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MODULE 2:
BIOCHEMISTRY

Ser Loisse R. Mortel, RPh, MS

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I) PROTEINS AND ENZYMES

General Properties:
• Chiral (L-amino acids are dominant in humans)
• Amphoteric
o Acidic due to -COOH group
o Basic due to -NH2 group
o When both are charged, they cancel out and
the net charge is 0 = zwitterion
o Isoelectric pH (iPH/ PI) = pH where all amino
acid molecules are zwitterionic
• There are 20 proteinogenic amino acids
• Essential amino acids:
PVT TIM HALL

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Enzymes are often proteins (apoenzymes) that are

bound to nonproteins that activate them (cofactors):

Levels of organization:
LEVEL DESCRIPTION ATOMS CONNECTIONS
INVOLVED
Primary Sequence Peptide Peptide bond
bond atoms
Secondary Arrangement Peptide Peptide bond,
in space bond atoms amide H bonds
Tertiary Complete 3D Peptide Peptide bond,
arrangement bond amide H bonds,
atoms, residue H bonds,
residues hydrophobic,
electrostatic, Inorganic Metal ions
disulfide Cofactor
Quaternary Independent Same as Same as tertiary
subunits tertiary Organic Coenzymes

Denaturation: Leads to primary structure (only

Coenzymes are usually from water-soluble vitamins:
peptide bonds stay)
VITAMIN COMMON NAME
Hydrolysis: Leads to individual amino acids (no
C Ascorbic acid
bonds remain) B1 Thiamine
B2 Riboflavin
Enzymes – biologic catalysts B3 Niacin
• Converts reactants (“substrates”) into products at B5 Pantothenic acid
a faster rate than original B6 Pyridoxine
• Must bind properly with substrates at their active B9 Folic acid
site in order to properly catalyze the reaction B12 Cobalamin

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Classification of Enzymes
EC # Name Example
(examples)
1 Oxidoreductase
Oxidases
Reductases
Dehydrogenases

2 Transferase
Kinases
Other transferases

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3 Hydrolase
Esterases
Enzymes that
simply end in – ase

4 Lyase
Decarboxylases
Dehydratases/
anhydrases

5 Isomerase
Racemases
Mutases
Other isomerases

6 Ligase
Synthases
Synthetases
Carboxylases

Enzyme Kinetics
Michaelis-Menten kinetics:
• As the substrate concentration [S] for an enzyme
increases in number, the enzyme activity [V] is
supposed to increase too
• Follows an initial direct relationship (linear) – the
graph follows first-order kinetics
• All enzymes become occupied at some point – the
graph shifts to zero-order kinetics
• Maximum velocity (Vmax) – highest attainable
velocity of a substrate for an enzyme
• Michaelis constant (Km) – [S] required to reach
half of Vmax

Effect of temperature and pH:

• Alterations in both temperature and pH
can denature enzymes -> reduction in
their activity
• Enzymes must be kept at their optimum
pH and temperature to work well

• Human normal body temp: 37 oC

• Human normal blood pH: 7.35 – 7.45

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Lineweaver-Burk plots for enzyme inhibition: Inhibition Vmax Km

a) Competitive b) Noncompetitive c) Uncompetitive
Competitive Same Increases

Noncompetitive Decreases Same

Uncompetitive Decreases Decreases

II) CARBOHYDRATES
Monosaccharides Disaccharides – popular examples include maltose,
Further subclassified by functional group type and sucrose, lactose
number of carbons Example Components
Maltose Glucose + glucose
Functional group Sucrose Glucose + fructose
o Aldose - aldehyde Lactose Glucose + galactose
o Ketose - ketone
Polysaccharides – includes glucans (ex. starch,
glucose and cellulose) fructans (ex. inulin) and
glycosaminoglycans

Linear form
• Fischer projection
• Shows the chiral carbons
• Exhibits optical isomerism
Number of carbons
Class Common Examples
Triose Glyceraldehyde, dihydroxyacetone
Tetrose Erythrose, threose
Pentose Ribose, xylose, arabinose
Hexose Fructose, glucose, galactose
Heptose Sedoheptulose, mannoheptulose

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Isomers from linear structures:

• Epimers - different in only 1 chiral carbon
• Enantiomers - mirror images (all chiral carbons switched)
• Diastereomers - different in 2 or more chiral carbons, but not exactly mirror images

Cyclic form
• Involves linkage between penultimate OH and carbonyl group
• Haworth projection

o Pyranose and furanose

o Anomers – differ at the anomeric carbon

Carbohydrate Derivatives

• Sugar alcohols - result from reduction of sugars

• Sugar acids - result from oxidation of acids
• Sugar linkages - glycosidic bonds

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III) LIPIDS
• A family of structurally diverse compounds
whose similarity is only their water-
insolubility/immiscibility.
• Lipophilic = Hydrophobic
Uses:
• Cell membranes (phospholipids)
• Storage of energy in bulk (triacylglycerols)
• Vitamins (A, D, E, K)
• Hormones (steroidal)
Types:
Fatty Acids
Saturated
• Long chain carboxylic acids
• Contain only C-C single bonds
• Content of most important physiologic lipids
• Compact molecules
• Have relatively high melting points
Physical Properties: Unsaturated
1. Polarity/Solubility – increased number of • Contain at least one C-C double bond
carbons increase lipophilicity • Not as compact as saturated fatty acids
2. Melting point – reduced compactness due to cis • Have relatively low melting points
double bonds reduces IMF and thus lowers
melting point

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A) SAPONIFIABLE LIPIDS LIPID HEAD

Phosphatidic acid None
1) Triglycerides Phosphatidylcholine (PC) Choline
• Triester of glycerol (a trihydric alcohol) with Phosphatidylserine (PS) Serine
three fatty acids Phosphatidylethanolamine (PE) Ethanol-
• Neutral lipids amine
• May be simple or mixed Phosphatidylinositol (PI) Inositol
• Storage lipid (in adipose)
3) Sphingolipids
• Use a sphingosine backbone
• Structural lipids (found primarily in nerve tissue)
SPHINGOLIPID HEAD GROUP
Ceramide None
Cerebroside Glu/ Gal
2) Phospholipids Globoside Oligosaccharide
• Similar structure to triglycerides Ganglioside Polysaccharide
• Acidic lipids (with sialic
• Structural lipids (found primarily in cell acid)
membrane) Sphingomyelin Phosphate and
choline

4) Waxes
• Esters of fatty acid with a long chain,
monohydric alcohol

Ex.

B) NONSAPONIFIABLE LIPIDS

1) Fat-soluble vitamins (A, D, E, K)

2) Sterols - contain the
cyclopentanoperhydrophenanthrene (CPPP) ring
ex. Cholesterol, ergosterol

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Importance of cholesterol o Glucocorticoids (ex. cortisol)

• Regulates fluidity of cell membranes o Mineralocoticoids (ex. aldosterone)
• Precursor of bile acids o Sex hormones (ex. testosterone,
• Precursor of vitamin D estradiol, progesterone)
• Precursor of steroid hormones
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IV) NUCLEIC ACIDS
• Biomolecules responsible for the storage, Components of a nucleotide:
transfer, and expression of genetic traits. 1. Sugar (5C)
• Made up of millions of nucleotides. 2. Phosphate
3. Base (nitrogenous) – chemically either:
Types a) Purine (GA)
• Deoxyribonucleic acid (DNA) b) Pyrimidine (CUT)
• Ribonucleic acid (RNA)

Structure of a polynucleotide strand:

a) Composed of nucleotides bonded together by phosphodiester bonds between sugar and phosphates
b) Locks almost all 3’ and 5’ carbons from all nucleotides
c) One end of the strand has a free phosphate – it is called the 5’ end
d) The opposite end of the 5’ end has a free OH on 3’ – it is then called the 3’ end
e) The bases are held by the sugars and form a sequence (we write then as a sequence of letters)

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DNA vs. RNA Properties of the double helix:

PROPERTY DNA RNA • Base pairing
Sugar Deoxyribose Ribose C base-pairs with G, A base-pairs with T
Bases A, G, C, T A, G, C, U • Anti-parallel
Common Double- Single-stranded The 5' to 3' direction in the two strands
form stranded go in opposite directions
Purpose Starting point Bridges DNA and • Handedness
of all genetic proteins in the Rotates either clockwise (right-handed)
material central dogma
or counterclockwise (left-handed)

The Central Dogma of Molecular Biology

1) Replication (DNA Synthesis)

An origin of replication is recognized in the DNA.
A replication bubble will then expose the two
separate individual strands to enzyme action.
The leading strand follows this sequence, but the
lagging strand goes the opposite.

• Leading strand will need one primer at the

origin and will proceed in a straight manner.
• Lagging strands need several primers. In the
backward manner of elongation, lagging strands
• Helicase unwinds to make DNA readable form okazaki fragments.
• Topoisomerase compensates the physical
instability by relieving torsional strain. 2) Transcription (RNA Synthesis)
• Primase introduces the RNA primers. • The RNA polymerase starts the recognition by
• DNA Polymerase adds nucleotides to elongate closing on to the helix, then unwinding it when
the new chain. the promoters have been detected.
• That used strand is called the
template/antisense. The non-used or non-
template strand is the sense strand.

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Set-up for transcription:

3) Post-transcriptional Modification
The sequence of mRNA post-transcription (RNA
processing) is as follows:
1. Capping at 5’-end
2. Polyadenylation at 3’-end
3. Splicing (removal of introns)

4) Translation (Protein Synthesis)

• Occurs in the ribosome • The mRNA is processed by a ribosome, and each
• Involves a larger subunit (50S for prokaryotes, codon is matched by an anticodon from new tRNA
60S for eukaryotes) and smaller subunit (30S for • The tRNA brings amino acids which are connected
prokaryotes, 40S for eukaryotes) to each other

• The mRNA is grouped into codons, triplets of

bases that equate to a single amino acid.

• The elongation of amino acids goes on as long as

the codons of the mRNA are processed – unless
the next codon to be read is a stop codon (i.e.
UAG, UGA, UAA)

• The tRNA which translates the gene reads the

correct codon by a complementary anticodon.

• Once a stop codon is encountered, the translation

process is terminated

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5) Post-translational Modifications • Nonsense – mutation of a base causes the

The nascent proteins may undergo several formation of a stop codon
processes: • Missense – mutation causes changes in coded
a) Folding towards the native form amino acid
b) Modification by trimming away or residue
modification
c) Degradation for wrong sequence or wrongly
folded proteins (usually nonfunctional)
d) Glycosylation as tagging mechanism for
transport of fully functional proteins

Mutation – are changes in the base sequence of DNA

• Transition – pyr to pyr; pur to pur
• Transversion – pyr to pur
• Silent – mutation does not affect amino acid
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V) METABOLISM
• Sum of all chemical processes in the body
• Broken down into “pathways”
• A pathway usually has a rate-limiting step
which decides if a pathway will proceed or not
• ATP - energy unit of metabolism
o NADH (“reduced NAD+”) = 2.5 ATP
o FADH2 (“reduced FAD+”) = 1.5 ATP
o Other triphosphates (ex. GTP) = 1 ATP
• Anabolic - builds up – uses energy
• Catabolic - breaks down – produces energy

A) CARBOHYDRATE METABOLISM
1) Glycolysis
• Breakdown of glucose to two molecules of pyruvate
• Consists of two phases: Energy investment (steps 1-5) and energy payoff (steps 6-10)
• Rate-limiting enzyme: Phosphofructokinase (PFK)

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2) Fates of pyruvate:

a) Under aerobic conditions -> acetyl-CoA

b) Under anaerobic conditions -> lactate
c) Under anaerobic conditions in yeast -> ethanol
d) When a person is in the extended fasting state, some of the pyruvate is converted back to
glucose via gluconeogenesis in the liver

Lactose produced in the muscles during anaerobic metabolism can also be converted by the liver back to
glucose via gluconeogenesis – the pathway forms a cyclic path known as the Cori cycle:

3) Glycogen Metabolism
GLYCOGENESIS GLYCOGENOLYSIS
• Synthesis of glycogen • Breakdown of glycogen
• Requires formation of α1,4 and α1,6 bonds • Requires breakdown of α1,4 and α1,6 bonds
• Stores glucose in the blood to the liver • Release glucose in liver and muscle to the blood
• Triggered in well-fed states • Triggered in fasted states
• Rate-limiting enzyme: Glycogen synthase • Rate-limiting enzyme: Glycogen phosphorylase

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Summary of carbohydrate metabolism:

4) Pentose Phosphate Pathway

• Converts glucose-6-phosphate to ribose-5-phosphate while producing NADPH
• The ribose produced is used in nucleotide synthesis
Rate-limiting Enzyme: Glucose-6-phosphate dehydrogenase (G6PD)
Uses of NADPH:
• Cofactor for biosynthetic (anabolic) pathways
• Detoxification (in conjunction with glutathione:

5) Krebs Cycle
• Citric Acid Cycle/ Tricarboxylic Acid Cycle
• Converts acetyl-CoA to two molecules of CO2
• Central pathway for energy generation
• Rate-Limiting Enzyme: Isocitrate Dehydrogenase

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6) Electron Transport Chain

• “Oxidative phosphorylation”
• Occurs in the inner mitochondrial membrane
• Consists of four main complexes that are
coupled to ATP synthase (aka Complex V)
o Complex I: NADH-CoQ reductase
o Complex II: Succinate-CoQ reductase
o Complex III: CoQ-cytochrome c reductase
o Complex IV: Cytochrome c oxidase
• Converts reduced cofactors NADH and FADH to
NAD+ and FAD+ in exchange for ATP

PROCESS MOLECULES PRODUCED ATP YIELD (aerobic) ATP YIELD (anaerobic)

Glycolysis 2 ATP 7 2
2 NADH x 2.5 = 5 ATP (no NADH)
2 pyruvate to 2 acetyl 2 NADH x 2.5 = 5 ATP 5
2 x acetyl to 2 CO2 3 NADH x 2.5 = 7.5 ATP 20
(TCA cycle) 1 FADH x 1.5 = 1.5 ATP
1 GTP x 1 = 1 ATP
Total of 10 ATP / acetyl
Overall 32 per glucose 2 per glucose

B) LIPID METABOLISM
1) Fatty Acid Metabolism
• Occurs in the cytosol
• Rate-limiting Enzyme: Acetyl-CoA Carboxylase
• When synthesized, fatty acids are esterified into phospholipids and triglycerides
• Timing: Fed state (stimulated by insulin)

2) Lipolysis and Beta-Oxidation

• Triggered by the fasted state, with glucagon release or
insulin inhibition
• Initial hydrolysis of triglycerides by hormone-sensitive
lipase (HSL) into glycerol and fatty acids
Beta-oxidation (Fatty acid catabolism)
• Timing: Fasted state (stimulated by glucagon)
• Continuous/successive breakdown of fatty acids by 2
carbons at a time (acetyl-CoA)
• All acetyl-CoA will be oxidized in the TCA cycle
• Occurs in the mitochondrial matrix
• Rate-limiting enzyme: Carnitine palmitoyltransferase 1 (CPT1)
• Requires transport of fatty acids to the matrix (see right)

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3) Fates of HMG-CoA

3a) Mevalonate Pathway

Pathway for cytoplasmic synthesis of sterols and terpenes
• Stimulated in the fed state (by insulin)
• Rate Limiting Enzyme: HMG-CoA Reductase (converts HMG-CoA to mevalonate)
• NOTE: HMG-CoA reductase is the target of statin drugs

3b) Ketogenesis
Production of ketone bodies in the mitochondrion
1. Acetone
2. Acetoacetate
3. Beta-hydroxybutyrate
• Ketone bodies serve as the emergency fuel of the brain in these cases
• Timing: Fasting/ Starvation
• Key Enzyme: HMG-CoA lyase (converts HMG-CoA to acetoacetate)

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• Ketosis – shift from dependence on glucose to dependence on ketone bodies

• Ketonemia – elevated ketone bodies in blood
• Ketonuria – elevated ketone bodies in urine
• Ketoacidosis – excessively elevated ketone bodies, causing acidification of blood pH

CONCERN INSULIN GLUCAGON

Blood Sugar REDUCED INCREASED
Timing FED FASTED
Cell glucose uptake ↑ -
Glycolysis ↑ ↓
Gluconeogenesis ↓ ↑
Glycogenesis ↑ ↓
Glycogenolysis ↓ ↑
Lipolysis ↓ ↑
Ketogenesis ↓ ↑
Fatty acid synthesis ↑ ↓

C) NUCLEOTIDE METABOLISM
• Purine and pyrimidine nucleotides are synthesized separately
• Methods of synthesis:
a) De novo (made from scratch)
b) Salvage (recycled from bases made de novo or from those obtained in food)

Ribose-5-P

De novo Salvage

Purines Pyrimidines

IMP OMP

AMP GMP UMP

CTP dTMP

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D) AMINO ACID METABOLISM

LIST OF SOME AMINO ACID DERIVATIVES
Amino Acid Products
Tyrosine • Catecholamines
• Thyroid hormones
Tryptophan • Serotonin
• Melatonin
Histidine • Histamine
Serine • Choline
Glutamate • GABA
Glycine • Heme

Nitrogen Disposal
• Excess amino acids are not stored
• Nitrogen balance – either positive (intake > output) or negative (outtake > input)
• Ammonia is the initial waste product, but is converted to urea (final waste product) in the liver

3 MAJOR PROCESSES:
1. Transamination
2. Oxidative deamination
3. Urea cycle (Rate-limiting enzyme: Carbamoyl phosphate synthetase)

• When amino acids are transaminated, the nitrogen is deaminated in the liver into ammonia
• Ammonia in the liver is further converted to urea (final product of nitrogen disposal)
•

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VI) QUALITATIVE TESTS

1) PROTEINS/AMINO ACIDS
NAME OF TEST MAIN REAGENT FOR POSITIVE COLOR/ RESULT
1. Ninhydrin Ninhydrin Free amino acids Violet
2. Biuret Copper ions Peptide bonds Violet
3. Xanthoproteic Nitric acid Aromatic amino acids Yellow
4. Millon’s Mercuric ions Phenolic compound (tyr) Rose/salmon
5. Hopkin’s Cole Glyoxylic acid Indole (trp) Violet ring
6. Sakaguchi Alpha-naphthol Guanido group (arg) Reddish/wine
7. Nitroprusside Nitroprusside Thiol group (cys) Red
8. Fohl’s Lead acetate Sulfur containing compounds Brown or black ppt
(cys, met)
9. Pauly’s Diazotized sulfanilic acid Histidine, tyrosine Red

2) CARBOHYDRATES
NAME OF TEST MAIN REAGENT FOR POSITIVE COLOR/ RESULT
1. Molisch Alpha-naphthol Carbohydrates, in general Violet ring
2. Iodine Iodine reagent Starch Violet
3. Benedict’s Copper sulfate (alkaline) Reducing sugars; Brick red ppt
4. Barfoed’s Copper sulfate (acidic) Monosaccharides (Barfoed’s)
5. Seliwanoff’s Resorcinol Ketoses Cherry red
6. Bial’s Orcinol Pentoses Blue-green
7. Mucic acid Nitric acid Galactose Crystals
8. Osazone Phenylhydrazine Some specific sugars Crystals of different shapes

3) LIPIDS
NAME OF TEST MAIN REAGENT FOR POSITIVE COLOR/ RESULT T
1. Liebermann-Burchard Sulfuric acid, acetic anhydride Sterols Green color
2. Acrolein Potassium bisulfate, heat Glycerol Burnt fat odor
3. Test for phosphate Ammonium molybdate Phospholipids Yellow crystalline ppt

4) NUCLEIC ACIDS
NAME OF TEST MAIN REAGENT FOR POSITIVE COLOR/ RESULT
1. Dische Diphenylamine Deoxysugars Blue
2. Test for phosphate Ammonium molybdate Phosphate Yellow crystalline ppt
3. Murexide Potassium chlorate in HCl Purines Pink
4. Wheeler-Johnson Bromine, barium hydroxide Pyrimidines Green with bromine, purple
with added Ba(OH)2

REFERENCES
1. Stoker, H. S. (2012). General, organic, and biological chemistry. Nelson Education.
2. Champe, P. C., Harvey, R. A., & Ferrier, D. R. (2005). Biochemistry. Lippincott Williams & Wilkins.
3. Appling, D. R., Anthony-Cahill, S. J., & Mathews, C. K. (2016). Biochemistry: Concepts and connections. Pearson Education.
4. Campbell, M. K., & Farrell, S. O. (2003). Biochemistry. Thomson Learning. Inc., USA.
5. Various internet sources

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