require more energy if the breakdown of pyruvate to
acetyl CoA is coupled to FAD formation or to NAD+ formation? Show your setup and work.
(b) Does the answer change if pyruvate is present at 1 mM while the other species are still present at 1 M.
96. Where do the reactant/product of triose phosphate isomerase derive from (directly) and what do they
get converted to catabolically.
97. (a) Why should citrate negatively regulate (discourage) the phosphofructokinase-1 reaction? What is
the general name for this phenomenon?
(b) Why should fructose-1,6-bisphosphate stimulate the pyruvate kinase reaction? What is the general name
for this phenomenon?
98. We discussed three branching catabolic fates of pyruvate. Draw the three reaction (reaction sequences),
including cofactors and enzymes.
99. (a) Why does the absence of alcohol dehydrogenase produce even more scurrilous behavior than if the
person has the enzyme?
(b) How is the blocked catabolic intermediate related to the typical role of pyridoxal phosphate in
enzymatic catalysis?
100. (a) Why does “carbonation” accompany ethanol production?
(b) How does the staff of life of the western world benefit (and us as a consequence)? [Bread, not beer!]
101. Dihydrolipoamide acetyl transferase uses a coenzyme we did not discuss earlier. (a) What is it and
how does it function in “fueling” the Krebs Cycle?
(b) What other coenzyme is also involved in this process? How?
102. (a) What “symport” reaction accompanies import of pyruvate into the mitochondrion and what
enzyme catalyzes the reaction?
(b) Does the pH of the cytoplasm increase or decrease as a result?
103. List the reactions, coenzyme(s), cofactor(s) and enzymes involved in the two “oxidative
decarboxylation” reactions of the Krebs Cycle.
phosphorylation” reaction of the Krebs Cycle.
290 Study Guide
105. List (only once each) all of the “energy conserving” compounds formed by the Krebs Cycle
accompanied by the number of “ATP equivalents” finally accrued after oxidative phosphorylation of 1
molecule of each of these compounds.
106. (a) How do fumarase and malate dehydrogenase “fix” a carbonyl group on succinate in the production
of oxaloacetate (OAA).
(b) What crucial 2 carbon compound is then “fixed” to OAA?
(c) How is the product used in food flavoring?
gluconeogenesis in mammals?
108. What is “protonmotive force” and what enzyme complex uses this phenomenon as the driving energy
for ATP synthesis in oxidative phosphorylation? (Contrast it with electromotive force, emf, voltage.)
109. How does electron transport drive production of the protonmotive force?
110. (a) How many reactions does each round of β-oxidation of a fatty acid require?
(b) What are the products of one round of β-oxidation and what is the tally in terms of ATP equivalents of
energy conserving products?
111. (a) Draw the coupled cofactor regeneration cycles that siphon off reducing equivalents then fix them
into coenzyme Q in reactions that are coupled to the first oxidative step of fatty acid β-oxidation.
(b) Write down the names and a short definition for the cofactors involved in this “siphon”.
112. Which three steps of the Krebs Cycle do the first three steps of the fatty acid β-oxidation cycle
resemble? Draw (horizontally) the three analogous reactions from (i) β-oxidation, (ii) the Krebs Cycle, and
(iii) the three generic chemical transformations that occur beneath the four analogous substrates.
113. Draw the analogous (but backward) [reduction-dehydration-reduction] series of reactions of fatty acid
synthesis starting from reactants and going to products beneath your three reaction series from problem 4.
Point the arrows in the opposite direction drawn for problem 4.
114. Draw the reactions for the condensation substep in acyl-carrier protein-mediated fatty acid synthesis.
Study Guide 291
Final Test Review (with answers)
1. Name the molecules drawn below. Be as exact with your nomenclature as you can.
(a) L-Methionine (b) L-Serine
O H
H
C C CH2 O
CH2 S CH3
O C C CH2 O
NH3 O H
NH3
(c) L-Histidine (d) L-Glutamate

H
H N H R2 H O H O
O
C C
C C CH2 C R1 C C CH2 CH2 C
O N C N O O
NH3 C H NH3
H
H
(e) L-Leucine (f) L-Asparagine
O H H
O H O
C C CH2 C CH3
O C C CH2 C
NH3 CH3 O
NH3 NH2
(g) L-Proline (h) L-Aspartate
H H O H
O O
H C O
N C C C CH2 C
H 2C CH2 O O
NH3
C
H2
(i) L-Lysine (j) L-Valine

H
O H H
O
C C CH2 CH2 CH2 CH2 NH3 CH3
O C C C
NH3 O CH3
NH3
(k) L-Isoleucine (l) L-Cysteine

H H
O H
O
C C C CH2 CH3
C C CH2 SH
O
NH3 CH3 O
NH3
2. The van der Waals interaction describes the relation between interatomic distances, electronic charge,
solution dielectric and free energies.
3. (a) Protein quaternary structure defines the relation among subunits in a multisubunit lattice.
(b) Protein primary structure defines the amino acid sequence.
(c) Protein tertiary structure defines the packing of helices, sheets, turns, etc.
(d) Protein secondary structure defines the motifs formed by short-range interactions between amino acids.
4. A hydrogen bond interaction involves polar O, N or both and the atom for which it is named, and
constitutes one of the important protein stabilization elements.
292 Study Guide
5. Name the following protein structure: β-sheet.
R3 R8 Strand 1
H O
O R1 H
H
C C N R7 Strand 2
R4 N C C
H H O R2
O H
R5 R6 Strand 3
6. Edman degradation is used to determine the sequence of a protein based on sequential chemical
reactivity.
7. A chaotropic agent induces denaturation of proteins by disturbing the hydrophobic effect.
8. Name the following protein structure: α-helix.
H
O
H
9. Name the following protein structure: disulfide bond.
R1 CH2 S S CH2 R2
10. A Ramachandran plot is a graph of the conformational torsion angles φ and ψ for the residues in a
protein or peptide, a map of the structure of the polypeptide backbone.
11. A zwitterion has two charges which neutralize each other.
12.The hydrophobic effect is the primary “force” of protein structural stabilization.
13. The initial rate is the characteristic speed of an enzyme’s kinetics extrapolated to the time when a
defined amount of substrate is added to the enzyme solution.
14. An act of catalysis does not change an enzyme and lowers the transition state free energy of the
associated reaction.
15. The maximum velocity of an enzymatic catalysis reaction is the rate achieved when it is saturated with
substrate.
16. The Lineweaver-Burk (or double reciprocal) equation defines parameters that are used to characterize
the kinetics of an enzyme.
17. Km is the substrate concentration when V0 = Vmax/2, or Michaelis-Menten constant.
Study Guide 293
18. A Michaelis complex is the enzyme-substrate combination formed during an enzyme catalysis event.
19. The catalytic rate constant of an enzyme is abbreviated as kcat.
20. Competitive inhibition of enzyme catalysis occurs when an inhibitor binds to the active site of the
enzyme.
21. Uncompetitive inhibition of enzyme catalysis occurs when the inhibitor only binds to the enzyme-
substrate complex.
22. The steady state approximation postulates that a constant input feed of substrate is supplied whose rate
equals that of product formation.
23. Two internal factors that limit the velocity of an enzymatic reaction are _________ and _________.
[hydrophobic effect, H-bonding, disulfide bonds, van der Waals forces, ionic bonds (salt bridges) or dipole-
dipole interactions (actually underlying all of the others)]
24. Two external factors that limit the velocity of an enzymatic reaction are _________ and _________.
[pH, solvent polarity, temperature, salt concentration(s) and types, presence of chaotropes, osmolytes,
others]
25. What amino acid and functional group in the esterase site of acetylcholine esterase reacts with the
substrate? serine, hydroxylate
26. Pyridine aldoximine methiodide (PAM) reactivates acetylcholine esterase, functioning as a

nerve gas antidote.
27. What kind of reaction produces the reactivated enzyme? nucleophilic substitution
28. The bisubstrate-enzyme ping-pong reaction is used by transaminases in the exchange of an amino group
for a carbonyl group between two progressively binding substrates.
29. An enzyme cascade works by amplifying an initial signal via several linked protease cleavage reaction
stages. (e.g., blood clotting)
30. A zymogen is a protein that is converted from inactive to active forms by a covalent modification,
typically protease cleavage.
31. A decrease in the activity of an enzyme as a result of binding of a product from the reaction in question
or subsequent reactions is referred to as feedback inhibition.
32. Allosterism involves binding of a regulatory molecule at a site other than the active site.
33. Kinase and phosphatase reactions, involving phosphate addition and removal respectively, regulate both
glycolysis and the Krebs Cycle.
34. Cyclin kinase regulates entry and exit from mitosis by catalyzing a covalent modification reaction.
35. Which two amino acids are modified in the reactions catalyzed by the enzyme in question 35?
tyrosine, threonine.
294 Study Guide
36. Two examples of reversible factors that control the catalytic capability of an enzyme are:
______________, ________________
[noncovalent modifications, pH and pKa changes, [salt] changes, possibly others]
37. Two two examples of irreversible factors that control the catalytic capability of an enzyme are:
______________, ________________
[covalent modification, proteolysis, irreversible inhibitors, possibly others]
38. The Arrhenius equation accounts for the temperature dependence of the rate of a reaction.
39. List the two “chemical modes of catalysis”. acid-base, covalent
40. List the two “binding modes of catalysis”. proximity effect, transition-state stabilization
41. A nucleophile attacks an electropositive site in its role in a chemical (enzymatic) reaction.
42. A common process used to produce the species in problem 41 is: acid-base catalysis.
43. The most common amino acid used by enzymes to carry out acid-base catalysis is histidine.
44. A catalytic triad of amino acids is typically present in (enzyme class name) serine proteases.
45. The amino acids collaborate to accomplish acid-base catalysis.
46. The most typically cited currency of energy in metabolism is (abbreviation) ATP.
47. Mg2+ is typically required to achieve optimal activity with (answer 46)-cosubstrate enzyme reactions?
48. A coenzyme is either a loosly bound cosubstrate or strongly bound prosthetic group.
49. The heavy metal molybdenum is used to facilitate the biochemical reaction in xanthine oxidase, a key
enzyme in purine catabolism.
50. When ATP used in some biochemical applications it yields AMP and pyrophosphate.
51. The (vitamin) nicotinamide is required to synthesize coenzyme NAD+ for use in metabolic redox
reactions.
52. The other key redox coenzyme is abbreviated FAD.
53. The coenzyme pyridoxal phosphate often forms a Schiff base with the ε-amino group of a lysine
residue in the enzyme.
54. What chemical group does coenzyme A typically carry in the course of its biochemical function?
acetate
55. The biotin-avidin noncovalent binding interaction is used to capture ligand-binding entities in the
“affinity capture” technique.
56.The coenzyme N5, N10 methylenetetrahydrofolate is required to incorporate the methyl group into
thymidine, a necessary prerequisite for the production of DNA.
57. Our understanding of this function can be used in a strategy for (treatment technique)
anticancer chemotherapy.
58. The coenzyme bound carbohydrates UDP-galactose and glucose are required to synthesize lactose?
Study Guide 295
59. Cis-retinal functions in transducing the signal of a photon of light into a chemically recognizable form?
60. The two important straight-chain forms of carbohydrate structure are the ketose and aldose.
61. The two important ring forms of carbohydrates are the pyranose and furanose.
62. The two important ring conformations of β-D-glucopyranose are the chair and boat.
63. The cyclohexane ring containing the compound inositol triphosphate is released by phospholipase C in
the phospholipid signal transduction mechanism.
64. The acronym NAG is used to abbreviate the name of the compound N-acetyl glucosamine.
65. The key polysaccharide in starch is amylopectin.
66. The key polysaccharide in the liver is glycogen.
67. The antibiotic penicillin selectively inhibits cell wall peptidylglycan synthesis in bacteria.
68. Extra-cellular surface carbohydrates regulate the osmotic pressure around cells.
69. Phospholipase C produces two different second messengers in the phospholipid signal transduction
pathway. The lipid-containing second messenger is diacylglycerol.
70. The compound chondroitin sulfate lubricates cartilage and skeletal joints.
71. Saturated / unsaturated (circle one) fatty acids of the same length have a lower melting temperature (Tm).
72. Lipid Tm values monitor the transformation from liquid crystal to dispersed forms.
73. Lipid bilayers are composed of two face-to-face monolayers while lipid micelles form a biphasic sphere.
74. The most popular model for a biological membrane is called the fluid mosaic model.
75. The four nucleic acid bases in RNA are adenine, guanine, cytosine and uracil.
76. The two normal base pairs in DNA and RNA are called Watson-Crick base pairs.
77. The glycosidic bond in a nucleoside connects the base to the sugar.
78. The absorbance at 260 nm can be used to determine if 2 single strands of DNA or RNA form a double
helix.
79. The face-to-face interaction between nucleic acid bases is called base stacking.
80. Counterions bind all nucleic acids and are required to neutralize the phosphodiester phosphates.
81. Protein complexes called histones serve this counterion function in the case of most chromosomal DNAs.
82. G•C / A•T (or A•U) (circle one) base pairs are less stable than G•C / A•T (or A•U) (circle one) base
pairs.
296 Study Guide
83. Two differences between A and B forms of DNA are

A-form B-form
1. ___________________________ versus ___________________________.
2. ___________________________ versus ___________________________.
3’ endo sugar conformation 2’ endo sugar
base pairs tilted 20° from helix axis bp’s perpendicular to helix axis
central axial cavity in the helix base pairs cross center of helix
shorter, squatter helix longer, narrower helix
84. The 2’-hydroxyl group catalyzes alkaline hydrolysis of RNA, a good example of anchiomeric
assistance in a non-protein biomolecular mechanism.
85. An antisense oligonucleotide functionally inactivate a mRNA for use in translation by a ribosome by
forming a double helix with it and precluding tRNA anticodon binding.
86. Name the two most prevalent of the four classes of RNA. ribosomal RNA and transfer RNA
87. Two distinctive features of most eukaryotic mRNAs are

___________________________ and ___________________________
7 +
[m G (5’-5’) cap, monocistronic, contains introns and exons, poly(A) tail]
88. A DNA probe is used to detect the presence of a specific complementary nucleic acid sequence.
89. Restriction endonucleases are required to produce, manipulate and clone specific pieces of DNA?
90. The two functional ends of transfer RNA are the anticodon and amino acid acceptor.
91. The three most central catabolic pathways of intermediary metabolism are
glycolysis, Krebs Cycle, and electron transport/oxidative phosphorylation
92. The four major compounds in which energy is captured in a chemically usable form by metabolic
reaction pathways are ATP, NADH, FADH2, and Coenzyme QH2.
93. The mass action ratio (Q) corrects for deviations from standard state concentrations (1 M).
94. Three (number) steps in glycolysis control most of the flux through the pathway under actual cellular
conditions?
95. What do the reactions in problem 94 have in common? They are metabolically irreversible.
96. In contrast, the rest of the reactions are near equilibrium.
97. The kinetics of an enzyme reaction are most easily controlled when KM is approximately equal to the
actual concentration of the reactant.
98. The enzyme triose phosphate isomerase converts dihydroxyacetone phosphate into glyceraldehyde-3-
phosphate.
99. When citrate negatively regulates (discourage) the phosphofructokinase-1 reaction, the general name
for this phenomenon is feedback inhibition.
Study Guide 297
100. When fructose-1, 6-bisphosphate stimulates the pyruvate kinase reaction, the general name for this
phenomenon is feed-forward activation.
101. The three possible catabolic fates of pyruvate are acetyl CoA, ethanol and lactate.
102. The enzyme alcohol dehydrogenase converts acetaldehyde to ethanol.
103. Dihydrolipoamide acetyl transferase uses the coenzyme lipoic acid in fueling the Krebs Cycle?
104. What symport reaction accompanies import of pyruvate into the mitochondrion and what enzyme
catalyzes the reaction? (enzyme name) pyruvate translocase
105. The two oxidative decarboxylation reactions of the Krebs Cycle are catalyzed by isocitrate
dehydrogenase and
α-ketoglutarate dehydrogenase.
phosphorylation” reaction of the Krebs Cycle. succinyl CoA synthetase
107. The enzymes fumarase and malate dehydrogenase “fix” a carbonyl group on succinate in the
production of oxaloacetate.
108. What crucial 2 carbon compound is then “fixed” to OAA? acetate
gluconeogenesis in mammals? alanine and lactate
110. What energy sources are used to produce the protonmotive force? NADH, CoQH2, FADH2 (indirectly)
111. What enzyme complex uses this phenomenon as the driving energy for ATP synthesis in oxidative
phosphorylation? fo f1 ATP synthase (ATPase accepted, but not synthetase)
112. How does electron transport drive production of the protomotive force? exports H+ from
mitochondrion (which creates a gradient, making them predisposed to flowing back in).
113. How many reactions does each round of β-oxidation of a fatty acid require? four (oxidation #1,
hydration, oxidation #2, thiolysis)
114. What are the products of one round of β-oxidation and what’s the tally in terms of ATP equivalents of
energy conserving products? 1 CoQH2, 1 NADH, H+, 1 acetyl CoA, 1 fatty acid (minus 2 Cs)
115. A set of coupled cofactor regeneration cycles siphon off reducing equivalents then fix them into
coenzyme Q in reactions that are coupled to the first oxidative step of fatty acid β-oxidation. Write down
the names of the four cofactors involved in this siphon. CoA, FAD/FADH2, Fe-S2+/3+, CoQ/CoQH2
116. Which three steps of the Krebs Cycle do the first three steps of the fatty acid β-oxidation cycle
resemble? succinate dehydrogenase, fumarase, malate dehydrogenase
INDEX
Index Terms Links
A205 30
A260 [ds, Tm, ε260] 127 128 238 239
271 287
A280 30 257
A605 [Tm] 103 128
α-adrenergic [receptors] 124
α-amino [acids, group] 26 46
absorbance [Beer-Lambert,
molar, photoisomerization,
spectroscopy] 21 22 30 83
91 127 128 205
230 238 239 257
270 273 287 295
abzyme 73
acceptor [end, species, stem] 49 70 71 146
147 165 173 178
186 241 244 277
296
acetaldehyde [acetic (acid), ethanol] 13 82 172 178
249 297
acetaminophen 108
acetic (-ate) [acid, choline] 12 13 61 85
123 244 249 250
251 279 294 297
acetoacetate [decarboxylase] 76 84 229
acetoacetyl [acetoacetyl-ACP,
CoA, ketoacyl-ACP] 197–198 201 255 280
acetone 76–77
acetyl-ACP [ketacyl] 197 198 255 280
acetyl-CoA [carboxylase,
lactate, NADH] 85 174 198–199
This page has been reformatted by Knovel to provide easier navigation.

Index Terms Links
acetylcholine [anionic,
esterase, receptor, release] 61 118 225 265
282–283 293
acetylsalicylic [acid] 108
acetyltransferase 249
acid-base [catalyzed (-sis), ionization] 12 14 71 74
76–77 226 262 265
284 294
acidic (-ity) [neutral, pKa, tartness] 23 75 89 90
110 200 216 250
251 259
acidosis 177 182
aconitase (-ate) [aconitate,
rearrangement] 68 180 181
ACP [acetyl, CO2, CoA,
malonyl, reductase,
regulation, synthase,
transacylase] 85 197–199 255 280
actin [filaments] 5 115
activate [protein,
transcription] 64 70 122 145
212 275
activated [acetyl, coenzyme, Gp, protease] 17 56 63 64
66–68 85 90 120–122
124 146 153 154
183 198 206 212
225 228 249 262
263 265
activates [ gluconeogenesis,
glycogen, phosphate, PKC,
plasminogen] 17 64–65 67 81
118 119 121–124 141
149 183 185 230
233 275
active [MPF, site, transport,
zymogen] 5 10 27 41
43 49 55 61–69
71–73 75 78–80 83

Index Terms Links
active [MPF, site, transport, zymogen] (Cont.)

87 89 92 108
110 116–118 121 143
146 170 181 183
185 186 192 209
210 225 226 234
249 252 265 266
271 275 283 293
activity [assays, range, rate] 27 28 43 45
51 52 58 65
67 69 74 75
81 97 101 107
108 120 123 159
177 183 185 210
226 263 284 293–294
acyltransferase 179
adenine [ade, dinucleotide] 1 4 82–83 90
125 131 155 213
228 236 274 295
adenosine [monophosphate, triphosphate] 1 4 81 119
131 274
adenosine-3’, 5’-diphosphate 237
adenosylcobalamin
[methylmalonyl] 88
adenylate [cyclase] 121 147 275
α-D-glucose 97
α-D-Glucose-1-phosphate 68
α-D-glucose-6-phosphate 261
α-D-glucosamine [NAG] 260
adipose [tissue] 198
A-DNA 133–135 239
ADP-ribosylation
[nucleotide] 70 124
adreneline [regulates] 124
adult [hemoglobin] 49 194
aerobic [metabolism] 178 186

Index Terms Links
affinity [capture,
chromatography, -labeling] 27 28 48 49
58 65 86 100
106 229 266 294
agarose [bead, gel] 86 155
aging 141
α-helix (-ices) 34 35 37 46
112 114 220 222
281 292
AIDS 2 62 228
α-ketoglutarate [αKG, dehydrogenase] 63 84 164 172
180 181 183 193
251 297
alanine [A, ala] 20 21 23 31
37 63 84 113
184 185 251 297
albumin [BSA] 30 162
alcohol [aldehyde,
dehydrogenase, glucose,
pKa, side-chain, sugar, xylitol] 9 22 74 82
91 94–96 102 110
175 178 215 247
249 258 289 297
aldehyde [acetaldehyde, ketone, regenerated] 9 74 76 84
91 95 101 215
249 280
aldimine 83
aldol [condensation] 278
aldolase 94 169 174 176
208 246 278
aldose [monosaccharides, pyranose] 93 94 227 230
285 295
aldoximine [methiodide] 61 225 283 293
algae 209–210
aliphatic [alanine, aromatic, side-chain] 15 20 75 215
259

Index Terms Links
alkaline [basic, hydrolysis,

pH, phosphatase, solution] 23 30 74 126
129 132 152 157–158
162 238 259 287
296
alkaline-labile [digoxigenin-11-deoxyuridine] 161
alkane [alkenes, chain, linkage] 15 18 39 102
164 165
alkene [chain] 15 102 165
alkoxide 152
allantoin (-ate), (-icase) [urea,
urease] 213
allosteric (-ism) [ activator,
binding, control, effector,
feedback, inhibitor,
regulator, site] 13 45 49 65
66 70 124 170
176 183 194 212
225 263 277 283
293
allysine 44
alpha [α, carbon, helices] 18 20 35 264
269
alternating [base-sugar, CG - field gel, phosphodiester,
purine-pyrimidine, sugar] 125 134 135 155
270
alternative (-ly) [folded, splicing] 40 57 58 71
149–150 178
alveoli 19 49
amide [bond, C=O, cation, hydrogen
imide, linkage, nitrogen, phosphate] 9 14 33 35
36 71 74 77
100 250 258
amine [group, hydrogen, nitrogen
pKa, secondary, tertiary, transfer] 9 20 35 63
71 74 84 215
aminoacyl [tRNA] 41 147 149

Index Terms Links
aminoacylate (-ed) (-ion)

[charged] 147 149
amino [terminus] 79
aminotransferase 84 185
+
ammonia [ H3N-] 211 213
ammonium [cation, NH4+, sulfate] 9 20 25 27
29 39 211 259
272
AMP [cAMP, cyclic, phosphodiesterase] 81 121 124 169
174 176 185 211
228 265 275 294
amphibians [freshwater] 213
amphipathic (-ity) 18 39 102 111
257
amplified (-ication) 64 159 225 263
283
R
Amp 156
amylase 98
amylopectin [starch] 98 231 233 286
295
amylase [unbranched] 98
anabolic 165 174
anaerobic [conditions,
glycolysis, metabolism] 177 178 186
anaerobically [metabolized] 178
analog (-s) [methotrexate] 73 124 229
anaphase [telophase] 6
anchiomeric [assistance] 296
anchor (-ed) (-s) [CHO, extracellular,
glycosphingolipids, membrane-bound, proteins] 100 111 114 115
236 268 269
angles [Cα, rotation] 33 37 96 134
258 271 292
anhydrase [zinc] 19 81 89 228
anhydrides [hemiacetals] 215
animal [cell (-s)] 2 3 73

Index Terms Links
anion (-s) 14 19 28 39
61 92 100 115
198
anion-exchange [protein] 115
ankyrin [glycophorin, spectrin, anchors] 115
anneal (-ed), (-s) [hybridization, primer] 13 127 153 159
160–161 239 241 274
annexins 106
anode 171 187
anomer [down, up] 95
anomeric [linkages] 97
antenna [complex, pigments] 36 91 204–206
anti [conformation, glycosidic, syn] 132–135 270–272
antichaotrope [salt] 39
anti-cancer [chemotherapy] 285
anti-digoxigenin [immunoconjugate] 162
anti-inflammatory [activity] 97
antibacterial [agent] 100
antibiotic (-s) [penicillin, resistance, valinomycin] 20 99 117 141
157 295
antibodies [abzymes, characterizing, circulate,
immunological] 50 73 100 145
151 233
antibody [production, recognition, repertoire, tags] 32 50 73 101
214
antibody-binding [antigen] 161
anticancer [chemotherapy] 227 294
anticodon [arm, binding, D, loop, sequence, trinucleotide] 132 146–148 241 272
296
antidote [pyridine] 61 225 265 283
293
antifreeze 93
antigen [binding,
digoxigenin] 7 50 73 97
101 151 161 162
antiparallel [β-sheets, chains, strand-strand] 34–37 130 137
antiport [exchanges, glutamate-aspartate] 119 193 250 280
antiprotozoan [drug] 58

Index Terms Links
antisense [binding duplex, mRNA, oligonucleotide,

RNA, strand, triplex] 8 136 137 238
240 271 287 296
ApaI [digestion] 155
apolar hydrocarbon 104
apoprotein [holoprotein, rhodopsin] 45 91
apoptosis [programmed] 153 213
aptamer 138
arachidonate (-ic acid) 102 107–108 199 267
272 273
arachidonoyl [CoA] 199
arginine [arg, guanidinium, leaving, R] 3 22 23 25
30 41 68 80
113 124 141 211
212 239 258 266
arginase [arginine] 212
argininosuccinate [lyase, synthase] 211 212
argininyl 256
argonaut [ribonuclease, siRNA-dependent] 153
arm [acceptor, anticodon, TψC] 146–148
aromatic [side-chain] 15 21 30 75
215 257
Arrhenius [equation] 70 226 283 294
ARS 141
arterial [walls] 107
arteries 106
arthritis 62 151
artificial [catalysts, chromosomes, sweetener] 26 32 73 141
ascorbate (-ic) [acid] 90 96
aspartame 26
aspartate [asp, carbamoyl, glutamate, transaminase,
transcarbamoylase, translocase, α-ketoglutarate] 22 49 63 66
67 71 75 76
81 84 113 119
193 194 211 212
214 249 260–262
aspirin [acetyl, acetylates, acetylsalicylic] 97 107 108
ATCase-catalyzed [reaction] 66 67

Index Terms Links
atherosclerosis [cardiac, ingestion] 106 108

atmospherically (-ic) [bound, O2] 130 210
α-tocophorol 90 91 104
Atorvastin [Lipitor] 203
ATP (-s) [equivalents] 81 169 182 211
247 290
ATP-binding [domain,
enzymes] 28 34
ATP-dependent
[carboxylation,
decarboxylation] 86 201
ATPase (-s) [ions, Na+, pumps] 116 119 173 186
191 244 252 277
297
autoimmune [diseases, antibodies] 62 151
autonomous [replication] 141
autophosphorylation [tyrosine] 124
avidin (-biotin), (-binding),
(-agarose) [affinity, bead, protein] 86 161 227 229
axial [cavity] 133 239 296
AZT [3-azido-2] 62
A•T [pair] 125 130 238 287
295
A•U [base] 130 131 238 287
295
α-ketoglutarate [succinyl] 183
B1 [thiamine] 87
B12 [cobalamin] 88 208 228
B6 [Schiff] 83
backbone (-s) [amide, atoms,
backbone, conformations,
phosphates, zig-zagged] 35 36 104 129
132 134 137 139
140 146 152 270
292

Index Terms Links
bacterial [acetyl-CoA, cells, membranes] 4 7 99 100

104 154 182 199
233
bacteriophage [DNA, lambda] 7 131 155
bacteriorhodopsin [ATP, protons, pumped] 112 113 116 192
bacterium [DNA, Micrococcus, plasmids, Thermus] 7 101 112 154
157 159
bactoprenol [undecaprenyl] 110
baking [brewing] 249
β-alanine 85
BamHI 156
base (-base) [interaction,
stacking] 238 271 287
base-catalyzed [hydrolysis] 129 158
base-pair (-ed) [stacking] 138 239
base-sugar [juxtapositions] 134
β-barrel (-s) [β-sheet, domain (-s)] 36 50
BCA [assay] 30 257
β-carotene [oxidative] 91 204
β-D-galactose 232
β-D-glucoside (-pyranose) 111 112 231 285
295
B-DNA (-form) [A-DNA, Z-
DNA] 132 134 135 139
140 239 296
β-D-ribose 230
bediate [translocation] 44
beer 178 247 289
Beer-Lambert [law, relation] 21 22 127 257
behenate 102
Benedict [test] 101
bent [DNA] 136
benzamide [inhibits] 266
benzene [alkanes, lignins] 15 97
benzoate [PABA] 87
benzyl [β-carbonyl] 9 76 258
benzylic 97
β-estradiol [sodium] 109

Index Terms Links
beta [oxidation, sheets] 35 196

BglII [site] 156 274
β-hydroxybutyrl-ACP
[acetoacetyl-ACP,
dehydrase, dehydration] 197 198 255
+
bicarbonate [anion, Na ] 19 86 89 198
bifurcated [H-bond] 77
bilayer [channel-forming,
dipole, head, stability,
surfaces] 14 105 106 113
117 234 236 268
269
bilayers [vesicles] 103 105 106 115
231 286 295
bile [excretion, salt] 109 112 269
biocoating [materials] 98
bioconjugate 32 112
bioelectrochemistry 72 171–173
bioenergetics 72 167–170
biotin [biotin-avidin, carboxylase, cofactor,
ligand, vitamin] 86 161 179 198
199 227 229 273
biotin-avidin [affinity, noncovalent] 86 229 285 294
biotinylated [DNA] 32 86
bisphosphate [-1, 3-phosphoglycerate,
carboxylase, PFKase] 94 175 185 204
208 209 246 278
bisubstrate-enzyme [ping-pong] 62 225 283 293
Biuret [method, reaction, technique] 30 257
β-lactoglobulin 177
blood [buffering, cells, clot (-ting),
CO2, flow, glucose myoglobin] 18 19 45 48
50 64 73 89
97 100 107 110
112 124 138 159
177 185 225 283
293
blot [filter, filter-bound, hybridization, membrane] 161 162

Index Terms Links
β-mercaptoethanol [BME] 32 258

boat [conformation, form] 96 231 232 285
295
Boltzmann [constant, kinetics] 71 273
bone (-s) (-y) [fish, growth, muscle] 36 100 213 234
bovine [serum] 30 162
β-oxidation [cycle, fatty, pathway, yields] 70 181 253 254
280 290 297
bp [rise] 132–134 155
BPG [hemoglobin, inhibits,
oxyhemoglobin, stabilizes] 13 46 49
Bradford [assay] 30
brain [gray, glucose, ribose-5-phosphate, tissue] 97 102 163
branched [polymer] 98 233
bridge [reaction, Zn2+] 20 38 83 85
87 88 171 178
182 183 187 229
244 277
bromide [cyanogen, modifies] 10 32
BSA [blocked] 30 162
β-sheet (-s) 34 36 37 220
221 281 292
budding 8
buffer (-ed), (-s), (-ing)
[reservoir, solution] 19 27 28 30
89 159 162 234
butanoyl-ACP (-tenoyl-ACP),
(-tryl-ACP) [β-hydroxybutryl-ACP] 197 198 255
C-terminus (-i) [modified, N-terminus] 26 31 33–36 46

50 77 114 144
257 258
C2’ [endo] 239 270
C3’ [endo, sugar] 96 239 270
C4/CAM [pathways] 208–210
C5-methyl [group] 131
2+
Ca -binding [grooves] 110

Index Terms Links
caffeine [theophylline] 124

calcium [Ca2+, enters,
metastasis, released] 81 109 119 122
124 265
calmodulin 81
calorimetry [spectroscopy] 80
Calvin [cycle] 5 87 208–210
cAMP [activates, cyclic, cytidine, IP3, protein] 120 124 185 265
272–275
cancer [antisense, cells, chemotherapy, grows] 62 87 101 123
136 141 194 229
canonical [A-form, duplex] 132 133
cap [monocistronic, structure] 42 132 150 240
272 296
capillary [beds, electrophoresis] 48 195
capsid (-s) [organelles, protein, reverse, RNA, tail] 6–8 15
capture [ligand-binding, technique] 53 86 204 208
227 229 285 294
carbamate [carbamoyl] 211
carbamoyl [aspartate, phosphate] 66 67 211 212
260 261
carbonic [acid] 19
carbanion [abstracts, cleavage] 74 86 209
carbocation 71 201
carbohydrate (-based) (-s)
[analytes, chains, conjugates, polymers,
recognition, sequences, UDP-galactose] 1–3 50 51 70
90 93–101 104 111
200 209 210 231
233 268 286 294
295
carbon [fixation] 208 209
carbonate (-ion) [ATP, CO2] 19 178 211 247
289
carbonic [acid, anhydrase] 19 81 89
carbonyl [acyl, C=O, carbon, group (-s),
intermediate, oxygen] 9 17 33 35–36
38 71 77–79 130

Index Terms Links
carbonyl [acyl, C=O, carbon, group (-s),

intermediate, oxygen] (Cont.)
131 148 196 209
211 216 247 262
290 293 297
carboxybiotin 86 198
carboxyglutamate [waxes] 110
carboxykinase 184–185
carboxylase [ATP, pyruvate, RuBisCO] 86 94 110 179
184 185 198 199
204 208
carboxylic [acid] 9 215
carboxymonoester [phosphodiester] 129 270
carboxypeptidase [B] 31 63 258
carcinogen 123
cardiac [muscle] 45 106 108
cardiolipin [sphingolipid] 104
cardiotonic [steroids] 119
carotenoids [phycocyanin] 205
carrots [β-carotene] 91 204
cartilage [bone, cushioning] 100 231 234 286
295
cascade (-s) [regulates, stages] 64 124 225 263
283 293
caspases 214
catabolic (-ism), (-ally) [degradation, fates,
intermediate, pathway(s)] 44 70 163 165
185 228 242 247
277 288 289 294
296–297
catalysis [acid-base, binding, chemical, proximity] 1 17 53 55
56 64 65 68
70–78 80 83 84
101 168 224 226
228 232 240 247
262 265 282–284 289
292–294

Index Terms Links
catalyst (-s) [, donating, hexokinase] 1 51 56 73

77–78 180 181
catalytic [activity, antibodies, efficiency, groups,
mechanism, rate, reaction, site, subunit, transfer,
transformations, triad] 14 17 27 43
49 53–55 59 60
66 69 71–74 77
101 124 146 147
150 170 225 226
240 262 263 265
283 284 293 294
catalyze [activation, hydrolysis, intron,
metabolically, reactions, regulate, selective] 51 64 73 108
122 147 150 168
169 176 194 199
208 211 242 288
catalyzed [Eact, peptide,
reaction] 1 19 41 57
64 67 69 71–74
84 85 143 169
184 189 193 196
198 210 212 226
240 243 244 277
278 284 293 297
catalyzes [ADP-ribosylation, aldol, alkaline, chromosome,
DNA, formation, H+, hydrolysis, methylation,
phosphorylation, proteolysis, transfer] 1 66 89 120
124 126 143 152
154 155 158 169
177 181 183 185
198–200 203 208 247
266 273 278 279
282 289 296 297
catalyzing [ATP] 179 192 293
cathode 171 187

Index Terms Links
cation (-s) (-ic) [conditions,

charges, diethylaminoethyl,
gradients, type] 28 33 39 46
92 117 130 137
239 272
CD4 [HIV] 8
cDNA (-s) 31
CDP-diacylglycerol 200
cell-cell [adhesion] 2 233
cell-mediated [immunity] 50 64
cellular [adenosine, compartments, conditions,
development, DNA, energy, environments,
function, glutamate, malfunctions, membranes,
movement, niches, receptors, redox, respiration,
response (-s), RNA, salts] 2 4 8 18
20 27 48 92
97 102 115 116
120–124 146 157 167
169 170 173 182
194 212 225 242
275 288 296
cellulose [fruit] 99 210
center-to-center [distance] 16
central [axis, cavity, cobalt, dogma, domain,
hydrophobic, ion, Zn2+] 1 9 46 49
52 58 88 105
133 137 138 144
148 182 191 209
239 242 277 288
296
centrifuging 27
centriole [vesicles] 2
centromeres 141
cerebrosides [gangliosides, steroids] 102 273
CG 135 270
chair [boat, conformation, polymer, ring] 96 231 232 285
295

Index Terms Links
channel (-s) [blockers, Ca2+,-forming,

opening, releasing] 112 117–119 122 124
191 192 275
chaotrope (-s), (-ic) [action, osmolytes, salt, urea] 39 70 222 259
281 292 293
chaperone (-s) [assist, hsp70,
proteins] 41 233
chaperonin (-s) [complex] 41 42 106
charge [complementarity, distribution, gradient,
impermeable, migrates, relay, repulsion,
separation, transfer] 17 20 23–25 28
33 68 71 75
77 83 84 106
116 118 130 131
134 136 182 188
216 239 256 259
262 270 291
charge-charge [charge-dipole, interactions] 16 25 38 72
charged [buffer, DNA, groups, oxygen, proteins,
serine, side, tRNA] 14 17 28 38
46 49 78 105
116 149 186 262
checkpoints 6 69 263
cheese [yogurt] 177 249
chelated (-ion) [iron] 45
chemical [modes] 283 294
chemiosmosis (-tic) [theory] 192
chemotherapy (-eutic) 62 87 227 285
294
childhood [leukemia] 62 204
chiral (-ity) [carbon, center, compound, isomer] 10 11 51 93
95
chitin 9
chloroform 158
chlorophyll (-s) [antenna, Chl, P700, special] 81 204–207
chloroplast (-s) [ATP, electron-driven,
genome, NADH] 3–5 41 173 191
192 210 215 274

Index Terms Links
cholate 109
cholecalciferol 92 104 109
cholera [catalyzes] 124
cholesterol [biosynthesis, lanosterol, levels, lipids,
metabolism, solidifies, squalene, synthesis,
testosterone] 102 106 109 200
202–203 236 269
cholic [acid] 112
choline (-esterase) [glycerol] 61 104 231 234
268 273 286
chondroitin [sulfate] 100 231 234 286
295
chromatin [fiber] 144 213
chromatograms, (-graphic),
(-graphy) [column, elution, HPLC, plating] 27–30 86 100 229
257
chromosomal [DNA, ends, nucleic, typing] 127 131 141 143
155 238–240 287 295
chromosome (-s) [5’, condensation, end,
plasmids, YACs] 1 4 5 8
138 141 143 154
155 159 239
chylomicrons 109 203
chymotrypsin (-trypsinogen) 31 63 71 79
155 257
circular [dichroism, species] 101 142
circulatory [system] 44 48 89 124
cis-retinal [rhodopsin, trans- retinal] 91 227 230 265
285 295
cis-trans [isomerization] 91
citrate [acetyl, fructose-1- phosphate, fumarate,
isocitrate, negatively, pyruvate, SSC, synthase] 68 85 162 164
174 176 179–181 183
243 244 246 251
264 276–278 280 289
296
citric [acid] 163 165 179–182
citrulline 211 212

Index Terms Links
clearance [destruction] 100 231 233 286

clone (-ed), (-ing)
[expression, gene, plasmids, techniques] 7 31 39 154
156–159 238 272 288
296
closed-circular [DNA] 141
clot (-s), (-ting) [dissolution, factors, fibrin, formation] 64 73 100 107
110 124 138 225
283 293
cloverleaf 146 272
CoA-ACP [transacylase] 199
CoA-S-R 70
CoA-SH [carries, citrate, oxaloacetate,
succinyl-CoA, TPP] 67 70 85 179
181 183 242 244
245 249 250 264
277 279 280 288
coagulation 64 104
coat [cross-linkage, protein] 138 142 233 257
cobalamin [methylated,
transfers] 88 208
cobalt (-vitamin ) [B12] 88 228 265
codon (-s), (-anticodon) [encode, loop,
nucleotide, usage] 140 147 148 241
272
coenzyme (-s) [5-methyltetrahydrofolate, A,
biotin, carrier, cofactor(s),lipoic, NAD+, pyridoxal, Q,
redox, retinal, vitamins] 1 4 17 32
63 67 68 71
76 81–93 109 113
161 165 173 179
186 192 196 197
200 208 227 228
244 247 250 253
264 265 277 279
280 284 285 289
290 294 296 297

Index Terms Links
cofactor (-s) [3-phosphoadenosine-5-phospho-

sulfate, NADH, regeneration, vitamin] 45 51 67 70
81 86 88 90
178 188 200 228
247 249 253 265
289 290 297
collagen [fibers, helix] 2 37 44 34
90 99 100
colony [hybridization] 161
colorectal [cancer] 13
column [avidin, matrix, packed] 27–29 86 126 229
257
combinatorial [, binding, DNA/RNA, permutation] 126 145
compartment (-s) [entropy, proteins] 2 5 15 17
63 105 147 194
competitive, (-ly) [enzyme, inhibition, inhibitor,
noncompetitive] 59 73 203 224
264 282 293
complement (-arity)[antibody, base(s),
DNA, RNA, sequence, single-stranded] 7–8 45 50 126
127 131 136 141
146 150 153 154
161 241 271 296
condensing (-ed), (-ation) [enzyme, nuclear, reaction,
reduction, stage, step, theory] 5 27 130 180
197 198 200 202
239 253 278 290
configuration (-s) 10 17 20 38
70–73 105 149
conformation (-s), (-al) [accessibility, anti, boat,
change, heterogeneity,isomer, sugar,
torsion, transition] 33 35 37 40
41 49 60 68
71 96 119 131–135
139 231 232 239
258 270 272 285
292 295 296

Index Terms Links
conjugate (-s), (-d) [acid, base, peptidoglycans] 12 31 45 47

71 74 99 216
259
Coomassie [blue] 30
cooperative (-ity) [formation,
hemoglobin, ligand, melting, process, substrate,
titration, V0] 40 45 47 48
53 65 70 73
127 170
copper [ion] 171 190
copy [DNAs, number] 1 8 31 136
CoQ [CoQH2, Fe-S, NADH, oxidized, ubiquinone] 92 172 180 181
188–190 196 244 251
254 255
CoQH2 [CO2, energy, FAD, FADH2, Fe, NADH,
oxidative (-ized), phosphoryation, reduced,
succinate, ubiquinol] 4 92 163 165
172 173 180 182
186 189 190 196
244 251 252 254
255 277 280 297
core [nucleosome, particle] 89 105 141 144
234
coronary [heart] 109 203
corrin [ring] 88
counterion [bind, condensation, nucleic,
offset, theory] 130 139 171 238
270 287 295
coupled [chemical, electron, reactions, redox,
transcription-translation] 5 19 27 148
173 186 190 242
244 252 253 277
288–290 297
covalent [antibody-enzyme, bonds, catalysis, lipid-
carbohydrate, modification, protein-inhibitor] 14 16 61 63
65 70 71 76
78 83 102 114
148 162 179 203

Index Terms Links
covalent [antibody-enzyme, bonds, catalysis, lipid-

carbohydrate, modification, protein-inhibitor] (Cont.)
227 236 262 264
275 285 293 294
crassulacean [acid] 210
CREB [binds, protein] 43
Crick [base] 125 132
crista [matrix] 4
Crohn’s [disease] 62 87 151
crosslinks [desmosine] 32 44
cruciform [DNA, secondary] 145 138 272
crystal [formation,
membrane-like, states,
structure] 34 102 103 106
128 234 273 295
crystal-to-dispersed [equilibrium] 128
crystallography 37 136 138 147
Cu+1 257
current [amperage, flows, protocols] 28 149 171
cutting [experiments, sites] 140 154 156 157
cuvette [molar, pathlength] 21 127 257
cyanobacteria 209 210
cyanogen [bromide] 32
cyclase [inhibitory, signaling] 121 275
cyclic [adenosine, AMP,
pathway, phosphodiester,
reduction, ring] 60 69 95 121
124 150 164 179
180 185 188 239
265 274 275
cyclin (-dependent) [heterodimer, kinase,
monomer] 6 69 225 263
283 293
cyclohexane [epoxide, ring] 204 232 295
cyclooxygenase [COX-1, COX-2] 107 108
cysteine (-s), (-yl) [(cys, Cys-SH, cystine] 22 23 26 30
32 38 89 113
114 214 256

Index Terms Links
cystine 30 38
cyt [adenine, base, guanine] 125 188 190 236
cytidine (-s) [N1-methyl,
sequence, triphosphate] 66 131 134 148
272
cytidine-2’, 3’-diphosphate 267
cytochrome (-s) [a, a3, b3, b560, b566, bf, c, c1, CoQ, O2,
oxidase, protons] 92 172 188–191 205–207
244
cytokinesis [cytoplasmic,
division, prophase] 5 6
cytoplasm (-ic) [division,
NADHs, plasma, surface] 2 3 5 8
115 148 149 186
194 210 211 247
250 252 269 289
cytosine [cyt] 125 236 295
cytoskeleton (-al) [lipid, network, protein] 5 115 154 233
cytosol (-ic) [aspartate, enzyme, extracellular,
malate, mitochondria, NADH, nucleus,
oligosaccharide, organelles, protein,
tyrosine] 32 98 111 112
118–123 179 193 194
198 213 268
D3 [cholecalciferol] 92
Δ -(Δ -) desaturase
5 6
199
DAG [G-proteins, IP3, phospholipase, PKC] 108 120–124 233 234
275
D-arabinose 93
D-arm 148
dATP 159
D-β-hydroxybutanoyl-ACP 198
D-β-hydroxybutryl-ACP 197
dCTP [dGTP] 15
ddNTPs 31
dead-end [inhibitor] 61

Index Terms Links
decarboxylate (-ion), (-ed), (-ase) [acetaldehyde,

aconitase, mechanism, NADH, pyruvate,
rearrangement] 68 76 84 85
87 178 180 183
201 229 247 249
264 280 289 297
decoupling [reagent] 27 106
dehydrase [condensation] 197 255
denaturation (-renaturation) [equilibrium
melting, midpoint, studies, unfolding] 28 38–41 70 71
127 128 159 292
deoxyadenylate 133
deoxycholate 111 112 269
deoxyribonucleic [acids] 4
deoxyribose [alternating, central, conformations] 96 125 126 129
133 137 270
deoxythymidine (-s) [dT, monophosphate] 87 130 161
deoxyuridine [dU, monophosphate, triphosphate] 87 130 161
dephosphorylated (-ion) (-ed) 6 67 69 70
119 225 244
deprotection [H2O] 27
deprotonated (-ing) [imidazolium, pI, species] 12 19 22–24 47
74 75 256 259
D-erythrose [D-threose] 93
desmosine [allysine] 44
detergent (-s) [action] 105 109 111 112
269
determinant [antigens] 97
dextrorotatory [right-handed] 93 95
DFP 61
D-fructose [ketose] 94 265
D-galactose 94 265
D-glucose [fuel] 94 230 265
D-glyceraldehyde 93 230
dGTP 159
diabetes [mellitus] 44 124
diacyl [glycerol] 108

Index Terms Links
diacylglycerol [DAG, linked, lipid, phosphate] 18 90 100 104

114 122 123 265
273 275 295
dialysis (-yzes) [removes, tubing] 27 29
diarrhea 106
dicarboxylate [translocase] 193
dideoxy [NTPs, sequence] 31
dielectric [constant] 16 25 71 291
dietary [chylomicrons, excess, protein] 106 203 212
+
diethylaminoethyl [DEAE ] 28
differential (-iate) [binding, separation] 27 48 101 136
215
diffracted 34
diffraction [analysis, data] 34 125 132
diffuse [laterally, through] 106 117
diffusion (-limited) [lipid, movement, rates] 55 98 106 117
DIG-11-dUTP (-labeled), (DIG-dUTP) [blot, probe] 161 162
digesting (-ion) [food] 98 155 156 225
274 283
digestive [enzymes] 31 63
digitalis (-oxigenin) [ouabain] 119
digoxigenin-11-deoxyuridine (digoxigenin),
(digoxigenin-dUTP) [DG] 159 161
dihydrofolate [reductase] 62 87 229
dihydrolipoamide (-lipoic) [acetyl, acid,
acyltransferase, dehydrogenase,
hydroxyethylthiaminepyro phosphate] 172 179 247 249
279 289 297
dihydrouridylate (-uridine) 146 148
dihydroxyacetone [phosphate] 75–76 94 174 176
184 208 210 230
246 265 276 278
280 296
diisopropylfluorophosphate [DFP] 61
dimers [active, stacking] 69 140 145 170
dimethylallyl [carbocation, diphosphate] 201
dinucleotide [anticodon, FAD, NADH, repeating] 1 4 82 83
270 272

Index Terms Links
diol [reduced, ubiquinol] 90 92 96 209

254 266
dione (-to-diol) [transformation] 90 92 254 266
dioxide [blood, CO2] 19 89 164 165
178 208 280
dipeptide 26 33
diphosphate [linkage] 90 138 150 200–202
265 273
dipole (-s), (-induced), (-dipole) [attractions,
direction, interactions] 14 16 38 72
138 216 239 259
293
disaccharide (-s), (-repeat) [two, unit] 97 100
discontinuous [replication] 141
disease (-s) [chemotherapy, model, progression,
symptom, temperature-sensitive] 44 58 62 87
106 109 121 136
151 153 203 214
disordered [coil, dissolved, domain,
entropy, pKID, protein] 38 43 105 234
disperse (-d) [fatty, forms, lipid, liquid, ordered] 102 103 105 128
273 295
dispersion [forces] 138 216 239
dissociation (-association), (-ing) [equilibrium, events] 44 117 127 128
271
disulfide (-s) [bond, bridges, formation, oxidation] 3 32 38 43
50 89 94 182
220 223 281 292
293
dithiothreitol [disulfide] 32 94
D-lactose 265
D-mannose 94
DNA-binding (-protein)
[complex, domain, site] 86 89 100 136
145 227 229
DNA-dependent [nuclease] 138

Index Terms Links
DNAs [amino, cDNAs,

computers, RNAs, sugar, triplex] 31 126 127 130
131 134 137 139
145 154 155 158–160
162 238 287 295
DNS [test] 101
dNTPs [DIG-dUTP] 159 161
dodecyl [sulfate] 18 39 194 257
dodecylsulfate [SDS] 162
double-reciprocal [plot] 54
double-stranded [DNA, nucleic, viral] 8 127 139 147
155
D-ribofuranose (-pyranose) 96
D-ribose 93 265 266
D-ribulose 94
D-threose 93 94
dTMP 87 229
DTT [concentrations] 32 94 258
dTTP 62 159
dU 130
dUMP [dTMP] 87 229
duplex (-es) [DNA, denaturation, dissociation,
equilibrium, formation, melting, RNA, sequence,
stability, template] 86 127–129 134 136–139
141–143 146 147 149
154 160 162 239
241 270–274
E0 (E0’) [acetyl, V] 173 189 242 245

288
ε260 [b] 127
ε-amino (-monium) [group] 38 75 76 84
227 285 294
EcoRI [duplex, fragment,
HpaII, reaction, site] 156 157 274
Edman [degradation, technique] 30 32 220 222
281 292

Index Terms Links
effector (-s) [binding,

enzyme, Vmax] 49 65–67 120 124
168 170 263 277
efficacy 58
efficiency 57 170
eicosanoid (-s) [biosynthesis,
prostaglandins] 97 102 107 108
199
elastase 63
elastin 44 100
electrochemical (-istry) [cells,
reaction] 171 186 187 252
electromotive [force] 171 173 187 247
290
electron-transport (-transfer)
[chain, reactions] 165 186 193 196
254
electronegative (-ity), (-ively)
[charged, electron, phosphodiester,
polyanionic, proteins] 14 110 130 239
262
electronic (-ally) [charge,lobes, overlap] 16 71 138 291
electrophile (-s) 62 71 226 284
electrophoresis (-etic) [band,gels, pattern,
SDS, SDS-PAGE, two-dimensional] 18 27–30 39 140
154 155 194 195
257
electrostatic [attraction, interactions, repulsion] 72 135 139 268
elongase (-ation) [reaction, stage] 198 199
elute (-d ), (-ion) [chromatograms, profiles] 28 86 257
emf [voltage] 187 247 290
enamine-ketamine [tautomerism] 33 77 265
enantiomers 95
endo [C2’, C3’, conformation, sugar] 96 133–135 239 270
296
endonuclease (-s) [5’, cutting, enzymes, recognition] 100 154 156 157
159 238 272 274
288 296

Index Terms Links
endoplasmic [reticulum] 2 3 41 44
122 149 215
endorphins 273
enol-keto [contribution,
tautomerism] 33 86 258
enolase 169 175 176
enolization [carboxylation, keto-enol] 209
enolpyruvate [attacks] 86
enoyl-ACP [reductase] 197 255
enoyl-CoA [hydratase] 196
enthalpy [accrued, associated, change] 15 38 102 105
216 259
entropy (-ic) [contribution, cost, energy] 15 17 38 72
105 139 216 259
envelope [fuses, membrane-embedded, nucleus,
proteins, rough] 2 3 6–8 96
enzymatic [cascades, catalysis, cleavage,
oxidation, reaction, synthesis] 54 64 86 91
130 155 167 224
225 228 247 262–265
282 289 292–294
enzyme-catalyzed [reactions] 51 52 62 66
75 141 196 225
226
enzyme-substrate [combination, complex,
interactions] 72 78 293
enzyme-substrate [complex] 293
enzymes [active, kinetics] 75 292
epimers 94
epinephrine [adrenaline] 124
equilibrate (-ed) [two-phase] 18 269
equilibria [hemoglobin,
standard] 23 48 60 167
equilibrium [association,
concentrations, constant,
denaturation-renaturation,
Ka, kinetics, poise] 1 12 17 19
49 53 54 56

Index Terms Links
equilibrium [association,
concentrations, constant,
denaturation-renaturation,
Ka, kinetics, poise] (Cont.)
57 72 82 98
113 127 128 167–170
216 224 226 242
244 270 277 278
282 288 296
ER [70S, activates, lumen] 3 41 124
erythrocyte [cytoskeleton,
membrane] 112 115
erythromatosis [SLE] 151
erythrose [-4-phosphate] 208 209 272
Escherichia [coli] 7 156
ester [amide,
phosphoanhydride] 9 26 79 110
123
esterase (-atic) [degrades,
inhibitor, reacts, site] 61 118 225 265
273 282 283 293
esterification [lipolysis] 163 197
esters (-ified) [amides,
promote] 104 124 215 241
ETF-ubiquinone [reductase] 196 254
ethanol [acetaldehyde,
anaerobic, glycolysis] 13 82 87 165
172 174 178 215
247 249–250 259 279
280 289 297
ethanolamine [inositol] 100 104 268
ether (-s) [anhydrides, ester, H2O] 9 215 259
ethylene [glycol] 93
eukaryotes [acetyl, CO2, inositol] 5 141 150 166
182 198–200

Index Terms Links
eukaryotic [cells, chromosomal, DNA,

genes, mRNAs, one-gene- per-mRNA, organelles,
plasma, topoisomerase] 2 6 41 111
131 137 144 148
150 155 182 213
238 240 272 287
296
evolution (-ary) [conservation,
improvement, origins] 4 136 140
excited [energies,-state] 1 72 91
+
excreted (-ion) [NH4 , nitrogen] 58 212 213 269
exercise [lactate] 177 179 253
exon (-s) [catalyzed, coding,eukaryotic,
poly(A), sequences] 150 151 240 272
296
exonuclease [action, activity] 159 240
exoskeletons 99
expression [cassette, parent,
plasmid, purification] 39 80 92 136
139 141 153 156–158
161 188 224
extinction [coefficient] 21 127 257
extracellular [binding, domain, fluids, hormone,
interface, lumen, matrix,
signal, space, surface] 2 4 100 106
111 112 116 118–123
185 231 233 268
269 275 286 295
eye (-s) 10 91 205 265
eyesight 91
F0 (-F1), (F1) [ATP, ATPase] 166 173 186 191

244 252 277 297
FAD [coenzyme, CoQ, CoQH2,
dihydrolipoamide, FADH2, PLP] 82 83 88 172–173
179 180 188–190 196
228 242 245 250

Index Terms Links
FAD [coenzyme, CoQ, CoQH2,

dihydrolipoamide, FADH2, PLP] (Cont.)
251 254 264 277
284 288 289 294
2+/3+
FAD/FADH2 [dehydrogenase, Fe-S ] 51 52 227 254
297
FADH2 [acceptor/donor,
CoQ, CoQH2, Fe, NADH] 83 92 172 179
190 196 228 242
244 245 254 264–266
277 280 284 288
296 297
Faraday (-s) [constant] 116 172 188 252
farnesyl [anchor, anchor, diphosphate, group,
pyrophosphates, squalyl] 114 202 236 268
269
fat [soluble] 91
fatty [acid, acyl, aldehyde] 18 85 88 102
104 107–108 110 114
128 158 163–165 181
196–199 231 234 236
249 253–255 270 273
280 286 290 295
297
2+ 3+
Fe (Fe ) 45 81 89 172
196 254
Fe-S [centers, clusters, CoQ,cyt] 88 188 189 207
254
feed-forward [activation,
regulation, stimulation] 65 246 278 297
feedback [inhibition] 49 65–67 194 225
246 263 278 283
293 296
fermentation [CO2] 177 249 280
ferredoxin [Fd, P700*, spinach] 172 206 207
ferritin 89
fetal [hemoglobin, O2] 49 194
fiber [attachment, diffraction] 132 141 144

Index Terms Links
fibrin [cascades, clot,

degraded, plasmin] 64
fibrinogen [fibrin] 64 110 138
fibroblasts [synthesizes] 2
fibrous [polypeptide] 115
fidelity 159
filaments [intermediate] 5
filter [crosslink, viral] 161 162
filter-bound [viral] 161
filtration [chromatography, gel] 28 29 257
finger (-s) [coenzymes, protein] 81 89 143 144
228 265
fingernails (-print) [x-ray] 34 37
first-order [kinetic] 53
Fischer [projections] 95
fish [amphibians] 108 213
fission [yeast] 69
fit (induced-) [concept, model, revisited] 53 65 73 89
139
fixing (-ation) [mechanism, ribulose] 3 5 208–210 244
277
flavin (-s) [adenine, coenzymes] 83 92
flavodoxin 207
flavoprotein 196 254
flavor (-ing) 97 247 251 290
flexibility [allysine] 44
fluid [behavior, mosaic, serum] 98 111 233 235
236 268 286 295
fluorescent (-cence) [affinity-labeling, quenching] 106 132 161
flux [standard, through] 168 169 177 242
245 277 288 296
FMNH2 [FMN] 172 188 189
focal [center] 91
F0F1 [ATPase] 279
folate [7, 8-dihydrofolate, coenzyme, derivative,
PABA] 62 87

Index Terms Links
folded [biomolecule, chain, Hu, intermediates, pKID,

protein (-s), state, structures] 15 34 38–43 46
129
folding [intermediates, mature, pathway (-s),
protein] 15 38–44 146
folic [acid] 87
food (-s) [additive, digestion, flavoring, processing,
source, substrates] 1 90 94 98
140 177 210 225
247 251 283 290
force [emf, exports field, microscopy, NADH, pmf] 38 71 106 167
171 173 186–188 239
244 247 252 272
277 279 290 297
fractional [saturation] 48
fractionate (-ion) 29 111
free-energy [change] 113 168 170 188
freeze-fracture [electron] 106
fructose [-1-phosphate 6-
phosphate, -6-phosphate,
glucosyl, ribopyranose] 97 174–176 208 265
fructose-1, 6-bisphosphate 75 174 176 177
184 185 243 244
246 276 278 280
289 297
fructose-2, 6-bisphosphate 124 174 176 177
185
fructose-6-phosphate [fructose-1-phosphate] 176 184 185 209
244 276 280
fructoside [glucose] 97
fumarase [fumarate, malate] 180–181 247 251 254
290 297
fumarate [hydratase, malate, succinate] 164 172 180 181
183 188 190 211
212 244 251 254
276 280
furanose 96 227 230 266
285 295

Index Terms Links
GABA 105
galactosyl [glucose] 90 94 98 230–233
265 285
gall [bladder] 269
gangliosides
[sphingomyelins] 102
gap [junctions] 61 142 143
gas [antidote, chromatography,
constant, law] 18 27 57 61
187 225 250 265
283 293
2+
gated [Ca ] 122
G•C [base, content, pair, ratios] 125 127 131 135
238 239 287 295
GDP [ATP, GTP, phosphoenolpyruvate] 121 122 180 181
184 251
gel [bead chromatography, electrophoresis, filtration,
network, permeation] 18 27–29 39 86
103 140 154 155
159 162 194 195
257 274
gene [encodes, expression, inactivation, inserted,
intron, ori, products, sequence, silencing, SMG,
therapy] 30 39 58 80
89 92 131 136
139 141 149–150 153
156–158 161 195 240
genes [encode] 7 43 50 109
137 141 142 145
149 150 153 240
genetic (-s) (-ally) [code, damage, diseases,
engineering, mutations, operator, phenotype] 1 4 5 20
31 44 58 73
126 131 136 141
142 145–148 154 158
215

Index Terms Links
genome (-es) (-ic) 7 8 31 58

128 142 153 155
195 274
geranyl [diphosphate] 201 202
G-G [base] 136
Gibbs [equation, free] 1 15 38 57
113 167 172 216
globin (-s) [polypeptide] 45 46 49
globular [proteins, structure] 34 45
globule [rearrangement] 40
glucagon [activates, binds,
fructose-2, 6-bisphosphate, regulates] 123 124 177 185
212
glucocorticoid [hormones] 212
glucokinase 174 176
gluconeogenesis (-ic)]
[glycogen, pathways, PEP,
PFKase-2, pyruvate] 68 84 124 163
165 168 169 177
179 184 185 210
247 251 278 290
297
glucosamine [GlcN] 97 114 295
glucose [biosynthesis, brain, catabolism, cyclization,
fuel, glc, hexokinase, lactose, metabolism,
phosphate, transporter, UDP-galactose] 49 73 81 90
94 95 97 98
105 116 124 131
163 165 169 174
176–178 182 184 185
210 228 230–233 244
265 277 285 294
glucose-1-phosphate 68 98 124
glucose-6-phosphatase 184
glucose-6-phosphate (-Pi)
[AMP, ATP, fructose-6- phosphate, glucose,
isomerase] 81 98 124 169

Index Terms Links
glucose-6-phosphate (-Pi)
[AMP, ATP, fructose-6- phosphate, glucose,
isomerase] (Cont.)
174 176 228 276
280
glucuronate (-ic), (-yl) [acid, glycerol] 96 100 272
glutamate (-s) [aspartate, enters, glu, -glutamyl,
malate, mitochondrial, nitrogen, react, residue, -
semialdehyde, α-ketoglutarate] 17 22 62 63
75 84 87 110
113 164 193 212
251
glutamate-aspartate [translocase] 193
glutamic [acid/glutamate] 76
glutamine [gln, glutaminyl] 22 26 113
glutamyl [phosphate] 164
glutathione [oxidized, reduced] 89 172 182
glycan [phosphoethanolamine] 114
glyceraldehyde (-s) [-3- phosphate, dehydrogenase,
DHAP, glycerate] 11 13 75 76
169 174–176 178 184
208 209 246 273
278 296
glycerate 13
glycerol [-3-phosphate, backbone, esterified,
glyceraldehyde, phosphate] 13 96 104 105
108 159 165 169
234 270 272
glycine [CO2, gly, residues] 21 26 37 113
272
glycoconjugates [lactose, ribose] 93–101
glycogen [catalyzes, conformational,
metabolism, phosphorylase, stores,
synthase] 68 98 123 124
163 165 177 231
233 264 286 295
glycolipids [proteoglycans] 98

Index Terms Links
glycolysis [ATP, calcium, catalyzed, enolpyruvate,

ethanol, gluconeogenesis, intermediate, Krebs,
pathway, pyruvate, sugar] 49 67 68 70
75 81 85 86
94 98 124 163–165
168–170 174–179 182 184–186
225 228–229 233 242
244 246 250 264
266 277 278 283
288 293 296
glycolytic [activity, enzyme, reactions] 29 49 168 169
185 278
glycophorin [anion] 115
glycoprotein (-s) [lipid, peripheral, proteoglycans] 98 99 111 114
268
glycosaminoglycan (-s) [polymer] 100 234
glycosidic [bond, torsion] 97 98 125 131
133–135 148 235 237
270 271 274 287
295
glycosphingolipids [membrane-bound,
proteoglycans] 111 236 268
glycosylation [myristoylation] 70
glycosylphosphatidyl-inositol
[proteins] 114
glyoxylate [allantoicase] 213
golgi [apparatus, sacs] 2–4 100 106
G-protein [Gp, phospholipase, transducin] 91 122 124
gradient (-s) [active, cytosol, energy,
oxygen-evolving, P700*] 4 14 17 82
105 116–119 166 173
178 186 188–192 194
205 206 244 252
272 279 297
gramicidin 117
grana (-al) (-um) [lumen,
stacks, thylakoid] 5 206
GroEL (-EL) (-GroEL) [cap, complex] 42

Index Terms Links
groove (-s) [hydrogen] 15 89 110 133

136 139 144 270
ground (-state) [equivalents, state] 1 57 206
group-transfer [potential] 71 86 90
growth (-controlling) [inhibition, factors,
movement, proteins] 36 87 120 123
124 275
GTP [GDP, hormone-inhibited, stimulatory,
UTP] 51 121 122 124
163 165 180 182
184 244 251 277
280
GTPases 120 124
G•U [base] 140
guanase 213
guanidinium [chloride, guanidine, pKa, SDS, urea] 23 39 41 70
105 215 256 258
259
guanine [adenine, cytosine, gua, nucleotides,
triphosphatases, uric, vitamin] 88 120 124 125
132 135 213 236
295
guide [gRNA, siRNA] 153 240
gyrase 142
H2O-octanol [partition] 113 269

H2O2 [H2O] 213
HaeIII 156
hairpin [duplex, primer, turns] 128 137 138 146
148–150 240
half-cell 172
0
half-reaction (-s) [E ’] 171–173 187
handedness [right] 135
Haworth [projections] 95 265 266
H-bond (-s) (-ing) [distance, disulfide, interfaces, salt] 14 16 70 77
131 268 293

Index Terms Links
HCO3 [H+] 19 198 199 201

212
head [group (-s)] 7 39 104 106
178 234 268
heart [attack, disease, medication, muscle] 97 109 119 124
203
heat [shock] 41 273
heavy [chains, metal, transition] 50 227 228 294
helical [axis, density, DNA] 1 35 133 141
239 270
helix [antiparallel, axis, conformation, duplex, left-
handed, long, pitch, right-handed, triplex, width] 35 37 125 127
128 132–136 139 147
153 159 238–240 270
272 287 295 296
helper [viruses] 142
heme (-s) [hemoglobin, ironprotoporphyrin
O2 binding, prosthetic, ring] 45 46 88 204
hemiacetal (-s) [acetals, hemiketals] 95 215 265
hemiketal (-s) 95
hemimethylated 155
hemoglobin [allosteric, apoprotein, binds, F, Hb,
oxygen, oxygenation, P50, regulation, transfer,
transports] 13 34 45–49 65
73 186 194 259
hemopheliacs 64
Henderson-Hasselbalch
[equation] 12 19 259
heparin (-Sepharose)
[affinity] 90 100
hepatitis 153
HETPP [CO2,
hydroxyethylthiamine] 87 179 249–250
hexanyl-ACP 198
hexokinase [ATP, glucokinase, glucose,
phosphofructokinase-1] 34 73 165 169
170 174 176 184
244 277

Index Terms Links
hexose [stage] 174 176

HindIII (-II) [inserted, SMG] 156 157
histidine [his, imidazole/imidazolium,
ligands, nitrogen] 17 22 45 72
76 81 89 113
181 226 262 284
294
histidyl 256
histone (-s) [code, H1, methylation, mRNAs,
octamer, proteins] 141 144 150 238–240
267 272 287 295
HIV [capsid, coat, particle, protein, provirus, reverse,
RNA, virus] 6–8 62 138 142
HLA [class, human] 7
HMG-CoA [reductase] 200 201 203
Hofmeister [series] 39
Holliday [junction] 145
holoenzyme [70S] 148
holoprotein [myoglobin] 45
homocysteine 88
Hoogsteen [base, variant] 131 132 136 271
hormone (-s) (-al) [activate, binds, glucagon, GTPases,
hormone, -inhibited, offsets, protein, signal-
transduction, -stimulated, stimulus] 104 109 110 120–122
177 185 194 212
273
host [bacteria, cell] 7 8 141 142
154
HpaII [cut, EcoRI, site] 156 274
HPLC 30 31
HS-ketoacyl-ACP [KAS] 255
hsp10 (-60), (-70) [GroES] 41 42
human [arteries, disease, erythrocyte, fetus,
immunodeficiency, leukocyte, plasma, retinol-
binding, serum] 6 7 34 36
49 106 112 136
153 194 195 233
253

Index Terms Links
humoral [immune] 50 145

hyaluronate 100
hybridize (-d), (-ation) [analyses, DNAs, reaction,
structure] 17 33 127 159
161–162 273
hybridoma [cells] 151
hydratase [FAD] 181 196 254
hydration [cage, oxidation] 19 39 139 180
196 209 254 297
hydride [proton] 13 82 83 87
216 264
hydrocarbon [chains, interior, layers, tails] 104 105 234 249
257 268 269 279
hydrodynamic [sizes] 28
hydrogen-bond (-ed) [molecules, pattern] 14 148
hydrolase (-s) [amylase, class] 51 52 67 70
98 273
hydrolysis [deacylation] 30 74 79 119
122 126 129 132
152 157 158 168
183 238 273 287
296
hydrolyzed (-es), (-ing)
[peptidoglycans] 100 232 233 275
hydropathy (-icity) [plot, scale] 112–114
hydroperoxidase 107
hydrophilic (-ity) [face, regime, regions, residues,
sequence, sulfate] 14 18 113 114
130 138 257
hydrophobic (-ity) [core, effect, environment, face,
interactions, residues, tail, water] 15 18 21 38
39 41 44 71
72 75 105 111
113 114 131 136
138 139 145 158
216 220 223 234
239 257 259 269
272 282 292 293

Index Terms Links
hydroxide [ions] 152 239

hydroxybutanoyl-ACP 198
hydroxyethyl (-thiamine- pyrophosphate)
[pyrophosphate] 87 179 249
hydroxyl [carbonyl, group, hydrogen, oxygen] 9 17 22 62
67 75 78 90
93 94 97 108
109 129 147 152
200 239–241 258 262
274
hydroxylate (-ion) [amide, nucleophile] 14 17 33 71
77 90 92 239
293
hydroxylysine 99
hypervariable [switching] 50
hypochroism (-micity) 239 270
hypoxanthine [H2O] 88 213
Ibuprophen 108
imidazole (/imidazolium ) [nitrogen, pKa,
protonation- deprotonation, ring] 22 76 78 79
131 256 271
imide [acid-base] 14
imine (-ium) 77 131
imino [A, tautomer] 130–132
immune (-ity), (-ized)
[animal, cell, response, system] 7 50 64 73
124 145 151
immunoconjugate (-s)
[colorless, detection, prehybridization] 32 161 162
immunodeficiency [virus] 6 7
immunoglobulin [G, gene- switching] 50 138
impermeable [charge] 106
indole [ethyl, ring] 21 256
induced [assembly, dipole, fit, induced] 65 73 105 106
139 216 263
induced-fit [mechanism] 80

Index Terms Links
infect (-s) (-ed) (-ion)

[bacterial, cell, tissues] 7 8 62 153
195
inflammation [pain] 107–108
inhibition (-based)
[allosterism, competitive, constant, hemoglobin] 49 57–62 65–67 87
121 154 176 185
194 203 224 225
246 278 282 283
293
inhibitor (-s) [catalytic, inactivate, reversible] 27 49 59–61 65
66 70 73 87
107 118 183 224
263 264 293 294
initial [C-terminal, lysate, rate, signal, slope, stage,
steps, substrate, telomere, template, velocity] 29 32 53 64
74 76 78 143
159 164 196 211
224 225 255 282
283 292 293
initiation [codon, complex, transcription, translation] 148 156 157 198
inosinate 146
inositide (-s) [diphosphate, insect] 273
inositol [choline, diacylglycerol, diphosphate, gal,
glucuronate, group, protected, triphosphate] 90 97 100 108
114 200 231 232
234 265 268 272
273 275 285 295
inositol-phospholipid
(-P-lipid) [Ca2+, DAG, G-
proteins, PIP2, signal (-ing)] 108 122 124 275
insert (-ion) [site] 80 126 141 157
insulin [binding, maturation,
monomers, receptor,
represses, response,
tyrosine] 44 123 124 177
212

Index Terms Links
integral [membrane, proteins] 111 112 114 186

191 236 268
integrase [HIV, RNA] 7 8
interatomic [distance, overlap] 15 16 34 221
281 291
interleukin 124
intermembrane [space] 4
intervening [loops, sequences] 32 65 114 146
150 240
intron (-s) [exon, intervening, lariat, removal] 31 150 151 240
272 296
inverted [repeat, two-fold] 54 138 154
iodoacetic [acid] 32
iodoalcohols 101
ion [channels, concentration,
conduction, Cu2+, diffusion, donates,
exchange, flow, gradients, hydrogen, transporters] 28 38 79 116–120
137 152 171 172
216
ion-transporting [ATPases] 116
ionic [bonds, conditions, iron, strength] 14 17 38 45
71 111 138 216
293
ionizable (-ation) [groups, residues] 23 25 45 256
ionophore (-s) [kill, valinomycin] 20 117
2+
IP3 [activates, Ca , DAG, IP3-gated,
phospatidyl, Pi, signal] 108 120 122–124 233
234 275
IP-STP 108
2+
iron [cofactor, Fe ] 45 46 81 89
190 265
iron-sulfur [center, clusters, proteins] 81 89 254
iron-protoporphyrin [IX] 45
irreversible (-ly) [covalent, inhibitors, reaction] 32 51 61 63
70 108 168 169
199 226 242 244
277 283 288 294
296

Index Terms Links
isoacceptor [tRNA] 147

isocitrate [aconitase, CO2,
dehydrogenase, α-ketoglutarate] 68 124 164 172
180 181 183 251
265 276 297
isoelectric [point, focusing] 23 194
isoenzymes 84
isoleucine [Ile] 20 21 113
isomer (-s) [classification] 20 51 52 141
196 231 285
isomerase (-s) [racemase] 51 52 75 76
91 169 174 176
201 208 210 243
246 278 289 296
isomerization (-s) [vision] 91 124 192 264
isopentenyl [diphosphate] 200–202
isoprene (-ium)
[condensation, units] 102 202 204 269
isoprenoid (-s) [chain] 109 114 269 273
isopropyl 11
isotope (-s) 10 208
isozyme (-s) [cytosolic, forms] 108 193–196 199
joints 100 231 286 295

juvenile [hormone] 110 273
K-1 (k-1) 70 71 187 244

263
k1 (k0) 51 52 54–57 61
70 278
Ka 19 49 86 127
KAS [synthase] 255
kcat [V0] 54–57 70 224 263
278 282 293
kcat/KM 55 73
Kd 127

Index Terms Links
keratin [skin, sulfate] 34 100

ketamine 33 77
keto (-enol) [tautomerism] 33 139 209 258
ketoacyl-ACP [reductase,
synthase] 197 198 255 280
ketone (-s) [bodies, pKa] 9 90 95 163
215
ketose (-s) [aldose] 94 227 230 265
285 295
KI [KM, slope] 59 60
kinase (-s) [ATP, DAG, domain, phosphatase,
phosphofructokinase-1, pyruvate, signal, threonine] 6 51 52 65
67–70 71 73 121–124
163 169 170 175–177
183–185 201 208 225
233 243 244 246
263 273 275 277
278 283 289 293
297
kinetic (-s ) [analysis, equation, plot, rates,
reaction, regulation, studies] 1 51–53 55 57
58 62 63 70
75 82 83 224
226 242 273 277
282 288 292 296
KIX [domain] 43
KM [activated, curve,
diffusion, E, inhibited, log [S], slope, V0] 49 52–57 59 60
62 65–67 70 170
224 242 245 263
277 278 282 288
292 296
KpnI [digestion] 155
Krebs [cycle] 63 67 68 70
84 85 88 89
124 164 178–183 185
190 196 212 225
228 229 244 246

Index Terms Links
Krebs [cycle] (Cont.)

247 249 250 253
254 264 277–280 283
289 290 293 296
297
kT 70
L-3-hydroxyacyl-CoA [CoA, dehydrogenase] 196 254

label (-s) (-ed) (-ing)
[detection, digoxigenin, duplex, probe] 30 31 67 106
159 161 162 215
226 235 241 257
262 268 273 284
287
lac [operon, repressor] 136 149
lactate [acetyl, dehydrogenase, glycolysis] 29 84 163 165
172 174 175 177
178 184 185 249
251 297
lactating [mammary] 198
lactic [acid, acidosis] 165 177 182
lactone [diol] 90 96
lactose [biosynthesis, cosubstrate galactosyl,
permease, sodium] 90 94 98 116
118 149 200 227
230–231 265 272 285
lanosterol 202 269
lariat 151
larval [development] 110
L-asparagine 218 281 291
L-aspartate 218 256 281 291
lattice (-s) [melt, protein] 14 47 48 65
234 291
laural [sulfate] 105
laurate (-ic acid) 102 267 272
laureate 31 192
LBHB [formation] 17

Index Terms Links
L-cysteine 219 281 291

lead [compounds] 58
leader [peptide, sequence] 41 148 149
leaflet (-s) [inner, protein] 106 112 114
leaves [diacylglycerol, wood] 63 76–78 81 90
119 123 210 232
250
leaving [group, potential] 31 32 39 61
76 90 165 197
200 239 258
lectin [binds] 101
left-handed [collagen, poly(dGdC), Z-DNA] 35 37 95 132
134
lemon (-s) [odor, scent] 104 110 273
leucine [leu, zippers] 20 21 113 145
272
leukemia 62
leukocyte [antigens] 7
leukotrienes [hormone-like] 102 107
levorotatory [left-handed, L-glyceraldehyde,] 20 93 95
Lewis [acid] 89
L-glutamate 217 281 291
L-glyceraldehyde [levorotatory] 20
L-leucine 218 281 291
LHC [PS, NADP] 206
L-histidine 217 281 291
life [cycles, science] 1–5 7–8 34 36
40 42 43 46
50 73 106 151
191 194 211 242
247 288 289
ligand (-binding) (-carrying) (-dependent) (/inhibitor)
(- lattice) [bead, binding, concentration, curve,
membrane, synthetic] 13 27 28 45–50
58 65 81 86
89 120 144 153
227 229 258 285
294

Index Terms Links
ligase (-s) [seals] 51 52 70 143

light (-dependent) (-driven) (-harvesting) (-sensitive)
[antennae, bacteriorhodopsin, chains,
coenzyme, energy, Fd, isomerization, P700, plant,
reactions, scattering] 5 21 34 36
50 91 93 95
103 113 116 127
154 166 192 204–207
210 227 285 295
lignins 97
lignocerate 102
limonene [lemon] 104 110
lineage [determination] 140
linear [DNA, double- stranded, equation,
glucose, pathway] 56 65 95 98
142 155 164 232
linearized [heme, target] 156 204
Lineweaver-Burk [equation,
plot] 54 56–57 292
linked [catalytic, pathway,
protease, reactions] 1 65 263 293
linker (-s) (-ers) [captured,
deoxyuridine, DNA, H1,
strand-to-strand] 43 86 125 144
161
links [glycolysis, liver] 85 264
linoleate 102 107
linolenate [cis] 102 103
linolenoyl [CoA] 199
linoleoyl [CoA] 199
lipases 51
lipid [absorption, addition,
aggregates, anchor (-ed),
anchor, assembly, bilayer,
capsid, chaperonins,
cholesterol, composition,
derivatives, head, liquid,
micelles, movement,

Index Terms Links
light (-dependent) (-driven) (-harvesting) (-sensitive)

[antennae, bacteriorhodopsin, chains,
coenzyme, energy, Fd, isomerization, P700, plant,
reactions, scattering] (Cont.)
properties, raft (-s),
soluble, tail, Tms, vesicle] 1 3 4 7
70 100 102–106 109–113
115 117 128 192
231 234 236 268
269 272 286 295
lipid-anchored [membrane,
protein, proteoglycans] 111 114 236 273
lipid-based (-containing) (-embedded)
[hydrocarbon, proteins] 99 111 249 279
295
lipid-carbohydrate
[bioconjugate, complex] 112 275
lipid-solvent [interface] 106
lipids [cholesterol, disperse,
membranes, phospholipids,
store, unsaturated, vitamins] 51 70 90 91
97 102–110 112 117
163 196–203 236 273
Lipitor 203
lipoamide 250
lipoic [acid] 172 249 250 279
297
lipolysis [polysaccharides] 163
lipoproteins 109 203
lipoyl (-E2) [ACP-SH, CoA-SH
dehydrogenase] 172 280
liquid [chromatography,
crystal, crystal-to- dispersed] 15 30 102 103,
105 106 128 234
236 268 273 295
liquid-monolayer [interface] 104
L-isoleucine 219 281 291

Next Page
Index Terms Links
liver [cells, glucagon] 2 68 153 177

198 211 233 264
295
L-lysine 219 281 291
L-malate [fumarase, H2O] 180–181
L-methionine 217 281 291
ln Q 167
loading [acetyl, stage] 197–198
logarithmic [Michaelis-Menten, plot, relationship] 13 47 53–54 167
London [dispersion] 138 216 239
lone [pair (-s)] 14 21 23 130
153 262
loop [envelops, region] 80 147–148 164
loops 112 114 146 269
low-barrier [hydrogen] 17 79
Lowry [reaction] 30
L-proline 218 281 291
L-serine 217 281 291
lubricates (-ant) [cartilage,
keratin] 100
lumen [adapted, aminoacyl,
LHC, PKC, stroma,
transfer] 3–4 41 44 122
124 191 206 275
lung (-s) [alveoli, bicarbonate,
pO2, surface, tissue, transplant] 19 45 48–49 104
153
lupus [erythromatosis] 151
L-valine 219 281 291
lyase (-s) [argininosuccinate] 51 52 212
lysed (-ate) 29 275
lysine (-yl) [deprotonation,
lys, ε-ammonium] 22 38 44 75
76 80 84 113
141 192 213 227
239 249 272 285
294
lysosome (-s) [peroxisomes, smooth] 2

Biochemistry Essential Concepts

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Biochemistry

Charles C. Hardin and James A. Knopp

New York Oxford

Oxford New York

Copyright © 2013 by Oxford University Press

For titles covered by Section 112 of the U.S. Higher Education

Published by Oxford University Press, Inc.

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Library of Congress Cataloging-in-Publication Data

Printed in the United States of America

Using Biochemistry: Essential Concepts

Chapter 1. Biochemistry: Subject Overview ……………………………………….……..……… 1

STUDY GUIDE ……………………………………………………………………….……….…… 215

Review Session for Chapters 21–25 ……………………………………………….....…………... 247

Index ………………………………………………………………………………………………..... 317

Chapter 1. Biochemistry: Subject Overview ……………………………………….……..………… 1

Chapter 2. Cell Biology Review ……………………………………………………………....……… 2

Chapter 3. Chemistry Review ……………………………………………………..………………….. 9

Chapter 4. Amino Acids ………………………………………………………………………….…... 20

Chapter 5. Proteins ……………………………………………………………………………….…... 26

Chapter 6. Protein Structure ………………………………………………………………….……... 33

Chapter 7. Ligand Binding and Functional Control …………………………….………….………. 45

Chapter 8. Enzymes …………………………………………………………………..……………….... 51

Chapter 9. Metabolic Enzyme Action ………………………………………………………………..... 70

Chapter 10. Coenzymes …………………………………………………………….…………….…...... 81

Chapter 11. Carbohydrates and Glycoconjugates …………………………………..……………...... 93

Chapter 12. Lipids ………………………………………………………………..………….……….... 102

Chapter 13. Membranes ……………………………………………………..……………………….... 111

13.2 Detergents ……………………………………………………..………………………….…….. 111

Chapter 14. Transport Through Membranes ……………………………………………………… 116

Chapter 15. Signal Transduction ……………………………………………………………….…… 120

Chapter 16. Nucleic Acids: DNA ………………………………………………………..……..……. 125

Chapter 17. RNA ……………………………………………………………………………..……… 146

Chapter 18. Biotechnology …………………………………………………………….…………….. 154

18.4 Polymerase Chain Reaction …………………………………………………………….………. 158

Chapter 19. Metabolism ………………………………………………………………..……………. 163

Chapter 20. Bioenergetics …………………………………………………………………….……… 167

Chapter 21. Bioelectrochemistry ……………………………………………………………….…… 171

Chapter 22. Glycolysis ………………………………………………………………………….……. 174

Chapter 23. The Krebs Cycle …………………………………………………..……………………. 180

Chapter 24. Gluconeogenesis ……………………………………………………..…………………. 184

Chapter 25. Electron Transport and Oxidative Phosphorylation …………………………….…… 186

Chapter 26. The Malate-Aspartate Shuttle and Proteomics ………………………………….…… 193

Chapter 27. Degradation and Synthesis of Lipids …………………………………………..……… 196

27.6 Regulating Cholesterol Levels …………………………………………………………………. 203

Chapter 28. Photosynthesis ………………………………………………………………………..... 204

Chapter 30. The Urea Cycle ………………………………………………………………….…….. 211

STUDY GUIDE ……………………………………………………………………….……….…….. 215

Chaotropic Agent ………………………………………………………………………….……. 222

Pyridoxal Phosphate; “Schiff’s Base” With … Lysine ……………………………….………... 227

Two Functional Ends of Transfer RNA ……………………………………………………….. 238

Biochemistry: Subject Overview

1.1 Central Themes

1.2 Central Dogma of Molecular Biology

replication Post-transcriptional Post-translational

Cell Biology Review

2.2 The Plant Cell

2.3 Selected Organelles

2.4 The Cell Cycle: Mitotic Cell Division