Bin Organic

MSCCH-603
M.Sc. III Semester

BIO-INORGANIC, BIO-ORGANIC AND
BIO-PHYSICAL CHEMISTRY
SCHOOL OF SCIENCES
DEPARTMENT OF CHEMISTRY
UTTARAKHAND OPEN UNIVERSITY
MSCCH-603
BIO-INORGANIC, BIO-ORGANIC AND

BIO-PHYSICAL CHEMISTRY
SCHOOL OF SCIENCES
DEPARTMENT OF CHEMISTRY
UTTARAKHAND OPEN UNIVERSITY
Phone No. 05946-261122, 261123

Toll free No. 18001804025
Fax No. 05946-264232, E. mail info@uou.ac.in
htpp://uou.ac.in
Expert Committee
Prof. B. S. Saraswat Prof. A. K. Pant
Department of Chemistry Department of Chemistry
Indira Gandhi National Open University G. B. Pant Agriculture, University
Maidan Garhi, New Delhi Pantnagar
Prof. A. B. Melkani Prof. Diwan S Rawat

DSB Campus, kumaun University Delhi University
Nainital Delhi
Dr. Hemant Kandpal Dr. Charu C. Pant

Assistant Professor Assistant Professor (AC)
School of Health Science Department of Chemistry
Uttarakhand Open University, Haldwani Uttarakhand Open University, Haldwani
Board of Studies
Prof. P.D. Pant Prof. B. S. Saraswat
Director, School of Sciences Professor Chemistry
Uttarakhand Open University Department of Chemistry
Haldwani, Nainital School of Sciences, IGNOU, New Delhi
Prof S.P. S. Mehta Prof. Viveka Nand

Professor Chemistry Professor Chemistry
DSB Campus, Kumaun University College of Basic Science & Humanities
Nainital GB Pant University, Pantnager
Dr. Shalini Singh Dr. Charu C. Pant

Programme Coordinator Department of Chemistry
Department of Chemistry School of Sciences,
School of Sciences, Uttarakhand Open University, Haldwani,
Uttarakhand Open University, Haldwani, Nainital Nainital
Programme Coordinator
Dr. Shalini Singh
Department of Chemistry
School of Sciences,
Uttarakhand Open University, Haldwani, Nainital
Unit Written By Unit No.
1. Dr. Vinod Kumar 04
Assistant Professor
Department of Chemistry, School of Sciences
Uttarakhand Open University, Haldwani
2. Dr. Deep Prakash 01, 02, 06, 09
Assistant Professor
Department of Chemistry, School of Sciences
Uttarakhand Open University, Haldwani
3. Dr. D.S. Dhami 07, 10
Assistant Professor
S.S.J. University, Almora
4. Dr. Mahesh Chandra Vishwkarma 03, 05
Assistant Professor
Department of Chemistry,
Govt. P.G.College, Bageshwar
5. Dr. Bhanu Pratap Guatam 08
Assistant Professor
Department of Chemistry,
Govt. P.G.College, Pithoragrah
Course Editor
1. Dr. Vinod Kumar
Assistant Professor
School of Sciences,
2. Dr. Deep Prakash

Assistant Professor
School of Sciences,
Title : : BIO-INORGANIC, BIO-ORGANIC
AND BIO-PHYSICAL CHEMISTRY
ISBN No.: :
Copyright : Uttarakhand Open University
Edition : 2022
Published by : Uttarakhand Open University, Haldwani, Nainital- 263139

CONTENTS
BLOCK-1 BIOINORGANIC CHEMISTRY
Unit 1 Metal Storage and Transport 1-48

Unit 2 Metalloenzymes 49-84
Unit 3 Metal-Nucleic Acid Interactions 85-118
BLOCK-2 Bioorganic Chemistry
Unit 4 Introduction 119-165

Unit 5 Enzyme & Mechanism of Enzyme action 166-208
Unit 6 Kinds of Reactions catalysed by Enzymes 209-242
Unit 7 Co-Enzyme Chemistry 243-262
Unit 8 Biotechnological Applications of Enzymes 263-298
BLOCK-3 BIOPHYSICAL CHEMISTRY
Unit 9 Bioenergetics 299-322

Unit 10 Biopolymer Interactions, Thermodynamics of 323-358
Biopolymer Solutions
BIO-INORGANIC, BIO-ORGANIC MSCCH-603
BLOCK I: BIOINORGANIC CHEMISTRY
UNIT 1: METAL STORAGE AND TRANSPORT

CONTENTS:
1.1 Introduction
1.2 Objective
1.3 Elements in living systems
1.4 Porphyrin ring
1.5 Metalloporphyrin
1.5.1 Some point noted about metalloporphyrin are
1.6 Role of iron in living system
1.7 metal storage transport and biominiralation ferritin, transferrin and siderrophores
1.7.1 Introduction
1.7.2 Storage of iron-ferritin
1.7.3 Transport of iron-transferrins
1.7.4 Siderophores
1.8 Introduction: Iron-containing proteins with porphyrin ligand systems
1.9 Haemoglobin
1.9.1 Properties hemoglobin
1.9.2 Biological roles of the hemoglobin
1.9.3 Function of hemoglobin in the human body
1.9.4 Structure of the hemoglobin
1.10 Self assessment question (SAQs)
1.11 Myoglobin
1.11.1 Structure of the myoglobin
1.11.2 The dioxygen-binding reaction
1.12 Cooperative effect
1.13 Bohr’s effect
1.14 Heme models
1.15 Photosynthesis
1.15.1 Introduction
UTTARAKHAND OPEN UNIVERSITY Page 1

1.15.2 Phase of photosynthesis
1.15.3 Role of photosystem I and Photosystem II
1.15.4 Mechanism of light dependent reduction
1.15.5 Generation of ATP via cyclic electron flow
1.15.6 The Dark reaction of photosynthesis, the calvin cycle
1.16 Nitrogen Fixation
1.16.1 Introduction
1.16.2 Type of nitrogen fixation
1.16.2.1 Physical nitrogen fixation
1.16.2.2 Biological nitrogen fixation
1.16.2.3 Basic requirement of nitrogen fixation
1.16.3 General structure feature
1.16.4 Specific structure feature
1.16.5 Mode of action of nitrogenase
1.17 Summary
1.18 SAQs types questions
1.19 Glossary
1.20 Reference
1.21 Suggested reading
1.22 Terminal question

1.1 INTRODUCTION
Living organisms store and transport transition metals both to provide appropriate
concentrations of them for use in metalloproteins or cofactors and to protect them selves
against the toxic effects of metal excesses; metalloproteins and metal cofactors are found in
plants, animals, and microorganisms. The normal concentration range for each metal in
biological systems is narrow, with both deficiencies and excesses causing pathological
changes. The storage of transition metals and the creation of transporter molecules are not
carried out by all types of cells in multicellular animals made up of a range of specialized cell
types, but by particular cells that specialize in these functions. Metals are constantly ionic,
although their oxidation states can change based on biological demands.
In order of decreasing abundance in living organisms, the transition metals iron, zinc, copper,
molybdenum, cobalt, chromium, vanadium, and nickel are important for biological storage
and transport. Despite the fact that zinc is not exactly a transition metal, it has many
bioinorganic characteristics with them and is treated as such in this chapter. Iron storage and
transportation are better understood than any other metal in the group. The goal of
biochemistry is to use chemical principles to explain biological structure and function.
Biomolecules are carbon-based compounds with various functional groups, therefore the
chemistry of living organisms is centered on carbon. Carbon makes up more than half of a
cell's dry weight. Hydrogen, oxygen, nitrogen, phosphorus, and sulfur are the other five most
prevalent elements. Although organic chemistry is more commonly linked with molecular
biology, inorganic elements and metal ions are equally crucial in biological processes. The
importance of platinum in anticancer medicines, metals as natural components of proteins,
metalloproteins, and mettaloenzyme in life processes are all significant.
In biology, alkali and alkaline earth ions, particularly Na+, K+, and Ca2+, have a role in
triggering cellular responses. Two of their critical activities are the fast influx of sodium ions
across the cell membrane to fire neurons and the control of intracellular activity by calcium-
binding proteins like calmodulin. Other activities that are critical for the organism's survival
include bone calcification, blood coagulation, acid-base equilibrium, fluid balance, and
osmatic control. Calcium, phosphorus, magnesium, sodium, potassium, chloride, and sulfur
are the seven major elements that make up roughly 70% of the inorganic substance in the
human body. Iron, copper, iodine, manganese, zinc, molybdenum, cobalt, fluorine, selenium,

and chromium are all important trace elements. Some other trace elements like, nickel,
vanadium and barium may be possibly essential in biological systems.
1.2 OBJECTIVE
The objective of this unit, you will be able to-
(i) What is the hemoglobin, its function and its structure
(ii) What is the myoglobin, its function and its structure
(iii) You will be able to function of ferrin and transferritin
(iv) After reading this unit you will be able to about photosynthesis process
(v) And end of this unit will be able to what is nitrogen fixation
1.3 ELEMENTS IN LIVING SYSTEMS

The living matter is made up of six elements: carbon, hydrogen, oxygen, nitrogen,
phosphorus, and sulfur (which make up roughly 90% of the dry weight of the human body).
Other physiologically significant elements are Ca, K, Na, Cl, Mg, Cu, Co, I, Zn, F, Mo, and
Se. Their body composition varies greatly, for example, Ca makes up about 2% of body
weight whereas Co makes up only 0.00004 percent.
1.4 PORPHYRIN RING

A porphyrin is a large ring molecule made up of four pyrroles (smaller rings made up of four
carbons and one nitrogen which is a heterocyclic compound (Fig.1.1). These pyrrole
molecules are linked by a chain of single and double bonds, forming a huge ring.
Fig. 1.1 Pyrrole ring

A tetrapyrrole is the technical term for four pyrroles linked together. The ring is flat in space
and has a reasonably even distribution of electrons around its diameter. As a result, a
porphyrin is classified as aromatics (Fig.1.2). A porphyrin molecule is highly stable in this
shape. Porphin is the model of a universal porphyrin.
Fig 1.2 Structure of porphyrin ring
The porphyrin pathway is found all across the biological world, acting as an assembly line for
nature's most prevalent colors in both plants and animals. Porphyrin molecules are ring-
shaped molecules that bind a variety of metal ions, each of which has a particular biological
function. Chlorophylls bind magnesium, which is essential for photosynthesis. Heme binds
iron to coordinate molecular oxygen and carbon dioxide transport, maintains the electron
transport chains required for cellular respiration, and aids numerous enzymes in catalysis.
Porphyrins bind nickel to produce coenzyme F430, which plays a crucial role in methane
metabolism in bacteria (Fig.1.3).
Vitamin B12 is produced when cobalt binds to a porphyrin derivative; a deficiency in the
vitamin can cause pernicious anemia and impair brain and nervous system function. These
porphyrin-derived pigments can be referred to as the "colors of life" since they are required to
maintain essential functions in virtually all organisms. Protoporphyrin, commonly known as
heme, is an iron-containing molecule that plays a vital role in many biological processes
binding of moleculer oxygen to its iron ion. Hememolecules' capacity to function depends on
the ability of certain proteins, known as hemeproteins, to attach to them. The remarkable
heme-iron coupling with gas molecules or particular amino acids is responsible for heme's
natural action.

Fig 1.3 Combination of porphyrin ring with different type metal ions.
Fig 1.4 Formation of heme group
Other metal-containing protoporphyrins are not as common as the iron-containing

protoporphyrin. Although there are many distinct types of hemeproteins (Fig.1.4) with
various functions, such as hemoglobins, myoglobins, cytochromes, and peroxidases.

Hemeproteins are involved in all major electron transfer (ET) chain processes in nature, the
most notable of which are our mitochondria's aerobic respiration system and chloroplasts'
photosynthetic system. The redox of the Fe ion within the heme (Fe2+ , Fe3+) shuttles
electrons in ET processes, and its redox potential is strongly reliant on the heme-binding site
within the protein.
key point
 Porphyrin is a macrocyclic compound. Structurally, porphyrin ring consists of four

pyrrole ring.
 Pyrrole ring is five membered heretocyclic compound consisting one nitrogen atom
and four carbon atom.
 In porphyrin ring four pyrrole ring connected with each other by methine (-CH=)
group.
 Porphyrin ring act as both diacidic as well as dibasic in nature.
 Porphyrin ring contain eleven (11) conjugative double bond.
 With a total of 26 pi-electrons, of which 18 pi-electron form a planar, continuous

cycle, the porphyrin ring structure is often described as aromatic.
 When porphyrin ring combine with any metal (Fe, Co, Mg,) is known as
metalloporphyrin.
 When Fe bind with porphyrin then it is kwon as heme, which is compound in red
blood cells, participate in the oxygen storage and transportation.
 The electronic transition in porphyrin ring is 𝜋 − 𝜋 ∗.
 The complexes which containing porphyrin ring colour appears due to 𝜋 − 𝜋 ∗

electronic transition.
1.5 METALLOPORPHYRIN
The metalloporphyrins include two important categories: the chlorophyll molecule and the
molecules carrying the heme group. The ability of chlorophyll to absorb light is related to the
conjugated polyene structure of the porphyrin ring. Magnesium ions that are coordinated to

the nitrogen atoms of the four pyrrole rings have at least two functions. They provide the
necessary structural rigidity and they increase the rate of conversion of the singlet-excited
state resulting from photon absorption into the triplet state that enables the transfer of the
excitation energy into the redox chain. The two main functions of heme iron-containing
proteins are the transport of oxygen and the mediation of electron transfer reactions. The
heme group is in all cases associated with a protein molecule, as in hemoglobin, myoglobin,
cytochromes, and enzymes such as catalase and peroxidase.
1.5.1 Some point noted about metalloporphyrin are:
i. Metalloprotein are cyclic structure or closed ring structure which is a micro cyclic
compound.
ii. Metal ion that their size match with the cavity, fit inside the cavity.
iii. Matelloporphyrin ring the number of conjugated double bond is 11.
iv. Mattelloporphyrin ring is planar, rigid and conjugative hence it is aromatic compound
because is obey huckel rule.
1.6 ROLE OF IRON IN LIVING SYSTEM

Iron is crucial for every kind of living organism, including plants, bacteria, animals and
humans, to transport oxygen (through the haemoglobin in animals and humans) and to
produce energy (through electron transfer in the mitochondrial respiratory chain). In living
system iron is found in following types of compounds.
(a) Compound containing one or more heme group e.g. haemeglobin, myoglobin,
cytochromes, cytochrome P450.
(b) Iron sulpher protein e.g. rubredoxin, ferridoxin, nitrogenase.
(c) Di-iron oxo bridged compound e.g. haemerythrene, ribonucleotide etc.
1.7 METAL STORAGE TRASNPORT AND

BIOMINIRALISATION FERRITIN, TRANSFERRIN AND
SIDERROPHORES

1.7.1 Introduction
In living organisms, particular organic surfaces of proteins and/or lipids are responsible for
the synthesis of minerals with certain shapes and compositions. Biomineralization is a natural
process that occurs in many places, including the ocean (Ca is involved in shell formation).
The development of the ferritin core is another example. Ferritin is the primary non-haem Fe
storage protein in mammals. Remember that the majority of Fe is found in haemoglobin and
myoglobin. When ferritin is completely loaded, it comprises roughly 20% Fe by mass. No
additional iron can enter the cell after the ferritin is saturated. Techniques exist in organisms
to not only store and accumulate metal ions, but also to utilise them later. To transport iron,
various complexion agents are utilised within the organisms.The iron binding protein
transferrins transports iron through the bloodstream of higher animals. Transferrins transfer
iron from the site of production of other iron-containing compounds, such as haemoglobin
and chromosomes, to the porphyrin ring, where it is inserted by enzymes. Iron storage is
important because it is required and utilised in significant amounts by cells, and inorganic
iron is stored in intestine mucousal cells linked to the intracellular protein ferritin.
1.7.2 Storage of iron-ferritin
(i) Role of ferritin
The French scientist Laufberger discovered ferritin in 1937 when he extracted a novel protein
from horse spleen that contained up to 23% iron by dry weight. Iron is stored mostly in
ferritin, an iron storage protein. For the production of haemoglobin, myoglobin, and
cytochrome, ferritin release tron is required. This is important because unbound free iron is
extremely toxic and causes cellular damage by promoting the production of free radicals.In
mammalian tissues such as the liver, spleen, and bone marrow, ferritin is a significant iron
storage.
(ii) Structure of Ferritin
Inside and outside of cells, ferritin is an iron-binding protein. Apoferritin, as shown in the
picture (Fig. 1.5), creates a roughly spherical container within which ferric iron is kept as the
ferrihydrite mineral ferric iron (Apoferritin refers to the iron-free form of the protein; the
iron-containing form is termed holoferritin or simply ferritin).

There are 24 subunits in the apoferritin shell. The two sorts of subunits are H and L. The ratio
of these subunits varies a lot depending on the tissue type, and it can alter a lot under
inflammatory and infectious situations. H-subunit-rich tissue ferritins (found primarily in the
heart and kidney) to L-subunit-rich tissue ferritins (found predominantly in liver and spleen).
Each apoprotein molecule has a diameter of around 450,000 d. The L monomer is made up of
174 amino acids and has a molecular weight of 174. The H monomer has a molecular mass of
21,000 d and is made up of 182 amino acids.
Fig 1.5 Structure of ferritin
1.7.3 Transport of iron-transferrins
Several structurally important and related Fe-protrins, all of which are glycoproteins, make
up transferrins. Their molecular weights (molar masses) are approximately 80 k Da. New
haemoglobin is produced in the bone marrow, while old red cells are destroyed in the spleen
and liver. Transferrins [as a class of protein, serum protein, lactoferrin (milk), and
ovatransferrin (egg)] are iron binding proteins that transport iron in higher animals via the
bloodstream to the site of synthesis of other iron-containing compounds such as
haemoglobin, cytochrome, and others, where it is inserted into the porphyrin ring via enzyme.

Key point
 Transferrin binds Iron is in +3 oxidation stste.
 Fe2+ is oxidation to Fe+3 by ceruloplasmin ( a blue copper protein).
 Transferrin binds Fe3+ (from stomach) and enters into blood and transport
Fe3+ to bone marrows. In bone marrow, Fe3+ is reduced to Fe2+ and is
delivered to ferritin. Before and after binding in transferrin, Fe in +2 oxidation
state when binds to transferin, Fe in +3 oxidation state.
 Transferrin delivers ions when it is reduced to Fe+2 in bone marrow, Fe+2 is

further oxidation by ceruloplasmin (ferroxidease) to Fe+3 and pickup by ferritin
( ferritin find in bonemarrow, spleen and livers.
 Ferritin contain two compound,
I. Apoferritin- 24 protein chain each with 175 amino acid.
II. Iron core- it contain 4500 Fe+3 ions.
 Ferritin release iron for the synthesis of hemoglobin, myoglobin and

cytochromes.
 Transferrin is the best scavengers of iron in the animals.
1.7.4 Siderophores
Despite the fact that iron in its ferric form is poorly soluble, it is necessary for microbial
growth. As a result, most bacteria produce and release siderophores, which function as high-
affinity chelators that enable iron into their cells. The production of siderophores is one of the
most common strategies used by bacteria, fungi, and plants (from the Greek: "iron carriers").
Siderophores (400–2000 Da) are natural iron chelators with a low molecular weight. They are
produced in the presence of an iron shortage to scavenge iron and form soluble iron(III)
complexes. They are involved in pathogenic bacteria taking iron from host proteins because
they allow organisms to access insoluble iron forms. Siderophores (400–2000 Da) are natural
iron chelators with a low molecular weight. They are produced in the presence of an iron
shortage to scavenge iron and form soluble iron(III) complexes. They are involved in

pathogenic bacteria taking iron from host proteins because they allow organisms to access
insoluble iron forms. Iron is an essential component for almost all microorganisms.
In mammals, the serum proteins ferritin and transferrin bind and transport iron. Because
microorganisms are unable to bisynthesize high-molecular-weight complex proteins, they
gather iron from their surroundings via siderophores, solubilize and transport iron (III) by
forming extremely stable octahedral complexes with Fe(III), and finally transport iron across
the cell membrane.
The following considerations should be taken into consideration:
Fig. 1.6 Structure of siderophores
 The siderophore is a chelating ligand that provides Fe(III) with a set of oxygen donors,
and the complexes are considered to have six coordinates (III).
 Based on their chemical structure and how they chelate iron, siderophores are divided
into various groups. A desferrichrome (Fig 1.6), for example, is a cyclic hexapeptide
with three consecutive amino acid residues carrying side chains that terminate in
hydroxamine moieties.
1.8 INTRODUCTION: IRON - CONTAINING PROTEINS WITH

PORPHYRIN LIGAND SYSTEMS

Iron is the most abundant transition metal found in biological systems with a percentage by
weight in the human body - for instance, 5.0 × 10−3 %. Therefore, it is not surprising that iron
- containing proteins and enzymes are found in huge numbers and varieties in all biological
species. First, one might classify iron - containing species in two categories: those containing
a porphyrin ligand system an iron containing heme moiety and those not containing
porphyrin ligands non heme iron containing proteins.
1.9 HEAMOGLOBIN
Haemoglobin (molecular wieght 645000 ) can be consider an oxygen carrier in animals. In
most of the animals, it is the pigment to provide colour of blood. The colour of blood is red if
haemoglobin is present with oxygen. In the absence of oxygen in haemoglobin the colour of
the blood is blue. It is due to the transfer of electron in between the π and π* orbital of the
ring and iron atom (Fig. 1.7). In human body near about 4 g iron is present. Out of thin
approximately 0.8 g is used to produce red colour in RBC by haemoglobin. Remaining iron is
store in the form of ferretin. Hemoglobin provides an excellence example of the Quaternary
structure of a protein (association of two or more peptide chains in the complete protein).
Hemoglobin is a collection of four heme units each bound to each of the four protein chains.
Thus, there are four sub-units in hemoglobin two each of two slightly different peptide
chains. These are held together by electrostatic and van der Waals force as well as hydrogen
bonding. In each subunit of hemoglobin the heme prosthetic group is held in possition by the
imidazole of histidine in the globin protein chain just as in myoglobin through a coordination
histidine nitrogen atom.
1.9.1 Properties hemoglobin
• There are 5 billion red cells/Ml, 280 million Hb molecules/red cell, and 4 heme groups Hb
molecule.
• Similar to Mb, each heme is tightly bound to a protein (globin) through about 80
hydrophobic interactions and a single coordinate bond between imidazole of the proximal
histidine and Fe(II).
• Molecular weight 64,500, tetramer.
• Human Hb has four chains, α2 β2, two identical chains labeled α, with 141 amino acids, and
two identical β-chains, with 146 amino acids.

Fig. 1.7 structure of hemoglobin
1.9.2 Biological roles of the hemoglobin
(i) Hemoglobin binds O2 to its heme iron at the lungs and delivers O2 to myoglobin, which
stores the O2 until it is required for metabolic oxidation.
(ii) A second task of Hb is to bring the CO2 by-product of oxidation back to the lungs to get
rid of it:

R−NH2 + CO2 R−NH−COO- + H+
CO2, also buffering, as a result of the reversible formation of HCO3.
CO2 + H2O HCO-3+ H+
The function of hemoglobin is to take up oxygen in the lungs, and transport it to the various
tissues in the body (Fig. 1.8).
Fig 1.8 Mechanism of transportation of oxygen through hemoglobin
Myoglobin, present in muscle tissues, takes up the oxygen from the hemoglobin and stores it
until it is needed for the production of adenosine triphosphate (ATP) in the mitochondria.
One oxygen molecule binds to the iron atom of the heme moiety at the sixth position which is
vacant in the deoxygenated form of these proteins.

1.9.3 Function of Hemoglobin in the Human Body
Following types function of the hemoglobin in human body are:
1. Hemoglobin is an oxygen carrier.
2. Hemoglobin is a carbon dioxide carrier.
3. Hemoglobin gives the red color to blood.
4. Hemoglobin maintains the shape of the red blood cells.
5. Hemoglobin acts as a buffer.
6. Hemoglobin interacts with other ligands.
7. Hemoglobin degradation accumulates physiologically active catabolites.
1.9.4 Structure of the hemoglobin
Hemoglobin is a conjugated protein. Its molecular weight is 64500. it is chromo protein. In

this globin and heme two substances are present. Heme is metalloporphyrin and globin
protein.
Heme + globin
Molecular formula of hemoglobin C34H32N4O4Fe. In the structure of hemoglobin two

carcoxylic and two ethylenic grioup present. All the four pyroole ring in heme are conneced
to ecah other by methine an (-CH=) group. All the four pyrrole ring has one methyl group
each and iron present in hemeglobin to the centre of the heme.
Hemoglobin comprises four subunits, each having one polypeptide chain and one heme group
(Fig 1.9 ). All hemoglobins carry the same prosthetic heme group iron protoporphyrin IX
associated with a polypeptide chain of 141 (alpha) and 146 (beta) amino acid residues. the
ferrous ion of the heme is linked to the N of a histidine.
The porphyrin ring is wedged into its pocket by a phenylalanine of its polypeptide chain. The
polypeptide chains of adult hemoglobin themselves are of two kinds, known as alpha and
beta chains, similar in length but differing in amino acid sequence. The alpha chain of all
human hemoglobins, embryonic and adult, is the same. A expanded view of the Heme group
reveals that it consists of an atom of ferrous iron (Fe2+) and a surrounding porphyrin
ring (four nitrogen-containing pyrrole molecules). The iron atom can reversibly bind with one

molecule of oxygen (O2). Hemoglobin (Hb) transports oxygen, in blood, taking dioxygen
from air in the lungs and delivering it to Mb in tissues. Hemoglobin is a multisubunit protein,
with two a and two p polypeptide chains.Life of the hemoglobin molecule is approximate 16
week.
Fig 1.9 Structure of hemoglobin
The main role of the protein chain around the hemoglobin molecule and it provide the
hydrophobic environment around of the Fe2+ ions and prevent it contact with water and its
oxidation.Hemoglobin is present in human body in the two form:
Deoxyhemoglobin form of hemoglobin without oxygen, the predominant protein in red

blood cells. Hemoglobin forms an unstable, reversible bond with oxygen. In its oxygen-
loaded form it is oxyhemoglobin and is bright red. In the oxygen-unloaded form it is
called deoxyhemoglobin and is purple-blue. In each chain there is an iron protoporphyrin IX

group held by a proximal histidine imidazole residue, as in Mb. In deoxy Hb, the iron lies 36
pm to 40 pm (Fig 1.10 ) out of the porphyrin ring plane but moves within 12 pm of the plane
upon binding of dioxygen.
Fig. 1.10 Dome structure or square pyramidal
In deoxyhemoglobin (deox-Hb) the iron is coordinated to only five ligand since oxygen is not
present. Thus in deoxy Hb and deoxy Mb has a pentacordinated iron (II) centre in the high
spin state [ iron (II) in d6], with high spin (the square pyramidal arrangementof heme in
myoglobin and hemoglobin can be considered an octahedron without the sex liigand) and
therefore, the iron atom will not fit into the hole of the porphyrin ring (Fig. 1.11).
Electronic configuration of iron in free state
Fe26 =1s2, 2s22p6, 3s2p63d6,4s24p0
Electronic configuration of iron in heme molecules
Fe2+26 =1s2, 2s22p6, 3s2p63d6,4s04p0

Fig 1.11 high spin model of hemoglobin
In the case of deoxy Hb, the iron atom contain four unpaired electrons (scheme) which are
repulsed by the non-bonded electron of the nitrogen atom ( nitrogen atom of pyrrole ring),
hence the iron atom in deoxy Hb molecules is 40 pm above the four pyrrole ring (porphyrin
ring). Deoxy hemoglobin are the paramagnetic in nature due to the presence of four unpaired
electron and their magnetic moment is 4.9 which measures by μ=√n(n+2) where n = Number
of unpaired electrons
μ=√4(4+2),
μ=√4(6) = 4.9
Oxyhemoglobin the molecule of oxyhemoglobin, like that of carbonmonoxyhemoglobin, is

found to have zero magnetic moment and to contain no unpaired electrons. Each iron atom is
accordingly attached to the four porphyrin nitrogen atoms, the globin molecule, and the
oxygen molecule by covalent bonds. In oxy Hb no unpaired present in the iron atom due to
presence of strong field ligand (O2), hence iron atom co-planner with the all four pyrrole ring
(Fig 1.12)

Fig. 1.12 Structure of the Oxyhemoglobin
Electronic configuration of iron in free state
Fe26 =1s2, 2s22p6, 3s2p63d6,4s24p0
Electronic configuration of iron in heme molecules
Fe2+26 =1s2, 2s22p6, 3s23p63d6,4s04p0
Fig 1.13. Low spin model of hemoglobin

In both Hb and Mb, there is a histidine situated in the Oxygen,-binding pocket on the side
away (distal side) from the coordinated imidazole base. X-ray and, for oxy myoglobin,
neutron diffraction studies indicate the formation of a hydrogen bond between the
coordinated dioxygen molecule and the N-H proton of the distal histidine residue. This Fe-O-
O-H-N unit is nicely accommodated, owing to the angular bend at the coordinated dioxygen
atom. In carbon-monoxide adducts of Mb and Hb, however, the X-ray data reveal a distorted
structure in which the Fe-CO angle is either bent or, more likely, tilted off the proximal
histidine N-Fe bond axis to avoid steric clash with the distal histidine. Thus nature has
cleverly tailored the iron- porphyrin centers in Hb and Mb to bind O2, rather than its toxic
surrogate CO, which often binds more tightly to metal complexes than does dioxygen.
Irreversible oxidation is another potentially catastrophic defect in the binding of dioxygen by

heme. When free heme in aqueous solution is exposed to dioxygen, it forms a -oxo dimer
known as hematin very instantly. The reactions are as follows, with the heme group ‘PFe (II)'.
The binding of the dioxygen molecule, as in haemoglobin, is the first step:
PFeII + O2 ⇌ PFeIIO2 ↔ PFeIIIO2 - (1)
The bound dioxygen now coordinate with a second heme, forming a μ-peroxo complex:
PFeIIO2 + FeII → PFeIII−O−O−FeIIIP (2)
Cleavage of the peroxo complex results in two molecules of a ferryl complex with the iron in
+4 formal oxidation state:
PFeIII−O−O−FeIIIP → 2PFeIII−O. ↔ PFeIV=O (3)
Finally, ferryl complex attract another heme to form hematin:
PFeIV=O + PFeII → PFeIII−O−FeIIIP (4)
Obviously, living systems must find a means to stop processes (1) to (4) otherwise, instead of
shutting electrons in the cytochromes or transporting dioxygen molecules in oxyhemoglobin
and storing them in oxymyoglobin, all the heme should be precipitated as hematin.
1.10 SELF ASSISMENT QUESTIONS (SAQS)

Question 1.
A. multiple choice

1. The molecular weight of hemoglobin
A. 44,450, B. 54,450,
C. 64,500, D. 74,450
2.The porphyrins are cyclic compounds formed through methylene bridges by the linkages of
pyrrole rings number.
A. 4 B. 3
C. 2 D. 1
3. Two alpha-chains of globin have identical amino acid composition of
A. 111 B. 121
C. 131 D. 141
4. Hemoglobin takes up the number of molecules of oxygen
A. 1 B. 2
C. 4 D. 6
5. Carboxyhemoglobin is formed by
A. CO B. CO2
B. H2CO3 C. HCN.
6. In the porphyrin ring four pyrrole ring connected with each other via
A. Methane B. Methyl
C. Methine D. Methylene
7. In oxy-hemoglobin, the iron centre is best described by which of the following
A. high-spin Fe(III) B. high-spin Fe(II)
C. low-spin Fe(III) D. low-spin Fe(II)
B. Fill in the bling
1. The absence of oxygen molecule hemoglobin is known as……….
2. The α-chains of hemoglobin contains…………. and β-chain contains……… amino acids.

3. Iron in the…………state is bound to the…………… atom of the pyrrole rings.
4. Iron is also internally linked to the nitrogen of the……….ring of ……….of the

polypeptide chains.
5. The main role of the protein chain around the hemoglobin molecule and it provide the
…………………………around of the Fe2+ ions and prevent it contact with water and its
………….
C. True and false:
1. Transferrin binds Iron is in +3 oxidation stste. True/False
2. Low spin form of hemoglobin is diamagnetic nature True/False
3. Life of the hemoglobin molecule is approximate 16 week True/False
4. Iron atom combine with the porphyrin ring it is called heme protein True/False
D. Match the following
i. Hemoglobin a. Iron storage
ii. Ferritin b. O2 transport and iron
iii. DeoxyHb c. Diacidic and dibasic
iv. Porphyrin ring d. square pyramidal
1.11 MYOGLOBIN (MB)

Myoglobin (symbol Mb or MB) is an iron- and oxygen-binding protein found in
the skeletal muscle tissue of vertebrates in general and in almost all mammals. myoglobin is a
pigment as hemoglobin. It is constituted by heme and globin. It is alpha halical neuclear
protein with 153 amino acids. The molecular weight of myoglobin is 17000. Myoglobin store
oxygen in the muscles tissue and release when oxygen is required. Myoglobin is the
monomer of the hemoglobin and contain Fe2+ ion in active site. Compared to hemoglobin,
myoglobin has a higher affinity for oxygen and does not have cooperative-binding with
oxygen like hemoglobin does. But at the core, it is an oxygen-binding protein in red blood
cells. In humans, myoglobin is only found in the bloodstream after muscle injury. Iron within
the heme group must be in the Fe+2 state to bind oxygen. If iron is oxidized to the
Fe+3 state, met myoglobin is formed. The total amount of myoglobin in an animal depends on

body weight, degree of muscle development, and the myoglobin concentration in muscle,
which varies between muscle types (red muscle is rich in myoglobin and white muscle is
myoglobin poor).
1.11.1 Structure of the myoglobin
Myoglobin (Mb) is the oxygen binding protein found principally in muscle tissues of
vertebrates. It consists of a single polypeptide chain of 153 amino acids called globin, made
up of seven a-helical and six non helical segments (Fig 1.14). Attached to the chain by
coordination to the imidazole ring of a histidine residue is the dioxygen-binding prosthetic
group, iron(II) protoporphyrin IX. The structure of sperm-whale myoglobin has been
determined in all three (deoxy, oxy, and met) forms.
Fig.1.14 Structure of myoglobin
Deoxy Mb has a pentacoordinate iron(I1) center in which the metal atom lies 92 pm out of
the plane of the four pyrrole-ring donor nitrogen atoms(sheme). It is displaced toward the
bonded imidazole group, which is called the proximal side of the heme. Upon binding of
dioxygen, the iron atom moves toward the FeN, planes. In oxy Mb, the dioxygen molecule is
bonded end-on to iron, forming a bent structure with a Fe-0-0 bond angle of 115.
1.11.2 The dioxygen-binding reaction
Hb and Mb, probably the most thoroughly investigated metalloproteins, have been the
subjects of innumerable spectroscopic, thermodynamic, and kinetic measurements. Their
optical spectra are dominated by intense porphyrin ring π to π* transitions in the 400-600 nm

range, known as the Soret, a, and bands that are sensitive to the state of oxygenation. From
resonance Raman spectroscopic studies of the coordinated dioxygen molecule and its '80-
substituted analogs, an O-O stretching band at - 1105 cm-1 has been identified. This value is
characteristic of coordinated superoxide (O2-) ion suggesting that MbO, and HbO, adducts
might best be assigned as iron(II1) complexes of this ligand. Magnetic coupling between
these ions leads to a diamagnetic, S = 0, ground state. Thus coordination of dioxygen to
deoxy Hb or Mb is accompanied by electron transfer to form a superoxide ion, which in turn
is stabilized by hydrogen bonding to the distal imidazole proton, as indicated above.
1.12 COOPERATIVE EFFECT

The phenomena where the binding of one O2 molecules to a sub unit encourage the binding of
O2 molecules to another sub unit is called cooperative effect.The cooperative effect describes
the ability of the four identical haemoglobin sub-units to change their conformation. The
cause of this change is the acceptance or release of an O 2 molecule by one of the sub-units,
which increases the ability of the other haemoglobin domains to accept or release oxygen.
Great importance to the physiological functions of Hb and Mb is the beautifully sophisticated
bioinorganic system devised by nature in which dioxygen binds to Hb in the lungs and is
transferred to Mb in tissues or is transferred to fetal Hb in the uterus of pregnant mammals.
At the heart of this system is the motion of iron toward the plane of the porphyrin ring upon
conversion of deoxy to oxy Hb, which serves as a trigger for cooperative binding of dioxygen
by the multisubunit hemoglobin protein. The protein is presumed to have two different
quaternary structures designated R, for relaxed, and T, for tense. The former has a high
affinity for O2,, similar to that of isolated subunits, whereas the latter, tense state has a
diminished O2, affinity. These two conformational states are in equilibrium with one another.
In the T state, prevalent when all four sub units are a ligated, inter sub unit interactions are
believed to constrain the proximal histidine to resist movement into the porphyrin-ring plane
and diminish the O2, binding constant.
1.13 BOHR’S EFFFECT

The Bohr effect is a phenomenon first described in 1904 by the Danish physiologist Christian
Bohr. The Bohr effect generally describes the effect of pH on the blood-O2 -binding affinity.
The affinity of hemoglobin (Hb) towards the O2 is pH dependent. The affinity of Hb towards

decreases with decreases in pH. Affinity of myoglobin (Mb) towards O2 is pH independent.
The Oxygen (O2) competitively and reversibly binds to hemoglobin, with certain changes
within the environment altering the affinity in which this relationship occurs (Fig. 1.15).
Fig.1.15 Partial pressure of the O2 (PO2 in mm Hg)
The sigmoidal shape of the oxygen dissociation curve illustrates hemoglobin’s propensity for
positive cooperativity, as hemoglobin undergoes conformational changes to increase its
affinity for oxygen as molecules progressively bind to each of its four available binding sites.
The Bohr effect describes hemoglobin’s lower affinity for oxygen secondary to increases in
the partial pressure of carbon dioxide and/or decreased blood pH. This lower affinity, in turn,
enhances the unloading of oxygen into tissues to meet the oxygen demand of the tissue.
1.14 HEME MODELS

One has studied that hem group I hemoglobin and myoglobin has the striking ability to bind
O2 molecule and its subsequent release without the iron atom becoming permanently oxidized
to the Fe(III) state. The following points are of significance:
 Before O2 binding, the Fe(II) centre is though to be located significantly out of plane of
the porphyrin unit called doming effect.

This results in a constraint beetween the globin portion of the protein and iron atom. The
movement of the iron atom of heme group on oxygen binding moves towards the plane of
the heme which acts as a trigger and sets in extensive structure changes in other subunits
in hemogobin.
 In both hemoglobin and myoglobin the protein bulk folds around the heme units(s). this
characteristic folding/protein structure around the heme unit leads to steric hindrance
which checks any hematin formation by prohibiting approach of two iron porphyrin
moieties.
In the synthetic models, bulk (like protein structure) has been added to sample iron porphyrin
systems to block approach to the reactive iron centers. The following points may be noted:
 Imidazole and its derivatives play a role of good mimics for the protein histidine residue
in the biological system
 In the picket fence porphyrins bulky hydrophobic substituent are attached to the
porphyrin core to give an upright fence around the Fe binding site. In the presence of 1-
methylimidazole which serves as a base in the axial position on the other side of the ring
from the pickets. The Fe(II) complex of this model porphyrin ring system binds O 2 is
bound in the cavity created by picket fence
Substituent while the unhindered axial site can be occupied by a suitable nitrogen base e.g.,
imidazole (Fig 1.16)

Fig.1.16 Synthetic models- picket fence models
1.15 PHOTOSYNTHESIS
1.15.1 Introduction
Plants produce their own food through a process called photosynthesis. Light energy is
converted into chemical energy by them. CO2 from the atmosphere and water are integrated
into organic compounds with the help of this chemical energy. Instead of photosynthesis, it's
sometimes termed CO2 assimilation. Photosynthesis is a crucial process not just in terms of
quality but also in terms of quantity. Photosynthesis transforms between 200 and 500 billion
tonnes of carbon every year. As a result, photosynthesis is a quantitatively important activity
as well. At the expense of solar energy, the light reaction of photosynthesis produces energy-
rich NADPH and ATP. These products are utilized in the carbon-assimilation process, which
reduces CO2 to produce carbohydrates and occurs in light or darkness. Green plants, algae,
and photosynthetic bacteria use photosynthesis to capture solar energy and use it to fuel the
synthesis of carbohydrates from carbon dioxide and water Fig.1.17.
Fig.1.17 Synthesis of carbohydrates from carbon dioxide and water
1.15.2 Phase of photosynthesis
There are two phases to the photosynthesis reaction:
(1) The light process, which produces NADPH and ATP using light energy.

(2) A carbon-assimilation or carbon-fixation process in which NADPH and ATP are used to
synthesise carbohydrate from CO2 and H2O.These reactions are frequently referred to as
"dark reactions" (Fig.1.18)
Fig.1.18 Dark reactions
1.15.3 Role of photosystem I and Photosystem II
Photosystem I (PSI) and photosystem II (PSII) are the two types of photosystems used by
green plants and algae ( PSII). PSI's reaction centre chlorophyll has an absorption maximum
of 700 nm and is therefore known as P700 (P for pigment), while PSII's reaction centre
chlorophyll has an absorption maximum of 680 nm and is thus known as P680. Other
electron carriers, particularly the cytochrome bf complex, connect the two photosystems (Fig
1.19 ). The different components from the so-called Z scheme (Fig.1.20) when organised

according to their redox potential because the overall form of the redox diagram provides a
look of z. Plastoquinone, plastocyanin, ferredoxin, and other highly mobile electron carriers
are used in these events.
Fig 1.19 Interaction of PSI and PSII during photosynthesis.
Fig 1.20 The Z scheme of photosynthesis.

1.15.4 Mechanism of light dependent reduction
In photosynthesis, electron transport pathways from H 2O to NADP+ are defined. The

absorption of light by PSII and PSII allows for this endergonic process. When a PSII
chlorophyll in a PSII reaction centre is in its ground, unexcited state, it has no inclination to
give up an electron. When a photon's energy reaches it through the antenna chlorophyll, it is
excited, and it has a high inclination to pass on its excited electron. In reality, it is a reducing
agent. The high-energy electron is transferred to plastoquinone (Q), a mobile quinone in the
thylakoid membrane with a structure similar to ubiquinone in the mitochondrial electron
transport chain. P680 is the P680+ cation as a result of this. Plastoquinone is converted to
plastoquinol(QH2) by absorbing two electrons and two H+ ions. It's worth noting the
following:
i. The light-driven splitting of H2O is catalysed by a Mn-containing protein complex,

resulting in the production of O2 (Fig. 1.21). This strong reductant transfers its electron to
NADP+ to create NADPH.
ii.The four electron abstracted from water do not pass directly to P680+, which can accept
only one electron at a time. Instead, a remarkable molecular device, the water splitting
complex,passes four electron one at a time to P680+.
iii.Reduced plastoquinone formed by PSII now passess electron into the cytochrome bf
complex. Pc is a copper containing protein that accept electron by the copper cycling between
Cu2+ and Cu+ state.
A Mn-containing protein complex catalyses the light-driven splitting of H2O, resulting in the
production of O2 (fig.1.21).This strong reductant donates an electron to NADP +, resulting in
NADPH.
Fig. 1.21

In PSI, an electron was excited out of P700+ and transferred to ferredoxin, resulting in the
reduction of NADP+. P700+, on the other hand, has lost an electron and is now P700 +, an
oxidising agent. It takes one electron from reduced plastocyanin (Pc) and returns to the
unexcited state. Remember how light excited one electron out of P680, the PSII reaction
centre pigments; this left P680+, which needed its electron restored so it could transition to
the ground state, ready for another photon to start a new round of reaction. Water is the
source of the electron (Fig.1.20).
1.15.5 Generation of ATP via cyclic electron flow
When ferredoxin has reduced virtually all of the NADP +, it gives an electron to the
cytochrome bf complex (Fig.1.22). The resultant proton gradient created by the H+ pump,
cytochrome bf complex, subsequently drives ATP production. ATP is generated during
photophosporylation without the formation of NADPH, and no O 2 is produced since PSII is
not engaged.
In summary, when electron transport through PSI AND PSII operates in a noncyclic mode,
the products are NADPH and ATP. The sole result of cyclic electron transport, on the other
hand, is ATP.
Fig 1.22. Cyclic electron flow

1.15.6 The Dark reaction of photosynthesis, the calvin cycle
The dark reaction, also known as the carbon-fixation process, converts CO2 into carbohydrate
using the ATP and NADPH generated by photosynthesis' light reaction. Sucrose and starch
are the end products. The metabolite 3-phophogycerate is converted to glucose by a sequence
of processes that are similar to glucogenesis in the liver, except that NADPH is employed as
the reductant instead of NADH (Fig 1.23)
Fig. 1.23 Pathways for carbohydrate synthesis during photosynthesis

The main carbon fixation step is catalysed by the enzyme ribulose bidphosphate carboxylase,
which is the most abundant single protein on the planet. It uses CO 2 to create 3-
phosphoglycerate. The calvin cycle (dark phase of photosynthesis) begins with the interaction
of CO2 and ribulose 1,5-bisphosphate, which produces two molecules of 3-phosphpglycerate.
The conversion of 3-phosphoglycerate to fructose and glucose 6-phosphate involves
comparable enzymes to those involved in gluconeogenesis, with the exception that
glyceraldehyde 3- phosphate degydrygenase in chloroplasts is selective for NADPH rather
than NADH. Several complicated reactions are used to regenerate ribulose 1, 5-bisphosphate
from fructose 6-phosphate, glyceraldehyde 3-phosphate and dihydroxoacetone phosphate.
Several stages in ribulose 1, 5-bisphosphate regeneration are similar to those in the pentose
phosphate pathway (Fig. 1.24).
Fig. 1.24 The Calvin Cycle

For every CO2 transformed into hexose, three ATP and two NADPH are used. Photosystem I
absorbs four photons, while photosystem II absorbs another four, resulting in two NADPH
and a proton gradient strong enough to cause the production of three ATP. The primary
carbohydrate reserves of plants are starch in the chloroplast and sucrose in the cytosol.
Rubisco adds CO2 to ribose 1, 5 bisphosphate under typical atmospheric conditions. It can
then add O2 if the CO2 content is low. Phosphoglycolate and 3-phosphoglycerate are
produced as a result of this process. Although the phosphoglycerate can be recovered and
utilised in biosynthetic reactions, this method produces CO2 and NH4+ ( wastage of metabolic
energy). As a result of the net outcome of th this processis to consume O 2 and release CO2 , it
is called photorespiration. Solar energy is captured using bipyridine Ru(II) complexes,
which act as a photochemical device and resemble green leaves. The following
considerations should be taken into consideration:
• Green plants employ chlorophyll as a photosensitizer, and the photosynthetic protein

complexes in green leaves capture light energy.
• As a result of the photoinduced electron, H2O molecules split into H2 and O2, and CO2 is
converted to sugar.
• Synthetic bipyridine Ru(II) complexes act as photosensitizers for amines such as dimethyl
aniline, which lose an electron that is captured by an Ir(III)-complexed ion connected to two
bipyridine Ru(II) complexes through two pyridine rings.
• As a consequence, Ir (III) absorbs two electrons from two dimethyl aniline molecules, and
Ir(III) is reduced to Ir(I).
• To recycle the electron-harvesting process, the Ir(I) state lowers CO2 to formate and then
oxidises back to Ir(III).
1.16 NITROGEN FIXATION

1.16.1 Introduction
Elemental nitrogen is highly irresponsible to ordinary chemical reaction. Nitrogen fixation is

the process by which atmospheric nitrogen is converted by either a natural or an industrial
means to a form of nitrogen such as ammonia. In nature, most nitrogen is harvested from the
atmosphere by the microorganism to form ammonia nitrites, and nitrates that can be used by

plant. The biosynthesis of all nitrogen- containing organic compound, such as amino acid and
nucleic acid required fixed inorganic nitrogen compound, so nitrogen fixation. is essential to
life. Being a part of nitrogen cycle, it is essential for agriculture and the manufacture of
fertilizer. It is also useful for the manufacture of all nitrogen chemical compounds, which
includes some explosives, pharmaceuticals, and dyes
Biological nitrogen fixation was discovered by Jean-Baptiste Boussingault in 1938.
1.16.2 Type of nitrogen fixation
There are two type of nitrogen fixation:
I. Physical nitrogen fixation
II. Biological nitrogen fixation
1.16.2.1 Physical nitrogen fixation
The physical nitrogen fixation can be take place by two method:
1.Natural nitrogen fixation: N2 and O2 of the air react to form nitric oxide under the influence
of lightening and thunder. The nitric oxide then again oxidized with oxygen to form nitrogen
peroxide.
The reaction are as follows:
N2 + O2 lightening → Thunder 2NO
2NO + O2 2NO2
When rain falls, NO2 combines with rain water to form nitrous acid and nitric acid.The acids
fall on the soil along with rain water react with the alkaline radical of the soil to form water
soluble nitrates and nitrite.
2NO2 + H2O HNO2 + HNO3; HNO3 + Ca or K
Salts Ca or K nitrates
The nitrates are soluble in water and are directly absorbed by the roots of the plants.
2- Industrial Nitrogen Fixation - In the industrial scale Ammonia is produced by direct

combination of nitrogen with hydrogen at high temperature and pressure. Further it is
converted into various kinds of fertilizer, such as urea etc.

1.16.2.2 Biological nitrogen fixation
The conversion of atmospheric nitrogen into the nitrogenous compound with the help of
living organism is called as biological nitrogen fixation. The process is generally carried out
by two main type of microorganism: those which live in close symbiotic association with
other plants and those which are free living or non-symbiotic.
A- Free living nitrogen fixing bacteria:
 Azotobacter, Beijerinckia and clostridium are saprophytic bacteria that perform nitrogen
fixation.
 These bacteria add approx 10-25 kg, of nitrogen/ ha/ annum.
 Many free living blue green algae also perform nitrogen fixation.
 They add 20-30 kg, of nitrogen/ ha/ annum.
B- Symbiotic Nitrogen Fixating bacteria:
 Several species of rhizobiom live in the soil but unable to fix nitogen by themselves.
 They undergo nitrogen fixation by only as symbionts in the association of roots of

legumes.
1.16.2.3 Basic requirement of nitrogen fixation:
Basic requirement of nitrogen fixation are-
Nitrogenase enzyme complex, protective mecahnism against oxygen- leghaemoglobin,

feerrodoxin, hydrogen releasing sysytem, constant supply of ATP, coenzyme and cofactor
like CoA, inorganic phosphate and Mg+2, Cobalt and molybdenum.
1.16.3 General Stuctural feature
 The nitrogenase complex, a highly conserved protein complex, is responsible for

biological nitrogen fixation. Molybdnum nitrogenase, Vanadium nitrogenase, and iron-
only nitrogenase are the three form of nitrogenase found in diverse nitrogen fixing
bacteria. Molybdnum nitrogenase has been explored and characterised in more depth. All
nitrogenase are made up of two protein: Fe protein dinitrogenase reductase and Mo-Fe
protein dinitrogenase reductase. During catalysis, electron move from a pair of ATP

molecules within component II to the Fe-S cluster, where N2 is reduced to NH3 (Fig.
1.25).
Fig. 1.25 Nitrogenase Complex
 The smaller protein has a molecular weight of 60000 and is often termed as iron protein.
It contains a Fe4S4 cluster and called reductase.
 The other protein has a molecular weight of 240,000 and is called the Mo-Fe protein.
This is an α2-β2 tetramer and contain two molybdenum atom, about 30 iron atom and
around 30 inorganic/ labile sulphur.
 The iron sulphur cluster seems to act as redox centers. A soluble protein free cofactor
containing molybdenum and iron has been isolated.
 Neither of the protein is separatly active, but on mixing them the activity is restored.
1.16.4 Specific structural feature
 The Fe- protein contains two identical subunit and a single Fe4S4 center is present in the
protein which is bound between the subunit by forming Fe-S bonds to two cystein
residues in each subunit. Furthermore, the single Fe4S4 centre is located at one end of the
molecule and this is the only poin of any significant contact between the two subunit.
The hydrolysis of ATP id carried out by these iron sulphur clusters, which operate as
redox center (Fig. 1.26)

Fig.1.26 P-cluster
 The Fe-Mo protein is thaught to be immediatly responsible for substrate reduction.it is

here in the FeMo protein where the actual conversion of N 2 to NH3 occurs at the active
site of this larger protein.in fact there are two type of centerwhich are present in the
nitrogenous FeMo protein and are designated P cluster and Fe-Mo center.
 FeMo protein has four P cluster that are similar to Fe4S4 but not ferredoxin cluster. The P
cluster has a double cubane structure. With all cystein ligands paired on one of the two
non bridged iron atom in one bound Fe4S4 cluster. Its not uncommon for a Fe4S4 to have
location of iron. The two Fe4S4 cluster are connected by two cystein ligand and two set of
fe atom in a face sharing arrangment. A disulphide unit connects the two clusters. During
nitrogenase cycle, this arrangment could be potentially redox active.
 The Fe-Mo is a novel iron- molybdenum cofactor. Ths cofactor is extremly insoluble
and extremly air sensitive substance which contain 2Mo; 6-8 Fe; and < 6S atoms
 FeMo is thought as the site of substrate binding and for the actual conversion of N 2 to
NH3 .
 X- ray crystallography was recently used to derive the structure of Fe-Mo of

molybdenum iron protein of nitrogenase, and the cluster core if composition Fe 3MoS8 is
represented by two cuboidal fragments. One of these components comprise five iron
atoms, whereas the other has three iron atom plus a molybdenum atom. Two S-2 ions and
an unknown ligand connect the two parts of the cluster.the fact that Mo is octahedrally
coordinated whereas the iron atom at the interface has led to the hypothesis that dinitrgen
is bound and activated for reduction at the clusters center by two or more iron atoms,
implying that dinitrogen is not directly coordinated to molybdenum (Fig. 1.27).

Fig. 1.27 Fe-Mo Protein
1.16.5 Mode of action of nitrogenase

The reduction of nitrogen to ammonia is an exergonic reaction.
N2 + 3H2 2NH3
The bond of N2 is very stable, so it can be understood that nitrogen fixation require a high
activation energy due to presence of triple bond. So, atmospheric nitrogen is almost
chemically inert under normal conditions. Biological nitrogen fixation however occurs at
biological temperature and at 0.8 atm of nitrogen. The high activation barrier is overcome via
several pathways and in part by binding and hydrolysis of ATP and represents the overall
process
N2 + 10H+ + 8e- + 16ATPN 2NH4+ + 16ADP + 16Pi + H2
The biological nitrogen fixation is carried out with the help of enzyme nitrogenase compl and
it requires six electrons to fix one molecule of N2. The extra two electrons are needed to
reduce 2H+ to H2.
N2 + 6e- + 6H+ 2NH3
N2 + 8e- + 8H+ 2NH3 + H2
A highly reduced version of Fe-Mo protein is responsible for nitrogen fixation, which
necessitates eight electrons: six for N2 reduction and two electrons are needed to produce one
molecule of H2. As a result, this protein in engeged in dinitrogen binding and reduction and
numerous additional iron-sulphur clusters in the protein are implicated in electron transport.

In comparison to other molybdenum-containing enzymes, the molybednum site in FeMo is
certainly unique.
Mechanism: Ferredoxin and Fe- protein transport electrons from the oxidation of pyruvate
to Fe-Mo protein. (Fig. 1.28 ) show a schematic diagram of the nitrogenase complex, which
is made up of Fe-Protein and Fe-Mo protein.
Fig.1.28 Nitrogen fixation by nitrogenase
During the conversion of N2 to NH3, the iron protein and nitrogenase component undergo
cyclic association and dissociation. Fe-Mo protein is the name given to the nitrogenase
component. Although neither of the teo protein can fix nitrogenon their own,they can be
separated and jpined to do so. As a result, a reduced Fe-Protein bind to the Fe-Mo protein and
transfer a singel electron. After which the oxidised Fe-protein dissociate from the Fe-Mo
protein in a repating cycle. Two ATP molecules must be hydrolysed for each cycle. The job
of ATP is to provide chemical energy and ATP hydrolysis cause conformational changes in
the protein, which help to reduce the activation energy of the nitrogen fixation process. The
Fe reduction proteins potential is shifted from 300-420 mV whwn two ATP molecule attach

to it. As a result, the reducing power of Fe-protein increases, making electron transfer to Fe-
Mo protein easier.
It is the iron- molybdenum protein which is thought to be the actual site of N 2 coordination,
since its mutants which have a defective Fe-Mo cluster co-factor can reduce acetylene alright
but N2 only poorly.
1.17 SUMMARY
The summary of the present chapter is:
 A Porphyrin is a large ring molecule made up of four pyrroles (smaller rings made up of
four carbons and one nitrogen, which is a heterocyclic compound). These pyrrole
molecules are linked by a chain of single and double bonds, forming a huge ring.
 In the absence of oxygen in haemoglobin the colour of the blood is blue and the colour of
the blood is blue due to the transfer of electrons between the π and π* orbital of ring and
iron atom. A tetrapyrrole is the technical term for four pryroles linked together. It is flat
in space and has a reasonably even distribution of electrons around its diameter.
 Haemoglobin (molecular wieght 645000 ) can be consider an oxygen carrier in animals.

In most of the animals, it is the pigment to provide colour of blood.
 Myoglobin is an iron- and oxygen-binding protein found in the skeletal muscle tissue of
vertebrates in general and in almost all mammals. myoglobin is a pigment as
hemoglobin. It is constituted by heme and globin. It is alpha halical neuclear protein with
153 amino acids. The molecular weight of myoglobin is 17000.
 Plants produce their own food through a process called photosynthesis. Light energy is
converted into chemical energy by them. CO2 from the atmosphere and water are
integrated into organic compounds with the help of this chemical energy.
 Instead of photosynthesis, it's sometimes termed CO2 assimilation.Elemental nitrogen is

highly un responsible to ordinary chemical reaction.
 Nitrogen fixation is the process by which atmospheric nitrogen is converted by either a

natural or an industrial means to a form of nitrogen such as ammonia. In nature, most
nitrogen is harvested from the atmosphere by the microorganism to form ammonia
nitrites, and nitrates that can be used by plant.

1.18 SAQs TYPES QUESTIONS

A. Multiple choice question
1. Myoglobin contains the number of porphyrine ring
A. 1 B. 2
B. 3 D. 4.
2. Myoglobin binding of O2 depends on …………
A. Hemoglobin B. O2 concentration and affinity of myoglobin for O2
C. Ka D. Kd
3. Myoglobin is a
A. Nitrogen fixation enzyme B. catalyst for epoxidation reaction
B. Component in photosynthetic system D. Di-oxygen binding metalloprotein
4. The active site of enzyme nitrogenase contains
A. Mo B. Mn
C. Fe D. Cu
5. In photosynthesis, the predominant metal present in the reaction centre of photosystem II is
A. Zn B. Mn
C. Cu D. Fe
6. The metals involved in nitrogenase are
A. Mo and K B. Mo and Fe
C.Fe and Mg C. Fe and K
7. The reduction of nitrogen to ammonia, carried out by the enzyme nitrogenase needs
A. 2 electrons B. 4 electrons
C. 6 electrons D. 8 electrons
8. The functions of myoglobin is
A. Storage of CO2 B. storage of O2

C. Storage of NO C. Storage of CO
9. Nitrogen is converted into NH3 by enzyme
A. Uriage B. Invertage
C. Nimenase C. Nitrogense
10. Which element is present in major amount in human body
A. Zinc B. Copper
C. Iron D. Titanium
B. Match the following
A. Nitrogenase i. Magnese
B. P.S II ii. Iron
C. Myoglobin iii. Molybdenium
D. Bohr effect iv. Christian Bohr
C. Fill in the Blanks:
1. Myoglobins are the…………pigments occurring in the…………cells of vertebrates and

invertebrates.
ii. The α-chains of hemoglobin A contains………..and β-chain contains…….amino acids.
iii. The porphyrins are……… compounds with…………structure.
iv. Iron in the…………state is bound to the…………… atom of the pyrrole rings.
v. Iron is also internally linked to the nitrogen of the……….ring of ……….of the

polypeptide chains.
Ans. Imidazole; histidine
D. True/False
a. Chlorophyll is magnesium-containing porphyrin and the photosynthetic pigment of plants.
True/False
b. Chlorophyll and heme of hemoglobin are synthesized in living cells by different pathways.
True/False

c. The affinity of hemoglobin (Hb) towards the O2 is pH dependent. True/False
d. Myoglobin is the monomer of the hemoglobin and contain Fe 2+ ion in active site.
True/False
Answers Key
Question 1:
A. 1 a 2. b 3.d 4. c 5. c
B. 1. DeoxyHb 2. 141, 146 3. Fe2+, nitrogen 4. cyclic, tetra pyrrole 5.

hydrophobic environment, oxidation
C. 1. True 2. true 3. true 4. true
D. i-b ii-a iii-d iv-c
Question 2 :
A. 1 A 2 B 3 D 4 A 5 B 6 B 7 D 8 B 9 C 10 C
B. A iii B i C ii D iv
C. i Respiratory, Muscle ii 141, 146 iii Cyclic, tetra pyrrole iv Ferrous, nitrogen
v Imidazole, histidine
D. a True b False c True d True
1.19 GLOSSARY
ET= Electron transfer
Hb = Hemoglobin
ATP = Adenosin triphosphate
Mb = Myoglobin
DeoxyHb = Deoxyhemoglobin
Oxy-Hb = Oxy-hemoglobin
His = Histidine
PsI = Photosystem I

PsII = Photosystem
NADPH = Nicotinamide adenine dinucleotide phosphate
CoA = CoenzymeA
1.20 REFERENCES
1. Kalsi P.S., kalsi J.P.(2006), Bioorganic, bioinorganic and supramoleculer chemistry, new
age international (P) Ltd. Publishers, london, new delhi, nairobi.
2. Knovich M. A. Storey, J. A., Coffman, L. G., Torti, S. V., Torti, F. M. (2009), ferritin
for the clinician, Blood Reviews, 23, 95–104.
3. Berenguera, M. A., Monachona, M., Joseph, E., (2018), Siderophores: From natural
rolesto potential applications, Advances in Applied Microbiology, Volume 106.
4. Rutschlin, S., Bottcher, T. (2019), Engineering siderophores, Methods in Enzymology,
ISSN 0076-6879.
5. Ponka, P., Tenenbein, M., Eaton, J. W., (2015), Iron, Handbook on the Toxicology of
Metals, Fourth Edition, 2, 879-902.
6. Bertini, I. Gray, H. B., Lippard, S. J. Valentine, J. S. (1994), Bioinorganic chemistry,
University Science Books Mill Valley, California University Science Books Mill Valley,
California, ISBN 0-935702-57-1:
7. Rosette M. Malone, R. (2008), Bioinorganic chemistry, 2nd ed. , ISBN 978-0-471-76113-
6 (pbk.).
8. Giardina, B., Messana, I., Scatena, R. and Castagnola, M. (1995) The Multiple Functions
of Haemoglobin. Critical Reviews in Biochemistry and Molecular Biology, 30, 165-196.
9. Marengo-Rowe, A. J., (2006), Structure-function relations of human hemoglobins, Proc
(Bayl Univ Med Cent), 19, 239–245.
10. Pauling, L., Coryell, C. D., (1936), The magnetic properties and structure of
hemoglobin, oxyhemoglobin and carbonmonoxyhemoglobin, PNAS, 22 (4) 210-216;

11. Everse, J., (2004), Elsevier Inc., volume 2, pp. 354–361,,
12. Shaanan, B., (1983), Structure of human oxyhaemoglobin at 2.1 A resolution, J. Mol.
Biol. 171, 31-59.
13. Perutz, M. F., Fermi, G., Shih,T. B. (1984), Structure of deoxyhemoglobin cowtown [his
HC3(146) beta Leu]: origin of the alkaline Bohr effect and electrostatic intractions
in hemoglobin, Proc. Natl. Acad. Sci. USA, 81, 4781-4784.
14. Feher, J., (2017), Oxygen and Carbon Dioxide Transport, Quantitative Human
Physiology. Elsevier. , 656–664.
15. Harvey, J. W., 2008, Iron Metabolism and Its Disorders, Clinical Biochemistry of
Domestic Animals (Sixth Edition), https://doi.org/10.1016/B978-0-12-370497.00009-X
1.21 SUGGESTED READING

1. Bertini I., Gary H.B., Lippard S. J., Valentine J. S. (1988), Bioinorganic Chemistry, First
south asian edition,
2. William H. E., Daphne C. E., Bioinorganic and molecular biology, fourth edition.
3. Ochia E. I., (1977) Bioinorganic Chemistry–An Introduction, Chapter 11, Allyn and
Bacon,Boston .
4. Lipscomb W. N., Sträter N., (1996), “Recent advance in zinc enzymology”, Chem. Rev.,
96, 2375–2433.
5. Christianson D. W., Cox J. D., (1999), Biochemistry, 68, 33–57 .
6. Liljas A., Kannen K. K., Bergsten P. Waara C. I., Fridborg K., Strandberg B., Carlborn U.,
Jarup L., Lovgren S., Petef M., (1972), Nature New Biology, Lond., 235, 131 .
7. Hay R. W., (1980) Inorg. Chim. Acta, 46, 115.
8. Lindskog S., Henderson L. E., Kannen K. K., Liljas A., Nyman P. O., Strandberg B.,
(1971), The Enzymes, P. D. Boyer, Ed., 3rd ed., p. 58.
1.22 TERMINAL QUESTION

Q.1 What the function of the ferritin and transferritine?
Q.2 Explain structure of the porphyrin ring with the help of the diagram.
Q.3 Give an account for the structure and its biological functions of myoglobin.
Q.4 How will you compares the myoglobin and aemoglobin.
Q.5 How metal ion complexes are helpful in biological system. Write down with special
reference to the Iron atom.
Q.6 What is the nitrogen fixation.Discuss the mechanism of Nitragenase.
Q.7 What is the metalloporphyrins ? Write down the structure of metalloporphyrins with the
example.
Q.8 What is the haemoglobin. Explain the structure of haemoglobin.
Q.9 Give the account for the biological function of haemoglobin in living.
Q.10 What is the cooperative effect. How it is helpful for the binding of oxygen in
hemoglobin.
Q.11 What is the Bohr effect. How it is varies with pH.

UNIT 2: METALLOENZYMES
CONTENTS:
2.1 Introduction
2.2 Objective
2.3 Carboxypeptidase A (zinc enzyme)
2.3.1 Structure of carboxypeptidase A
2.3.2 Mechanism of Carboxypeptidase A
2.4 Carbonic anhydrase
2.4.1 Mechanism of action of carbonic anhydrase
2.5 Catalases and Peroxidases
2.5.1 Define the biological role and the main properties of catalases
2.5.2 Defifine the main biological role of peroxidases
2.5.3 Mechanism and structural features
2.6 Cytochrome P-450
2.6.1 Structure of cytochrome P-450
2.6.2 The mechanism of oxidation of a substrate with cytochrome P-450
2.7 Copper enzyme
2.7.1 Superoxide dismutase (SOD) a copper enzyme
2.7.2 Structure of Cu-Zn superoxide dismutase
2.7.3 Mechanism Cu-Zn superoxide dismutase
2.8 Molybdenum oxaotransferasees-Xanthine oxidase
2.8.1 Structural features and mechanism
2.9 Vitamin B12
2.9.2 Application of vitamin B12
2.9.3 Vitamin B12 Deficiency
2.9.4 Food sources of Vitamin B12
2.10 Summary
2.11 SAQs types question
2.12 Glossary
2.13 References
2.14 Suggested Reading

2.1 INTRODUCTION
In unit first we have discussed about the functions and role of hemoglobin and myglobin in
human and other living organism and also discuss about other oxygen transfer and oxygen
storage agency's. In this unit we also covered nitrogen fixation and enzymes involving in
nitrogen fixation and what is the process of photosynthesis in the plant and type of the
photosynthesis. In unit second we want to discuss the metalloenzymes and functions of
metalloenzymes. Metal ions (metal cofactors) that are directly bound to the protein or to
enzyme-binding nonprotein components are found in metalloenzymes (prosthetic groups).
Metalloenzymes make up around a third of all enzymes discovered so far. Other
metalloproteins, in addition to enzymes, are involved in non-enzyme electron transfer
reactions (cytochromes), and can serve as storage (for example, ferritin for iron) or transport
proteins (e.g., transferrin for iron). Metal storage is reversible in the latter groups of proteins,
and the metal is only a transient component. In a larger sense, ribozymes, i.e. RNA molecules
with enzyme function, may contain structurally and/or functionally significant metal ions
(usually divalent metal ions like Mg2+) and are thus referred to as metalloenzymes.
Natural metalloenzymes are well-known proteins that include one or more transition metal
ions such as Fe, Cu, Zn, Ni, and Co. These metalloenzymes are capable of catalyzing a wide
range of biosynthesis and metabolic events. These metal ions serve mostly as Lewis acids or
redox-active sites. Furthermore, several enzymes have been used to manufacture important
chemical molecules in both laboratory and industrial-scale operations. In one prominent case,
nitrile hydratase, which possesses a Co(III) ion in the reactive site, has been utilized to
produce acrylamide in commercial quantities. Metal complexes comprising valuable metals
such as Ru, Rh, or Pd, on the other hand, have been used as catalysts in the creation of a wide
range of chemicals and drug precursors. Several research groups have looked into altering
such metal complexes to improve not only their individual catalytic reactivities, but also their
stereo- and regio- selectivities, as well as their substrate specificity.
2.2 OBJECTIVE
In this unit you will be able to learn the-
(I) What is the function of zinc enzymes like, carboxypeptidase and carbonic anhydrase and
how it is impoartant for all biological process of living object.

(II) Role of Iron enzymes in the life and main functions of catalase, peroxidase and
cytochrome P-450 in biochemical reactions.
(III) Role of copper enzymes - superoxide dismutase, Molybdenum oxatransferase enzymes-

xanthine oxidase in may oxidation-reduction.
(IV) Functions and catalatic activity of Coenzyme vitamin B12 in the biochemistry.
2.3 CARBOXYPEPTIDASE A (ZINC ENZYME)

Carboxypeptidase A is a zinc enzyme with a tetrahedral shape and a sp 3 hybridization. Its
active site contains Zn2+. The C-terminal peptide link in proteins and peptides is hydrolyzed
by carboxypeptidase A, releasing the C-terminal amino acid. Regarding carboxypeptidase A,
it's worth noting the following:
 In the active site of carboxypeptidase A, Zn2+ is present.
 Function catalyses the hydrolysis of the C-terminal end of peptide linkages or proteins.
 Tetrahedral structure with sp3 hybridisation.
 The enzyme carboxypeptidase A, which is secreted by the pancreas and used to speed up
the hydrolysis reaction, has a molecular mass of 34,800.
 This enzyme is made up of a single 307-amino-acid chain that folds into a compact,
globular form with helices and pleated sheet regions.
2.3.1 Structure of carboxypeptidase A
Carboxypeptidase A (CPA) contains a zinc (Zn2+) metal center in a tetrahedral geometry with
amino acid residues in close proximity around zinc to facilitate catalysis and binding. Out of
the 307 amino acids bonded in a peptide chain, the following amino acid residues are
important for catalysis and binding; Glu-270, Arg-71, Arg-127, Asn-144, Arg-145, and Tyr-
248. Figure 2.1 and 2.2 illustrates the tetrahedral zinc complex active site with the important
amino acid residues that surround the complex.
The zinc metal is a strong electrophilic Lewis acid catalyst which stabilizes a coordinated
water molecule as well as stabilizes the negative intermediates that occur throughout the
hydrolytic reaction. Stabilization of both the coordinated water molecule and negative

intermediates are assisted by polar residues in the active site which are in close proximity to
facilitate hydrogen bonding.
Fig. 2.1 structure of the carboxypeptidase A
Fig. 2.2 mechanism of carboxipeptidase A with substrate

The following point may be noted :
 A Zn2+ion is present at the active site of carboxypeptidase A and is held in place by a

complex formed with Glu 72, His 196, and His 69, as well as water molecules.
 The reaction that occurs when an enzyme catalyses the breakdown of a protein's C-
terminal peptide bond to liberate C-terminal amino acid is known as hydrolytic cleavage.
When Zn2+ binds to water, it becomes more acidic, making the nucleophile more like the OH
ion. The negative charge that arises in the transition state is stabilised by Zn2+, and the
negative charge is also stabilised by Arg 127. Glu 270 is a general-purpose base catalyst. The
substrate is held securely in place in the active site by Arg 145 and Tyr 248.
2.3.2 Mechanism of Carboxypeptidase A
Hydrolysis of the C-terminal peptide bond in peptides and protein and release the C-terminal
amino acid (Fig. 2.3)
Fig 2.3. Hydrolysis of C- terminal end of amino acid.
2.4 CARBONIC ANHYDRASE

Carbonic anhydrase is an enzyme that catalyses the conversion of carbon dioxide (CO2) to
carbonic acid. It is present in red blood cells, stomach mucosa, pancreatic cells, and renal
tubules (H2CO3). Carbonic anhydrase affects CO2 transport in the blood and so plays a
significant role in respiration. The uncatalyzed equilibrium takes a long time to reach.

The forward (hydration) reaction happens in erythrocytes (red blood cells) during CO 2 uptake
by the blood in tissue, whereas the backward (dehydration) reaction occurs when CO 2 is
released in the lungs. The carbonic anhydrase enzyme multiplies the rate of this equilibrium
by a million.
This enzyme has a molar mass of around 30,000 and a single protein unit of 260 amino acids.
The active site contains a Zn2+ ion that is tetrahedrally coupled to three histidine imidazole
nitrogen atoms (His-96 and His-119), as well as water molecules or hydroxide ions (Fig. 2.4).
It comprises additional amino acids that work via hydrogen bonding, proton transfer and
other mechanisms.
Fig. 2.4 Structure of carbonic anhydraase
As the pH rises, the forward and reverse reaction rates in the CO 2 hydration equilibrium
increase. In carbonic anhydrase, the Zn2+ ion is more acidic than in carboxy peptidase. The
inclusion of a neutral or liss basic histidine residue rather than a glutamase residue
contributes to the acidity of the Zn2+ ion. The three histidine residues are also puleed back,
causing the Zn2+ ion to become more electronegative and acidic as it approaches the fourth
position. The nucleophilic OH - then attacks the carbon atom of CO2 trapped in the
hydrophobic pocket near the Zn2+ ion, forming a temporary five coordinate Zn2+ ion in which
a carbonato oxygen from HCO-3 coordinates to the Zn2+ ion. Following rearrangement, the
HCO3- ligand is replaced by an H2O ligand that is coupled to the Zn2+ ion, resulting in the
regeneration of Zn-OH-, which then attacks another CO2 to complete the catakytic cycle.

2.4.1 Mechanism of action of carbonic anhydrase
The following point may be noted:
 The accepted explanation is based on the fact that the kinetics of the carbonic anhyydrase
reaction are affected by pH (faster at high pH and when influenced by a group with an
apparent pKa value of around 7.0).
 When compared to the value of 14.0 for free water, the pKa value for zinc coordinated
water is around 7.0, which is quite low.
Fig. 2.5 Cyclic mechanism of carbonic anhydrase
 Because water attached to the zinc ion is swiftly transformed into hydroxide ion, the
mechanism (Zn-hydroxide mechanism) begins with the creation of zinc bound hydroxide
ion (Fig. 2.5). The nucleophilic hydroxide ion is positioned perfectly to attack carbon
dioxide's carbon atom.

 The zinc ion also aids in the orientation of almost CO2 to the active site (CO2 is
noncovalently bound), resulting in a high concentration of OH -. As a result, the
involvement of Zn2+ in carbonic anhydrase is crucial in bringing the substrates close
together, which improves their reaction orientation.
 CO2 attaches to metal-hydroxide once it forms, and the nucleophilic attack of -OH
produces metal bound bicarbonate, which is then displaced by water.
2.5 CATALASES AND PEROXIDASES

Peroxide is a heme protein that catalyses the oxidation of a number of substrates by hydrogen
peroxide, including ascorbate, ferrocynide, and cytochrome c. Almost every living entity
contains the catalase enzyme. The disproportionation of hydrogen peroxide and organic
peroxides was catalysed by catalases. They also catalyse the hydrogenperoxide oxidation of
substrates. Cataleses have the largest turnover of any enzyme, with one catalyse molecule
converting millions of molecules of H2O2 to water and oxygen every second.
The structure and reaction processes of peroxide and catalyses are similar. Both have a
Fe(III) heme active site with a high spin, and the imidazole nitrogen of the residue occupies
the fifth coordination site. In the resting enzyme, a water ligaand occupies the sixth
coordination site.
The reaction is believed to occur in two steps:
[FeIII—P]+ + H2O2→ [O = FeIV—P+]+
[O = FeIV—P+]+ + H2O2 → [ FeIII—P]+ + O2
Where FeIII—P represents the enzyme's heme group and O=FeIV—P+ represents the
mesomeric form of O=FeV—P, indicating that Fe(III) is not entirely oxidised to Fe(V), but it
does receive one electron from the porphyrin ring. It indicates that Fe(III) is oxidised to
Fe(IV), and the porphyrin ring (P) is oxidised to porphyrin by one electron, and the porphyrin
ring with one unpaired electron becomes a radical cation.
Tyr-357 (tyrosinate at position 357) in the fifth (axial) position can increase the reactivity of
the iron core, assisting in the oxidation of Fe(III) to Fe(IV).Human catalase works best at a
pH of around 7. Several metabolic activities produce hydrogen peroxide, which is a toxic
byproduct. It must be transformed into H2O and O2 to avoid damage to cells and tissue. The π

to π* transition in the porphyrin ring gives oxidised and reduced versions of catalase their
colour.
2.5.1 Define the biological role and the main properties of catalases
The following points may be noted :
 Catalases are found in, Erythrocyte, All plants, most aerobic bacteria.
 Catalases have the short C-conformation, in which both an axial position on iron and the
porphyrin periphery are exposed.
 Catalases catalyze the decomposition of H2O2 into H2O and O2.
 Molecular weight 240,000.
 Catalases are made up of four identical subunits, each with one heme group at the active
site and high-spin Fe3+–porphyrin prosthetic groups.
 The enzyme's resting state is Fe3+.
 Catalases and peroxidases are closely related enzymes.
 In the presence of cyanogen bromide (BrCN), absorption variations between native and
modified catalase were barely evident, implying that there is no considerable change in
the tyrosine environment.
2.5.2 Defifine the main biological role of peroxidases
The following points may be noted:
 Peroxidases are enzymes catalyzing the oxidation of a variety of organic and inorganic
compounds by H2O2, also acting as dehydrogenases:
 Peroxidases and catalases are related enzymes; both are capable of promoting the
oxidation of H2O2. The mechanism of this oxidation involves a similar enzymatic
intermediate.
 Examples of peroxidases:
(i) Chloroperoxidase: halogenates organic substrates.
(ii) Lactoperoxidase: antibacterial, oxidizes NCS
(iii) Cytochrome P-450: hydroxylates organic substrates.

(iv) Cytochrome c peroxidase: reduces H2O2.
 All the peroxidases purifified from plants contain the heme group (Fe3+–protoporphyrin
IX).
 Horseradish roots and the sap of fifig trees are the richest source of plant peroxidases.
Cytochrome c peroxidase from baker’s yeast also contains Fe3+–protoporphyrin IX.
 Many of these enzymes are mixtures of isozymes of differing physical but similar
catalytic properties.
 Most peroxidases are glycoproteins.
2.5.3 Mechanism and structural features
 Beef liver catalase and horseradish b peroxidase have had their structures determined.
The active sites of both enzymes have a high spin iron group.
 Catalase's peroxidase activity is generally preferred in most in vivo conditions. Catalase

can be found in the blood, bone marrow, mucous membranes, kidneys, and liver, among
other places. It is involved in the oxidation of H2O produced by oxidases. As a result,
enzymes that create H2O2 are grouped together with enzymes that degrade it.
 The catalase axial ligands are phenolate (tyrosine's deprotonated phenolic oxygen atom)
and most likely a water molecule. The water molecule coupled to heme is assumed to be
stored in the cavity of the enzyme's active site at the sixth position. On the active side,
the phenolate moiety from a tyrosyl residue on the protein is attached to the heme and
kept far away from the cavity (which holds water at the sixth position on the heme).
During the catalytic activity of the enzyme, the water in the cavity at the active site is
replaced by H2O2.
 In horse radish peroxidase the axial ligand is an imidazole from a histidyl residue on the
protein.
Moreover, near thed active site of both enzyme are histidine and asparagine or arginine side
chains which are suitably oriented to make part in the catalytic cleavage of O-O by the
enzyme when H2O2 replaces water.

Fig. 2.6 Structure of catalase
Fig. 2.7 Enzyme catalase action

Hydrogen peroxide binds to the ferric center and a subsequent heterolysis of the O-O bond
takes place. This requires a prior positive and negative charge separation in the trasition state.
The amini acid side chains (histidine and asparagine or arginine) near the active site perfoem
this role. The basic imidazole group of histidine helps in the proton transfer from the oxygen
atom of H2O2 attached with the iron to the departing oxygen atom and the arginine residue
helps to stabilize the developing negative charge on the departing of oxygen atom. This
results in O-O bond cleavage and the formation of the common high valent oxo intermediate
known as compound 1. Compound 1 has tha ability to oxidize other soecies by two electrons.
[Recall that in biological systems, like in chemical systems, oxidation (loss of electron) is
always accompanied by reduction of an electron acceptor](Fig. 2.7).
Fig. 2.8
The highly oxidising intermediate, formerly known as "compound I," is an organic cation
radical that represents Fe(IV). In HRP, the radical is placed on the porphyrin ring, but in

bcytochrome c peroxidase, it is positioned on tryptophan-191, a peptide residue that is almost
loated.Fe-O bonding can take many forms, ranging from Fe(IV)=O (also known as' ferryl') to
Fe(IV)-O----H, in which the O atom is either protonated or attached to a donor group via a
hydrogen bond. Two one-electron transfems are used to reduce compound (I) to the resting
Fe(III) state, which can be accomplished with organic substrates or cytochrome c.In a variety
of biological activities that involve oxygen, the Fe(IV) intermediates catalyse the elimination
of harmful H2O2 [H2O2 (aq) + 2e-+ 2H+ (aq) →2H2O(l)] (Fig.2.8)
 Using H218O2, it was discovered that the dioxygen generated in the catalase reaction is
obtained from hydrogen peroxide. As a result, hydrogen peroxide disproportionation
could take place in two stages. The reaction's substrate, H2O2, is reduced to water in the
first step, and the resultant compound first represents the enzyme's oxidation (Fig.2.9).
Fig. 2.9
 The oxidation of H2O2 by compound I (the oxidation state enzyme) results in the
formation of dioxygen and water. Fig. Formate, nitrate, and ethanol, as well as hydrogen
peroxide, can all be oxidised by compound I. As a result, the iron centre in compound I is
highly oxidised, and the job of the tyrossinate axial ligand phenolate O -) in catalase is to
stabilise such a centre. Similar stabilisation may be given by the histidyl imidazole ligand
via deprotonation in the case of peroxidase.
2.6 CYTOCHROME P-450

Cytochrome P-450 is a family of cytochromes that can be found in plants, animals, and
microorganisms. It's called a pigment because its CO compounds absorb light at 450 nm. This
is owing to the π-π* (blue to red) transition, and this bond is known as the SORET bond.
Cytochrome P-450 aids in O2 cleavage and acts as a monooxygenase, facilitating the insertion
of oxygen atoms into the substrate. Oxygenases are enzymes that add oxygen to a food

source. A monooxygenase introduces one oxygen atom into the substrate, while a
dioxygenase inserts two oxygen atoms into the substrate. The C-H bond is transformed to C-
OH groups in the most significant compounds. Here are a few examples:
 Conversion of an hydrocrbon RH to ROH.
 Conversion of an aldehydeto the carboxylic acid.
 Conversion of alkene into epoxide.
 Conversion of Benzene into Phenol
 Oxidation of amine into amine oxides.
 Oxidation of sulphide into sulphoxides.
Fig.2.10 Different type catalytic reactions catalysed by cytochrome P-450

In the kidney, cytochrome P-450 enzymes convert insoluble hydrocarbons into water-soluble
R-OH molecules, which are then excreted in the urine.An organic substrate receives one
oxygen atom, which is then reduced to H2O.The active site of cytochrome P-450 is heme,
which is comparable to haemoglobin and myoglobin except for the following differences:
(i) Fe is present in Fe(III) state and it is low spin octahedral.
(ii) Sixth coordination site is occupied by H2O.
(iii) One S-atom of cysteine is coordinated to Fe(III) instead of hisdine in the Proximal
position.
The cytochrome P-450 enzyme have low spin octahedral Fe(III) active site.
Cytochrome P-450 belongs to a large group of heme-containing monooxygenases that

oxygenate a wide range of substrates. Such enzymes catalyse the dioxygen oxidation of
organic substrates and play a vital role in biosynthesis, metsbolism, and, most importantly,
the deetoxification of hazardous chemicals. Water insoluble aliphatic or aromatic
hydrocarbons, for example, are transformed into water soluble alcohols and expelled through
the urine.
Fig. 2.11 Epoxidation of benz[a] pyrene
More arenes (including benzene) are epoxidized in humans, and this process is catalysed by a
family of enzymes in the liver (cytochrome P-450). One of the compounds under
investigation is benz[a] pyrene, a result of the reaction of a variety of organic materials,
including tobacco, to produce an epoxide with carcinogenic characteristics.
2.6.1 Structure of cytochrome P-450
Cytochrome P-450 is a physiologically active protein that participates in a variety of

oxidation processes. Cytochrome P-450 (heme B type) is a protein with the prosthetic group
Fe(III) protoporphyrin IX.Cytochrome P-450, like myoglobin, has an oxygen-binding heme

unit, but instead of the axial histidine seen in myoglobin, a cystein thiolate residue is present.
A single polypeptide (-halical) chain exists in cytochrome P-450.
A heme group b molecule is sandwiched between two helices of two axial ligands, one of
which is a cystein ligand from a protein and the other of which is a water molecule. The
CYPs are hemoproteins with a single haem prosthetic group in the active site and 400-500
amino acid residues. Low spin (LS), in which the five 3d electrons are maximally paired, and
high spin (HS), in which the five 3d electrons are maximally unpaired, are the two spin states
of iron in the ferric form (Fe3+).According to spectral, NMR, and crystallographic evidence, a
water molecule creates a sixth axial ligand of the Fe3+ in the substrate-free form, maintaining
the LS state of the ion. When substrates bind to the enzyme, the iron-water molecule is
displaced, changing the Fe3+ coordination state from six to five, where the Fe3+ travels out of
the plane of the haem ring.
Fig.2.12 Structure of cytochrome P-450
Most of the information on P-450 is based on studies undertaken on an enzyme P-450

obtained from the bacterium Pseudomonas putida. This source (organism) makes use of
camphor as its only source of carbon, the stage being. The oxygenation at the C5 position.

2.6.2 The mechanism of oxidation of a substrate with cytochrome P-450
The molar mass of the cytochrome P-450 enzyme is around 50,000. Figure 1 depicts the
catalytic cycle for the action of the cytochrome P-450 enzyme.
The organic substrate enters a hydrophobic pocket of the protein at the Fe(III) center,
expelling water molecules from the iron axial coordination site to form Fe(III) complexes,
which are then reduced by another enzyme system to form high-spin Fe(II) complexes in the
second step.
In step third, a dioxygen molecule forms a bond with a Fe(II) center, similar to haemoglobin
and myoglobin, and one electron is transferred from Fe(II) to dioxygen, resulting in the
formation of a Fe(III)-superoxo complex.In step four, another electron is added to form the
Fe(III)-peroxo complex.In step five, the protonation of the Fe(III)-peroxo complex results in
the removal of one oxide ion as water, resulting in an oxyferryl complex.
Fig. 2.13 Cyclic mechanism of cytochrome P-450

One electron has oxidised from the-HOMO of the porphyrin ring, leaving it as a radical
cation. Fe(V) = O, or oxygen that has been double linked to Fe(IV).Step six involves
oxidising the organic substrate (R-H) to R-OH while simultaneously binding an H2O ligand
to the active site of the metalloenzyme, which now has a low spin Fe(III) center.
2.7 COPPER ENZYME

In yeast, copper is essential for iron intake, energy synthesis, and the protection against
oxidative stress. The bulk of phenotypes found during copper deprivation are explained by
three key copper enzymes in yeast: a multicopper oxidase, a copper heme oxidase, and a
superoxide dismutase.
2.7.1 Superoxide dismutase (SOD) a copper enzyme

Superoxide dismutase is a catalytic enzyme that catalyses the elimination of the harmful
superoxide anion, O2-, which is produced as a byproduct of oxidative metabolism. Superoxide
is converted to molecular oxygen and hydrogen peroxide by this enzyme. The subsequent
work of enzymes like catalase removes hydrogen peroxide, which is a potentially hazardous
chemical. As a result of their collaboration, SOD and catalase help to safeguard organisms
that use dioxygen from potentially hazardous byproducts of O 2 metabolism.
 Superoxide dismutase (SOD) is present in all aerotolerant organisms for the purpose of
minimizing the concentration of superoxide, O2, and thus providing protection against
oxygen toxicity.
 The SOD like, Ni SOD, either Fe or Mn SOD, which seem to be the same protein, and
Cu/Zn SOD.
 Fe or Mn SOD is found in the prokaryotes or the mitochondria of eukaryotic cells, while

Cu/Zn SOD is found in the cytoplasm of eukaryotic cells.
There are three type of superoxide dismutase :
(1) Copper- Zinc superoxide dismutase, CuZnSOD

(2) Manganese superoxide dismutase, MnSOD
(3) Iron superoxide dismutase, FeSOD
2.7.2 Structure of Cu-Zn superoxide dismutase
CuSOD (bovine superoxide dismutase) is present in the mitochondria of eukaryotic cells,

while the other two are found in bacteria (prokaryotes). Cu 2+ and Zn2+ ions are coupled to the
imidazole of a histidine residue, according to a CuZnSOD crystal structure determination
(fig. 2). The Cu2+ ion is a deformed square pyrimidal site bound to four imidazole histidine
nitrogen atoms and a water molecule, whereas the Zn2+ ion is tetrahedrally coordinated to
three imidazole histidine nitrogen atoms and oxygen from the asparatate residue.
Fig.2.14 structure of Cu-Zn superoxide dismutase
The molar mass of copper-zinc dismutase is around 16,000. The Cu2+ ion has been
demonstrated to be the functional one, whereas the Zn2+ ion serves as a structural stabiliser by
holding the bridging imidazole histidine residue in place.
Cu2+ is a more important ion that cannot be substituted by another metal while maintaining
activity. The Zn2+ ion, on the other hand, can be replaced with other divalent metals such as
Co or Cd while maintaining the majority of the activity.
2.7.3 Mechanism Cu-Zn superoxide dismutase
The following point may be noted:
 When Cu(II) and Zn(II) are removed from an enzyme, its activity is diminished. Only the
addition of Cu(II) reactivates the enzyme. As a result, the role of Zn(II) is only relevant
from a structural standpoint. Furthermore, Zn(II) occupies all four coordination sites in

Cu-ZnSOD. The displacement of His 46 by superoxide is thought to be the mechanism
by which it binds to Cu(II).
Fig.2.15 Mechanism Cu-Zn superoxide dismutase
 It has been proposed that the imidazole bridge breaks and reforms during each catalytic
cycle of the reaction.
 The pKa of the other nitrogen atom is lowered when a histidine ring nitrogen atom is
coordinated to Zn(II). As a result, it prefers to attach a proton rather than Cu (I).
 When a proton is transferred to a peroxide ion, which is then transformed to H2O2, the
bridge is reformed. This idea also creates a location at the Cu(I) centre for substrate (O 2-)
binding, eliminating the necessity for a potentially high-energy five-coordinate transition
state.
 The reaction is therodynamiccally favourable. The redox potential for the O 2/O2- coupleis
-0.33V and for the O2-/H2O2 couple +0.89V. Any metal atom capable of one-electron
redox chemistry between Mn2+ and M(n+1)+ states that has a potential between the limits -
0.3 ≤ Eº ≤ + 0.9 will be thermodynamically feasible to act as a superoxide dismutase. In

keeping with this, other single atom SOD’s e.g. both Fe and Mn SOD also exist in
nature.
2.8 MOLYBDENUMOXOTRANSFERASES-XANTHINE OXIDASE

It has been proposed that the imidazole bridge breaks and reforms during each catalytic cycle
of the reaction.
The pKa of the other nitrogen atom is lowered when a histidine ring nitrogen atom is
coordinated to Zn(II). As a result, it prefers to attach a proton rather than Cu (I).
When a proton is transferred to a peroxide ion, which is then transformed to H 2O2, the bridge
is reformed. This idea also creates a location at the Cu(I) centre for substrate (O 2-) binding,
eliminating the necessity for a potentially high-energy five-coordinate transition state.
Fig.2.16 Oxidation of xanthine to uric acid
Gout is caused by an excess of uric acid in the body, which can be treated with xanthine
oxidase inhibitors. The Mo(VI) site in xanthine oxidase performs the two-electron oxidation
of xanthine to uric acid and then self-reduces to Mo. (IV). The Mo(VI) site is regenerated by
transferring electrons to the Fe2S2 and FAD sites one at a time, making Mo(VI) ready for the
oxidation of the next equivelnt of xanthine. The electron flow can be depicted as follows:
Xanthine → Mo(VI) → 2Fe2S2 → FAD → O2
Fe2S2 sites in xanthine oxidase play the same electron-transfer role as the Fe2S2 ferroxine
play in photosynthesis.
2.8.1 Structural features and mechanism
Molybdenum is the only second-row transition metal that has biological use.
It has a unique value and experiences two electron transfer reactions, mostly between the
Mo(VI) and Mo(IV) oxidation states, as previously stated. It can also transfer an oxo atom to
a substrate, as seen in fig.2.17

MoVI (O2) + S → S ═O + MoIVO
Fig.2.17 Structure of xanthine oxidase
The metal is usually coordinated by a ligand called molybdopterin in all Mo enzymes (fig.
2.18). A pair of S atoms from a ditholene group that is covalently bonded to a pterin serve as
metal donors. A nucleoside base R, such as guanosine 5'-diphosphate, is linked to the
phosphate group. The pterin group could have a function in assisting redox reactions by
acting as an electron channel.
The mechanism of the reaction may be studied considering the following,
 One terminal molybdenum oxo (Mo=O) group, two thiolate-type sulphur ligands (pterin
dithiolene side chain), and one terminal sulphido group (Mo=S) group characterise the
oxidised form of the enzyme.
 The terminally attached sulphido group serves as a base to remove hydrogen from the
reactant xanthine, while an oxygen atom is transferred from the Mo(VI)=O unit to
xanthine.
 The Mo enzyme transfers an O atom directly to the substrate (xanthine). The O atom
transported to the substrate is thus not via the Mo-promoted assault of solvent water or
hydroxide on the substrate.
 The oxo group from the water molecule is repaired at the Mo centre via linked
deprotonation and electron processes.

Fig.2.18 Mechanism of xanthine oxidase

 The Mo enzyme transfers an O atom directly to the substrate (xanthine). The O atom
transported to the substrate is thus not via the Mo-promoted assault of solvent water or
hydroxide on the substrate.
 The oxo group from the water molecule is repaired at the Mo centre via linked
deprotonation and electron processes.
 Significantly, this oxygenation reaction varies from that of other Fe and Cu enzymes in
that the oxo group transported by Mo enzymes is generated from water rather than
molecular oxygen.
The Mo(VI) site [Mo cofactor (Molybdepterin)] in xanthine oxidase catalyses the two-
electron oxidation of xanthine to uric acid, which is then reduced to Mo. (IV). Loss of
electrons (one at a time) to the Fe2S2 and FAD sites regenerates the Mo(VI) species.
Although the Fe2S2 sites of the enzyme are not directly involved in substrate reaction, they
are critical to the enzyme's overall function. The Fe2S2 core of the enzyme has a role similar
to Fe2S2 ferrodoxine's simple electron-transfer job in photosynthesis.
2.9 VITAMIN B12

The only organometallic compounds found in nature are vitamin B12 and coenzyme B12.
Vitamin B12 was originally extracted from liver extracts, and it was discovered that human
permicious anaemia is caused by a vitamin B12 or B12 coenzyme shortage. The Co(III) ion is
linked to four N-atoms of a corrin ring in vitamin B12. The corrin ring is a modified porphyrin
ring with one fewer =CH- bridge connecting the two pyrrole rings than the porphyrin ring. As
a result, the corrin ring is less symmetric and saturated than the porphyrin ring. An imidazole
nitrogen and a cyanide ion occupy the fifth and sixth positions, respectively.
The cyanide ion, on the other hand, is absent in vivo, and the sixth place is occupied by a
loosely attached water molecule.Cobalt can be reduced by one electron to give vitamin B12
[Co (II) complex] or by two electrons to give vitamin B 12 [Co (I) complex] after
incorporation of Co (III) into the corrin ring, altering the reduction potential of cobalt.
Reduced ferredoxin can carry out these reductions in vivo. Because it is very nucleophilic, it
is easily alkylated.
Some reaction of vitamin B12 are given below (Fig.2.19)

Fig.2.19 Catalytic reaction of vitamin B12
The R sight is unoccupied in vitamin B12, and the 5-coordinate cobalt (I) atom is extremely
reactive. Cobalt is present in a +3 oxidation state in vitamin B12 and its various derivatives.
Because the spin octahedral field of Co (III) in these compounds is low (d 6→t26eg0), they are
diamagnetic and EPR inactive.
The red and brown colours of vitamin B12 and vitamin B12 are caused byπ-π* transitions in
corrin rings. Due to the presence of an unpaired electron in the dz 2 orbital, the latter is EPR
active. Vitamin B12r has a low Co(II) spin.Co(I) in vitamin B12 is also EPR active due to the
presence of two unpaired electronns, and its blue-green colour is due toπ-π* transitions.
Unfortunately, certain bacteria may methylate not only sulphur in organic molecules, but also
heavy metals including Hg, Sn, Pd, Pt, and Pd in aqueous solution, resulting in very
hazardous species like Hg (CH3) and Pd (CH3)4.
Adenosyl and cobalt form a direct cobalt-carbon bond when adenosine triphosphate (ATP)
reacts with vitamin B12s. B12 coenzyme is the chemical that results. It was the first time an
organometallic compound was found in live organisms. Co(III) is a coordinated carbon atom
of an adenosyl ligand that replaces the CN- ligand in coenzyme B12. This coenzyme catalyses
1,2 general type rearrangements.

Vitamin B12 with CN- removed is called cobalamin, thereforee, vitamin B12 is called
cyanocobalamin.
Coenzyme B12 takes a methyl or hydroxy methyl group (bound to Co) that can be transferred
to a substrate to add a carbon. As ribonucleic acid (RNA) is converted to deoxyribonucleic
acid, coenzyme B12 converts the -CH(OH) group to the -CH2 group (DNA).
The breaking of the Co-C bond is the precise mechanism of these reactions. It's worth noting
that given the right conditions, cobalt porphyrin analogues of vitamin B 12 can be converted to
the Co(I) state. As a result of the porphyrin ligand's failure to stabilise the Co(I), the corrin
ring has been chosen in place of the porphyrin ring in the evolution of B 12 cobalt complexes.
2.9.1 Structure and characteristic features of vitamin B12
 It is the only naturally occurring organometallic complex (a species with a direct metal to
carbon connection) in biology, and it is the only vitamin that contains a metal ion.
 The molecule is an octahedral cobalt (III) complex with corrin, a 15-membered 4-

nitrogen ring ligand.
 All of the side chains attached to corrin are acetamide and propionamide groups, with
one of these being an isopropanol phosphate residue attached to a ribose. Finally, the
molecule is terminated by a 5,6-dimethyl-benzimidazole, which coordinates.
 The majority of B12 coenzyme reactions are catalysed or initiated by homolytic cleavage
of the CO-C bond, which is catalysed or launched by homolytic cleavage of the CO-C
bond.

Fig. 2.20 Structure of vitamin B12
2.9.2 Application of vitamin B12
Vitamin B12 is a water-soluble vitamin found in meat, fish, and dairy products. It can also be
manufactured in a laboratory and is frequently combined with other B 12 vitamins.Many
components of the body, including the brain, nerves, and blood cells, require vitamin B12 for
proper function and development. The active form of vitamin B12 is methylcobalamin. The
most common type used in supplements is cyanocobalamin, which must be converted by the
body into the active form.
Vitamin B12 is widely used to treat vitamin B12 deficiency, cyanide poisoning, and excessive
blood homocysteine levels. It's also used to treat canker sores, cataracts, Alzheimer's disease,
osteoporosis, weariness, and a variety of other ailments, although most of these claims lack
scientific backing. The following are some processes performed by enzymes that require
coenzyme B12:
A secondary methyl group is interted into a main chain e.g., shown in (Fig.2.21 ) or an amino

Fig. 2.21
Group can isomerize from a primary to a secondary carbon. Dioldehydrase B 12 coenzyme

produces propiopaldehyde from 1, 2-propanediol. The initial product of vicinal 1, 2-
intercharge is a hydrate which loses water to give the product.
2.9.3 Vitamin B12 Deficiency
With age, it can become harder to absorb this vitamin. It can also happen if you have had
weight loss surgery or another operation that removed part of your stomach, or if you drink
heavily.You may also be more likely to develop vitamin B12 deficiency if you have:
 Atrophic gastritis, in which your stomach lining has thinned
 Pernicious anemia, which makes it hard for your body to absorb vitamin B12
 Conditions that affect your small intestine, such as Crohn's disease, celiac disease,
bacterial growth, or a parasite
 Alcohol misuse or heavy drinking can make it harder for your body to absorb nutrients or
prevent you from eating enough calories. One sign that you lack enough B12 may be
glossitis, or a swollen, inflamed tongue.

 Immune system disorders, such as Graves' disease or lupus
 Been taking certain medications that interfere with the absorption of B12. This includes
some heartburn medicines including proton pump inhibitors (PPIs)
suchas esomeprazole (Nexium),
lansoprazole (Prevacid), omeprazole (PrilosecOTC), pantoprazole (Protonix), and
rabeprazole (Aciphex), H2 Blockers such as cimetidine (Tagamet) and famotidine
(Pepcid AC); and certain diabetes medicines such as metformin (Glucophage).
2.9.4 Food Sources of Vitamin B12
You can get vitamin B12 in animal foods, which have it naturally, or from items that have
been fortified with it.Animal sources include dairy products, eggs, fish, meat, and poultry. If
you're looking for a food fortified with B12, check the product's Nutrition Facts label.
2.10 SUMMARY
The different essential aspects of enzymes are covered in the above unit. The following is a
summary of the unit:
 Carboxypeptidase A is a zinc enzyme with a tetrahedral shape and a sp3 hybridization.

Carbonic anhydrase is an enzyme that catalyses the conversion of carbon dioxide (CO 2)
to carbonic acid.
 Cytochrome P-450 aids in O2 cleavage and acts as a monooxygenase, facilitating the

insertion of oxygen atoms into the substrate.
 In yeast, copper is essential for iron intake, energy synthesis, and protection against
oxidative stress. The bulk of the phenotypes found during copper deprivation are
explained by three key copper enzymes in yeast.
 Superoxide dismutase is a catalytic enzyme that catalyses the elimination of the

superoxide anion, O2, which is produced as a byproduct of oxidative metabolism. SOD
and catalase help to safeguard organisms that use dioxygen from potentially hazardous
byproducts of O2 metabolism.
 Xanthine oxidase convert xanthinin into uric acid. Gout is caused by an excess of uric
acid in the body, which can be treated with xanthine oxidase inhibitors.

 The only organometallic compounds found in nature are vitamin B12 and coenzyme B12.
Vitamin B12 was originally extracted from liver extracts, and it was discovered that
human permicious anaemia is caused by a lack of B12 or B12 Coenzyme shortage. The
Co(III) ion is linked to four N-atoms of a corrin ring - a modified porphyrin ring with one
fewer =CH- bridge connecting the two pyrrole rings than the porphryin ring. Because it
is nucleophilic, it is easily alkylated and can be reduced in vitro using reduced
ferredoxin.
2.11 SAQs TYPE QUESTION

A. Multiple Choice Question
1. Which of the following mettaloenzyme converts oxygen to water
a. Hemoglobin b. Catalase
c. Cytochrome c-oxidase d. haloperoxidase
2. Zn in carbonic anhydrase is coordinated by three histidine and one water molecule. The
reaction of CO2 with the enzyme is an examples of,
a. Nucleophilc addition b. Electrophilic addition
c. Electron transfer d. Electrophilc substitution
3. Enzyme Carbonic anhydrase metal is present :
a. Fe b. Zn
c. Cu d. Co
4. The ligand present in Vitamin B12 is :
a. Porphyrin ring b. Corrin
c. Crown ether d. Phthalocyanin
5. Carboxypeptase contains :
a. Mg(II) and Hydrolysis CO2 b. Zn (II) and hydrolysis peptide bond
c. Mg(II) hydrolysis peptide bond d. Zn(II) and Hydrolysis CO 2
6. The metal ion present in the active site of carboxypepdase enzyme is :

a. Iron b. Copper
c. Molybdenium d. Zinc
7. A well known naturally occuring organometallic compound is :
a. Hemoglobin b. Cytochrome P-450
c. Vitamin B12 d. Superoxide dismutase
8. Xanthine oxidase convert xanthinin into :
a. Citric acid b. Uric acid
b. Acetic acid d. Cinnamic acid
9. Normal blood pH is
a. 7.3 b. 7.2
c. 7.4d. 8.4
10. Loss of electrons can be termed as ______________

a. Metabolism b. Anabolism
c. Oxidation d. Reduction
11. Which of the antineoplastic agent is metabolized by xanthine oxidase?
a. 6-mercaptopurine b. Vincristine
c. Chlorambucil d. 6-Thioguanine
12. Deffiency of Vitamine B12 is,
a. Asthma b. Noval covide-19
c. Penecius anemia None of these
13. Excess of uric acid
a. Gout formation b. Noval covide-19
c. Penecius anemia a. Asthma
14. Which of the following statements is not true about cytochrome P450 enzymes?
a. They ontain heme and Mg2+ b They called monooxygenases.
c.There are over 30 different Cyt P-450 d. They contain Fe3+ ion in active site

15. Which of the following groups is least susceptible to cytochrome P450 enzymes?
a. Terminal methyl groups b. Allylic carbons
c. Benzylic carbon atoms d. Quaternary carbon atoms
16. The enzyme carbonic anhydrase is of which type ?
a. Reversible b. Lyases
c. Unidirectional d. Isomerase
17. Carbonic anhydrase enzyme present in,
a. WBC b. RBC
c. Blood plasma d. Platelets
18. In production of which carbonic acid, Aspergillus niger is useful ?
a. Citric acid b. Acetic acid
c. Plamitic acid d. Butyric acid
B. Fill in the bling
(i) Cytochrome P-450 aids in O2 cleavage and acts as a…………………. , facilitating
the insertion of oxygen atoms into the substrate.
(ii) Vitamin B12 with CN- removed is called……………… , thereforee, vitamin B12 is
Called cyanocobalamin.
(iii) Superoxide dismutase is a catalytic enzyme that catalyses the elimination of the
harmful superoxide anion, O2-, which is produced as a byproduct of……………
(iv) Cataleses have the largest………………… of any enzyme, with one catalyse molecule
converting millions of molecules of……………… to water and oxygen every second.
(v) Carbonic anhydrase affects CO2 transport in the blood and so plays a significant role in
…………………………..
C. True/False
(i) The red and brown colours of vitamin B12 and vitamin B12 are caused by π-π* transitions
in corrin rings. True/False

(ii) Cytochrome P-450 called a pigment-450 because its CO compounds absorb light at 450
nm True/False
(iii) Carboxypeptidase A (CPA) contains a zinc (Zn2+) metal center in a octahedral geometry
with amino acid residues in close proximity around zinc to facilitate catalysis and
binding. True/False
(iv) The disproportionation of hydrogen peroxide and organic peroxides was catalysed by
catalases. True/False
(v) Coenzyme B12 takes a methyl or hydroxy methyl group (bound to Co) that can be
transferred to a substrate to add a carbon. True/False
(vi) Carbonic anhydrase is an enzyme that catalyses the conversion of carban monoxide (CO)
to uric acid. True/False
i. Xanthine oxidase a. Monoxygenase
ii.Carboxypepdase b. Uric acid
iii.Cytochrome P-450 c. 1-2 methyl shift
iv. Vitamin B12 d. Peptide bond hydrolysis
v. Catalase e. Disproportionation
Answer Key
A. 1 c 2b 3b 4b 5b 6d 7c 8b 9c 10 c 11 a 12 c 13 a
14 a 15 d 16a 17 b 18 a
B. i Monooxygenase, ii Cobalamin, iii oxidative metabolism, iv Turnover, H2O2,
v. respiration.
C. i True ii True iii False iv True v True vi False
D. i b, ii d, iii a, iv c, v e,
2.12 GLOSSARY
Glu = Glutamic acid

FAD = Flavin Adenine Dinucleotide
SOD = superoxide dismutase
CYP = Cytochrome
HS = High spin
LP = Low spin
Arg = Argene
CPA = Carboxypeptidase A
His = histidine
2.13 REFERENCES
1. Adman, E. T., 1991. Copper protein structures. Advances in Protein Chemistry, 42, 145–
197.
2. Anbar, A. D., 2008. Oceans. Elements and evolution. Science, 322, 1481–1483.
3. Barton, L. L., Goulhen, F., Bruschi, M., Woodards, N. A., Plunkett, R. M., and
Rietmeijer, F. J. M., 2007. The bacterial metallome: composition and stability with
specific reference to the anaerobic bacterium Desulfovibrio desulfuricans. BioMetals, 20,
291–302.
4. Bertini, I., Ciurli, S., and Luchinat, C., 1995. The electronic structure of FeS centers in
proteins and models. A contribution to the understanding of their electron transfer
properties. Structure and Bonding, 83, 1–53.
5. Sauer, Daniel F. (2019). Advances in Bioorganometallic Chemistry Artificially Created

Metalloenzyme Consisting of an Organometallic Complex Immobilized to a Protein
Matrix, 307–328.
6. Christianson, D., W., and Lipscomb, W., N. (1989) Carboxypeptidase A. American

Chemical Society, Vol (22): 62-69.
7. Hill HAO, Roder A, Williams R. J. P., (1970), The chemical nature and reactivity of
cytochrome P-450. Struct Bond, 8, 123-51.

8. Ortiz de Montellano P. R, De Voss J. J. (2005), Substrate oxidation by cyto-chrome P450
enzymes. In: Cytochrome P450: Structure, Mechanism, and Biochemistry, Ortiz de
Montellano PR, (Ed). Kluwer Academic/Plenum Publishers, New York, ; 3rd ed, pp
183–245.
9. Shannon R. D., Prewitt C.T., (1970), Revised values of effective ionic radii. Acta Cryst;
B26: 1046-8.
10. Poulos T .L., Finzel B. C., Howard A. J., (1986), Crystal structure of substrate-free
Pseudomonas putida cytochrome P-450, Biochemistry-US, 25(18), 5314-22.
11. Joseph J. Stephanos, Anthony W. Addison, (2014), Chemistry Of Metalloproteins, John

Wiley & Sons, Inc., Hoboken, New Jersey, 1-451.
12. Stephen J. Lippard, Jeremy M. Berg, (1994), Principles of Bioinorganic Chemistry,

University Science Books, , ISBN 0-935702-72-5, pg 318 [1]
13. Kumar A., (2014), OrganoMettalic and Bioinorganic Chemistry, Aaryushi Education
Ghaziabad, 1-176.

1. Bertini I., Gary H.B., Lippard S. J., Valentine J. S. (1988), Bioinorganic Chemistry, First
south asian edition,
2. William H. E., Daphne C. E., Bioinorganic and molecular biology, fourth edition.
3. OchiaE. I., (1977) Bioinorganic Chemistry–An Introduction, Chapter 11, Allyn and
Bacon,Boston .
4. LipscombW. N., SträterN., (1996), “Recent advance in zinc enzymology”, Chem. Rev.,
96,2375–2433.
5. Christianson D. W., CoxJ. D., (1999), Biochemistry, 68, 33–57 .
6. LiljasA., KannenK. K., BergstenP. WaaraC. I., FridborgK.,Strandberg B., CarlbornU.,
JarupL., LovgrenS., PetefM., (1972), Nature New Biology, Lond., 235, 131 .
7. HayR. W., (1980) Inorg. Chim. Acta, 46, 115.
8. LindskogS., HendersonL. E.,Kannen K. K., LiljasA., NymanP. O., StrandbergB.,

(1971), The Enzymes, P. D. Boyer, Ed., 3rd ed., p. 58.
2.15 TERMINAL QUESTIONS

Q1. What is the active metal in carborxypepdase A. What is the coordination number and
how is it satisfied?
Q2. Write a reaction that is catalyzed by vitamin B12 and propose a mechanism for such
reaction. What are the advantages of using vitamin B12?
Q3. Peroxidase, catalase and cytochrome-P450 all produce Fe(IV)=O as an important

intermediate during the reaction with the substrates. This being the case, why are they so
different in their biological activities?
Q4. Discuss about the diffence and similarities between cytochrome P-450 and other electron
or oxygen-transfer functions of other heme proteins.
Q5. Explain that cytochrome P-450 is a monooxygenase. Discuss about the special features
of cytochrome P-450 and its mechanism.
Q6. Explain with example the importance of metalloenzymes.
Q7. What are xanthine oxidase ? Discuss their role in formation of uric acid.
Q8. What is the Superoxide dismutase ? Describe the role of superoxide dismutase in
oxidative metabilism.
Q9. What is Carboxypeptidase A ? Discuss about the structure and mechanism of action of
carboxypeptidase A.
Q 10. What is carbonic anhydrase ? How it is helpful for the conversion of CO 2 into carbonic
acid.
Q11. Discuss the structural features and mechanism of peroxidase and catalase.
Q12. Carboxypeptidase A with a bound peptide chain is a good example of a supramolecule

explain.

UNIT 3: METAL-NUCLEIC ACID INTERACTIONS

CONTENTS:
3.1 Introduction
3.2 Objectives
3.3 DNA (Deoxyribose Nucleic Acid)
3.3.1 Primary structure of DNA
3.3.2 Secondary structure of DNA
3.3.3 DNA polymerization
3.3.4 Model of DNA polymerisation
3.3.5 Catalytic mechanism of DNA polymerase
3.3.6 Detailed description of DNA polymerization process
3.4 Classification of elements according to their action in the biological system
3.5 Na+-K+-ATpase
3.6 Biological metal-coordination sites
3.7 Magnetic resonance imaging (MRI)
3.8 Toxicity of metals: Hg,Cd, Pb, As and the chelate therapy
3.9 Summary
3.10 SAQs types questions
3.11 Terminal questions
3.12 Bibliography
3.1 INTRODUCTION
In Unit III we learn about DNA, RNA and role of metal ion in our living organism. In this we
can understand use of metal ions in medical field. The toxicrole of metal ion in our
environment. Metal ions are essential in many biological processes as catalyse

(enzyme).Components of our bodies Consider the elements that make up an average healthy
person's constitution (weighing 70 kg). Oxygen accounts for more than half of an average
man's overall weight. Table 1 lists the number of different elements in human bodies.
Table 1: Average concentration of various elements present in our body Elements Amount in
our body
Element Amount in our body
Oxygen 45.5 kg
Carbon 12.6kg
Hydrogen 7.0 kg
Nitrogen 2.10 kg
Phosphorous 700 g
Calcium 1050 g
Potassium 140 g
Sodium 105 g
Magnesium 35 g
Iron 4.2 g
Zinc 2.3 g
The rest of the metals, particularly Cu –0.11 g and Mn – 0.02 g, have a content of less than one
gramme. Metals make up only 2% of the human body, but they play a critical role in human
life. They bind to the substrate and orient it in relation to the active site's functional group,
resulting in the creation of a redox reaction site. Enzymes contain some metal ions. Some are
structural components, such as calcium in bones and teeth, while others are involved in
transport, such as Fe2+ in haemoglobin and myoglobin, and still others are involved in control
systems, such as Na+and K+ in nerve transmission.
3.2 OBJECTIVE
After studying this Unit, you shall be able to know:

 What is the Constitutional structure of DNA molecule? Which type of bases and sugar
are present in DNA and RNA biomolecule?
 Type interaction modes of binding and cleavageof DNA.
 We know about role of Metal ionin living organism and their ill effects.
 The metal complex used for diagnosis and chemotherapy.
3.3 DNA (DEOXYRIBOSE NUCLEIC ACID)

Deoxyribonucleic acid (DNA) is a very extensive macromolecule that holds genetic
instructions for all known living organisms' development and function. It contains information
in the form of a unique genetic code that can be passed through generations. Each DNA
molecule is tightly twisted and bundled into chromosomes, which are thread-like structures that
wrap around certain protein complexes (Fig. 3.1). Chromosomes are placed within the
membrane-bound nucleus of eukaryotes (cells with a nucleus), while they are housed within the
cellular cytoplasm of prokaryotes (cells without a nucleus). DNA is a polymeric biomolecule
made up of nucleotides, which are linear chains of monomeric units. The nucleotide is made up
of three parts: a phosphate, a pentose and a nitrogenous base. In DNA, the sugar is always 2 ’-
deoxyribose.
Fig. 3.1 Scheme show cell, chromosome, gene and DNA

2’ deoxyribose sugar
There are two types of nucleotide bases: pyrimidine and purine. Purines have a five-membered
imidazole ring fused to a six-membered ring, while pyrimidines have a six-membered
heterocyclic conjugated ring. Cytosine (C) and thymine (T) are two pyrimidine nucleotides
found in DNA. N1 links the pyridine bases to pentose.
Adenine (A) and guanine (G) are the purine bases found in DNA (G).
The purine bases are attached to pentose sugar by N9.

Fig. 3.2 Structure of nucleotide
3.3.1 Primary Structure of DNA
The fundamental structure of DNA is a single polynucleotide chain. The phosphate group on
one nucleotide's 5' carbon is joined to the hydroxyl (OH) group on another nucleotide's 3'
carbon to form a phosphodiester linkage, which results in a polynucleotide chain. The
phosphodiester linkage creates the sugar phosphate backbone of the polynucleotide chain. At
one end of the chain, a free 5' phosphate group exists, whereas at the other, a free 3' OH group
exists.
The fundamental structure of DNA is:
Nucleic acids do not contain equal quantities of each nucleotide, as demonstrated by Erwin
Chargaff and other biochemists in the 1940s. Chargaff extracted DNA from a variety of species
and hydrolyzed it into individual nucleotides. After that, paper chromatography was used to
separate the nucleotides. Chargaff claimed that for any given species, based on his experiments:
 A=T and G≡C
 Sum of purine bases (A+G) = Sum of pyrimidine bases (T+C)
 Ratio of A to T and G to C were close to one i.e. A/T=G/C ≈1
These facts arecalledChargaff’s rule.
3.3.2 Secondary structure of DNA

In 1947, William Astbury used the X-ray diffraction technique to study DNA fibres and
discovered a 0.34 nm repeating unit within DNA. Rosalind Franklin's work on purified DNA
samples (between 1950 and 1953) supported Astbury's findings and showed that DNA has a

helical structure. Using a combination of Franklin's data and Chargaff's guidelines. James
Watson and Francis Crick proposed the double helical model of DNA in 1953.The following
are the primary characteristics of DNA's double helix:
Fig. 3.3 Synthesis of polynucleotide chain of DNA
i) The DNA molecule is made up of two polynucleotide chains that coil around a central
axis to form a right-handed double helix with a diameter of 20Å.
ii) The two chains run opposite direction, that is one chain run in 5 ’ to 3’ direction, while
another run in 3’ to 5’ direction.
iii) The two anti-parallel polynucleotide chains are complimentary to each other in base
sequence, that is Guanine (G) in one strand cab base pair with cytosine (C) in other
strand, and similarly, Adenine (A) and Thymine are base4 paired. This is called
complementary base pairing.
iv) Hydrogen bonds between the nitrogenous bases of opposing strands and base stacking
interactions hold the two polynucleotide chains together.

v) The bases are flat structures which are stacked on one other, inside then helix (3.4 Å
apart). They are lying perpendicular to the helix axis.
vi) Each base pair is rotated 36o relative to next base pair around the helix axis.
vii) The hydrophilic sugar-phosphate backbone of the helix is on the outside and the base
pairs lying on the inside of helix.
viii) The base pairing of two stands creates alternating major and minor groove on
the surface of double helix. The major groove is wide, whereas the minor groove is
narrow and deep.
Fig. 3.4 The Wotson-Crick Model of B-DN
3.3.3 DNA Polymerization
The most crucial biological activity in all living species is DNA replication, which involves
copying a double-stranded DNA molecule into two identical duplicates.DNA replication is the
most important biological process that occurs in all living organisms by which a double
stranded DNA molecule is copied to produce two identical replicas. DNA Polymerase is the
enzyme responsible for DNA replication (Figure ). This enzyme catalyses the synthesis of
polynucleotide chains by adding nucleotides produced from deoxynucleoside triphosphates one
after the other. It normally acts in pairs to split a single DNA molecule into two identical DNA
strands. A DNA polymerase requires the following components for replication:

 ATP, GTP, TTP, and CTP are the triphosphate versions of four nucleotides.
 Single-stranded DNA is used as a template.
 A primer, an existing strand of nucleic acids with a free 3' end a primer.
 An existing strand of nucleic acids with a free 3' end
Fig 3.5 Human DNA polymerase (Pol), a key enzyme in the base excision repair (BER)
process, is depicted on the surface.
DNA is usually composed of two polynucleotide chains coiled around each other in the
form of doubler helix. DNA polymerization is the synthesis of polynucleotide chain by
addition of successive nucleotides. The enzyme that catalyzes the synthesis of
polynucleotide chain of DNA are known as DNA polymerases. DNA polymerases require
three components for DNA synthesis-template, primer and four nucleotides (dATP, dGDP,
dTTP, and dCTP). The template is a single stranded (ss) DNA that will direct addition
complementary to 3’ end template. The primer provides a free 3’ OH group that extended by
DNA polymerase by addition of nucleotides. A nucleotide is made up of three components-
a nitrogenous base, a pentose sugar and three phosphate groups.The DNA polymerase

catalyzes the synthesis of DNA by addition of nucleotides to the 3 ’OHgroup of primer. The
newly synthesized DNA have base complementary to the template DNA.
Fig. 3.6 DNA Polymerization by DNA polymerase
During DNA polymerization, the primer's 3' OH group assaults the incoming nucleotide's -P.
When the incoming nucleotide is joined to the primer's 3' -OH group, the pyrophosphate is
released. The hydrolysis of pyrophosphate into two inorganic phosphates by an enzyme known
as pyrophosphatase provides the driving power for this process.els of DNA polymerization
3.3.4 Model of DNA Polymerisation
After Watson and Crick's discovery of the double helix model of DNA, three models for DNA
replication were presented. (a) Conservative Model
(a) Conservative Model:
The parental molecule guides the production of one daughter molecule with both
parental DNA strands and another daughter molecule with DNA strands from all freshly
synthesised material in this scenario.
(b) Semiconservative Model:
In this model, the parental molecule's two DNA strands split, with each strand serving
as a template for the creation of a new DNA strand. The consequence of one round of
replication is two DNA double helices, each with one parental and one new strand.
(c) Dispersive Model:
The parental double helix molecule is first broken down into double-stranded DNA
segments, which are then randomly recombined in thismodel.
3.4.5 Catalytic mechanism of DNA polymerase

X-Ray the structure of various DNA polymerases suggest that they all have a similar DNA
polymerization catalytic mechanism. Two metal ions, generally Mg2+, are retained in position
and orientation at the active site of a DNA polymerase by interactions with two conserved

aspartate residues. The (P) group of this dNTP interacts with the 3' OH group of primary,
whereas the other metal ion (B) interacts with all three phosphate groups of this dNTP. The
metal ion (A) activates the primer's 3' OH group, allowing it to attack the incoming dNTP's -
(P) group nucleophilically. The metal ion (B) shields the negative charge that accumulates on
the Penta co-ordinate transition state, causing the pyrophosphate to be released.
Fig. 3.7 Human DNA polymerase (Pol), a key enzyme in the base excision repair (BER)
process, is depicted on the surface.
Fig. 3.8 Active sites of DNA polymerase

3.3.6 Detailed description of DNA polymerization process
The steps in the replication of DNA are as follows (Figure 7):
(1) Initiation: When an enzyme called helicase loosens the two strands of DNA molecule by
breaking hydrogen bonds between each nucleotide, DNA replication begins. The origin of
replication is the point at which a short section of DNA helix opens up, and the structure that
results is known as the Replication Fork. SSB proteins (Singlestranded DNA-binding
proteins) then bind to the unwound single strands of DNA, preventing them from breaking
and reannealing.
(2) Elongation: However, while the helicase splits the strands, RNA primase attaches to
each strand for a brief period of time and creates an RNA primer, which serves as a starting
point for DNA synthesis. DNA polymerase III begins creating a new complementary strand
by adding the reciprocal sequences of the DNA to a single unwinding polynucleotide strand
after the primer is in place. Because DNA polymerase can only add nucleotides in a 5'
(prime) to 3' (prime) direction, this process occurs in the opposite way.This means that bases
are added toward the origin of replication on the leading strand, but DNA is copied in the
opposite direction of fork movement on the lagging strand. As a result, the lagging strand is
produced in short, Okazaki-like pieces. The primer RNA fragments are then removed from
both strands by RNase enzyme, and DNA Polymerase fill in the gaps with the required
nucleotides. Through the activity of DNA Ligase, a single nick on the leading strand and
numerous nicks on the lagging strand are created, which are subsequently filled to produce
two continuous double strands of DNA.
(3) Termination: Termination is the final phase of DNA replication, which occurs when the
DNA polymerase enzyme reaches the end of the strands, where no further replication is feasible.
The RNA primer is removed from the last segment of the lagging strand, which is not duplicated.
Telomeres are regions of the genome that contain a repetitive non-coding sequence of
nucleotides. A portion of the telomere is lost at the conclusion of each replication cycle, resulting
in shorter strands after each cycle.Finally, enzymes such as nucleases "proofread" the new
double helix structures, removing any reduced nucleotides that occurred during DNA
replication.As a result, the removed bases leave a few gaps, which DNA Polymerase I eventually
repairs.

Fig. 3.9 DNA replication process
3.4 CLASSIFICATION OF ELEMENTS ACCORDING TO THEIR

ACTION IN THE BIOLOGICAL SYSTEM
Elements
Essential Trace Toxic

Nonessential
O,C, H, N, P, Na, K, I, Fe, Cu, Zn, Mn, Cd, As, Pb, Hg, Ni,
Al, Sr, Ba, Sn
Mg, Cl, Ca, S Co, Mo Cr
 Essential elements are absolutely essential or necessary for life processes.
 Trace elements are also necessary for life processes.
 Non-essential elements are not essential. If they are absent other elements may serve the
same function.

 Toxic elements disturb the natural functions of the biological system.
Essential and Trace elements
Only about seven elements are known to be needed for the efficient functioning of the human
body out of more than 100 identified elements. Essential elements include elements such as Na,
K, Mg, Ca, P, Fe, Mn, S, Zn, Cu, Co, Cr, Mo, Cl, F, I, and Se. They are said to as crucial since
the organism would not be able to thrive without them. Essential components are divided into
two categories based on their absolute quantities in the body:
 Macronutrients
 Micronutrients
The rest of the elements, which are only required in small amounts by the body, are referred to
as micronutrients. They are also known as trace elements because they are only needed in trace
levels in the body.
3.4.1 Metal ions in biological System
i. Sodium
Sodium is the principal electrolyte found in high concentrations in extracellular fluid (140
mmol/L) Na+ is the principal. It regulates the body's osmotic pressure. In the body, sodium is
mostly found in the form of chloride and bicarbonate, as NaCl and NaHCO 3, respectively.
Adults require between 1 and 3.5 grammes of salt each day. It enhances glucose and amino acid
absorption. In conjunction with chloride and bicarbonate, it maintains acid-base balance. It is
involved in the control of membrane potential.
The most frequent source of sodium in cooking is table salt (NaCl). Bread, cheese, carrots,
cauliflower, egg nuts, spinach, and other foods are among the other sources. A lack of sodium
causes headaches and abdominal muscle cramps. On the other hand High blood pressure is
caused by a high intake of table salt.
iii. Potassium
K+ is the principal cation of the intracellular fluid. It increases the activity of cardiac muscles.
Along with Na+ it maintains osmotic pressure of the body. It also maintains acid-base balance.
It increases the activity of the enzymes like pyruvate kinase. It also plays a prominent role in
blood coagulation and synthesis of ribosomes.

The good sources of potassium are chicken, beef liver, banana, orange juice, pine apple etc.
deficiency of potassium leads to depression and also affects the nervous system.
Table 2 The biological roles of metal ions
Metal Related compounds Action
Haemoglobin ( *H) Oxygen transport

Myoglobin( *H) Oxygen storage
Hemerythrin( *N) Oxygen storage
*
Oxygenases (( N) Insertion of oxygen atom into substrate
Hydrogenases Oxidation of H2
*
Cytochrome P-450 ( H) Insertion of oxygen substrate
Fe *
Catalase ( H) Catalyze oxidation of substrate by H2O2
*
Peroxidase ( H) Catalyze oxidation of substrate by H2O2
Cytochromes C (*H) Electron transport
Ferredoxins ( *N) Electron Transport
Ferritin ( *N) Iron Storage
Transferrin( *N) Iron Transport
Co Coenzyme B12 Methylation of organic compounds
Amine oxidase Oxidation of amine into aldehyde

Ceruloplasmin Transport of Fe from transferrin to ferritin, Cu
Cu
storage and transport
Hemocyanin Oxygen transport
Hydrolysis of peptide bonds

Carbopeptidase
Zn Catalyses the equilibrium
Carbonic anhydrase
CO2 + H2O=HCO3-+H+
Chlorophyll Photosynthesis
Mg
Phosphotransferase Phosphate hydrolysis
Mg, Mn Aminopeptide Catalyses the cleavage of amino acid
Fe, Mo Nitrogenase Nitrogen fixation
Oxidase Redox reaction involving O2 electron acceptor

Fe, Mo, Cu
Reductase Catalyses reduction reaction

Hydroxylases Oxidative degradation of organic compounds
Cu, Zn, Mo Superoxide dismutase Dismutation of O2 - into O2 and H2O2
Mg, Cu, Zn Phosphatases Removal of phosphate groups from substrate
Ni Urease Hydrolysis of urea into CO2 and NH3

*
H= Heme iron
*
N=Non-heme iron
iii. Magnesium
Magnesium is a macronutrient that the body need in substantial quantities. About 25 gms of Mg
found in the human body. 70 percent of the body's mg is found in the bones and teeth. Mg2+ is
a phosphatase enzyme activator and an important cation in intracellular fluid. Magnesium,
calcium, and phosphorous combine to produce a complex salt of bones. It's an important part of
chlorophyll. It also participates in the ATP hydrolysis process (universal source of energy).
Magnesium, like calcium, helps with muscular contraction, blood coagulation, lung function,
and blood pressure management. It is involved in a variety of life-sustaining processes.Role of
magnesium in enzyme action, energy production, Nerve conduction, muscle protein formation
nucleic acid stabilization and DNA synthesis.
Nuts, soybeans, and seafood are good sources of magnesium. Mg deficiency results in
neuromuscular dysfunction.
iv. Calcium
Calcium is the most abundant mineral in the human body. It is major constituent of teeth and
bones. About 90% of the body calcium is in the skeleton, when it is maintained as deposits of
calcium phosphate. Calcium and phosphorus are the principal minerals of bone and teeth,
where it exists as the double salt of calcium and phosphate, CaCO 3.nCa3(PO4)2 (n ranging from
2 to 3). These minerals lend hardness and strength to these tissues. A little calcium is scattered
in soft tissue like muscles and organs. Calcium helps in blood coagulation. It plays a prominent
role in muscle contraction. Ca acts as a cofactor of various enzymes like protein kinase, lipase,
adenylate, cyclase, etc. it also helps in nerve action. The chief sources of calcium are milk, egg,
nuts, beans, cabbage, cauliflower, etc. Deficiency of calcium leads to the disease rickets in
children (weakness of bones) and osteoporosis in adults. However, excess of calcium adversely
affects the body, giving rise to formation of stones.

THE calcium in plasma exist in three forms:
 Protein bound (35-50 % of plasma calcium)
 5-10% in complexes with organic acids and phosphate, and
 50-60% in ionised form.
v. Phosphorus
Phosphorous, in the form of phosphate, is present in the body. The total body phosphate content
is approximately 700 g. More than 85 percent is found in bones, with only about 15 percent
found in soft tissues and 1 percent found in extracellular fluid. 90% of daily dietary phosphate
absorption It is found in bone and teeth in conjunction with calcium. It is also a component of
DNA and RNA, which serve as the foundation for life and growth. Furthermore, it is required
for the phosphorylation-mediated regulation of enzyme activity. Vitamin D boosts intestinal
phosphate absorption by increasing the expression of the Na-P co-transporter in the small
intestine. The parathyroid hormone (PTH) reduces phosphorous reabsorption in the kidney and
thus increase its urinary excretion. The Ca:P ratio in diet affects the absorption and excretion of
phosphorous. If one is in excess in diet, the excretion of the other is increased. Phospholipid is
an important constituent of bone and teeth. It is also constituent of phospholipid, nucleic acids
and lipoprotein. It plays important roles in biological processes and also assist various
enzymatic reactions.
Phosphorus is found plentiful in most foods like milk, cheese, meat, etc. Deficiency of
phosphorus leads to poor mineralisation of bones and teeth and causes osteomalacia and poor
growth.
vi. Iron
Iron is a vital trace element for the human body. The average human body has 2.4 grammes of
iron. It's found in the active centres of proteins involved in O2 transport (like haemoglobin and
myoglobin) and electron transport (like cytochromes), as well as in the active sites of
metalloenzymes like nitrogenase, reductase, and hydrogenase. Essential (or function) iron and
storage iron are the two types of iron found in the human body.
 Essential iron: Essential iron is one which is involved in the normal metabolism of the
cells. It is further divided into three groups:

 Heme protein: Haemoglobin and myoglobin are two heme proteins which contains an
iron porphyrin prosthetic group attached to globin protein. Both of them are involved in
dioxygen transport.
Other Heme proteins are catalases and peroxidases. Catalase enzyme contains four heme
groups and is found in blood, bone marrow, mucous membrane, liver and kidney. It catalyses
the conversion of hydrogen peroxide into molecular oxygen and water.
Peroxidase, another heme protein found in milk, erythrocytes, leucocytes and lens fibres.
Cytochrome: Cytochromes are another class of iron containing compounds. Cytochromes are
chiefly found in mitochondria.
Iron containing enzymes: certain enzyme also use iron as co-factor like succinate
dehydrogenase, aconitase, ribonucleotide reductase, etc.
Storage iron: Storage from iron is ferritin and hemosiderin. Free iron is toxic while the iron
bound to0 ferritin is non-toxic. Ferritin, the storage protein of iron found in blood, liver, spleen,
bone marrow and intestine. Hemosiderin is derived from ferritin. It contains a larger fraction of
iron as compared to ferritin.
Dietary sources of iron: Foods rich in iron includes cereals, legumes, molasses, eggs, meat,
fish, etc. Iron also obtained from non-food sources like foods cooked in an iron skillet.
Deficiency of iron leads to anaemia. Symptoms of iron deficiency take year to develop and
include fatigue, weakness, and shortness of breath.
vii. Zinc:
Zn is the second most abundant dress material average Zinc content in the body is
approximately 2g.It is distributed in different part of a body like bones, teeth, skin, kidney and
muscle acceptor.It is essential for normal human body growth, wood healing and tissue
repairing. It regulates the function of insulin and also maintain the normal concentration of
vitamin A. It is the essential component of several enzymes. Important zinc containing
enzymes are superoxide dismutase, carbonic anhydrase and Carboxypeptidase. Superoxide
dismutase is aCu-Zn protein complex with two Zn2+ per molecule of the enzyme.It is present in
epithelial cells, red blood cells and brain cells. Carbonic anhydrase contains one Zn2+ per

molecule of enzyme. It is present in epithelial cells, red blood cells and parietal cells.
Carboxypeptidase is the hydrolytic enzyme present in pancreatic juice. Another zinc containing
enzymes are alcohol dehydrogenase alkaline phosphate, lactate dehydrogenase, glutamate
dehydrogenase, DNA and RNA polymerase.
Fighting off of cold or flu:
 Zinc supplements to reduce the cold symptoms such as age runny nose coughing and so
throat
 Zinc make skin Nails and hairy health skin hair healthy.
 Zinc helps in healing wounds.
 Zinc reduces the diabetic problems.
 Zinc increases production of testosterone and other male harmones. Therefore, it

reduces the male infertility.
 Zinc helps to preserve eye sight and improves memory.
 Zinc may help teenagers with pimples.
Symptoms of Zinc Deficiency
 Reduced growth of children
 Reduced mental retardation
 Slow wound healing
 Skin irritation
 Hair loss
 Loss of sense of taste
 Frequently infections
 Zinc work best with Vitamin A, Vitamin B6, insuline, Vitamin D, Vitamin E, glucose,
Mg and Mn.
Excess of zinc in the human body causes: Dysfuntion of central nervous system, Anaemia,
Diarrhoea, Dizziness, store stomach, Nausea, Vomiting, alcohol intolerance, electrolyte
imbalance and increase LDS cholesterol and lower HDL cholesterol.

Chief sources of zinc are egg, seafood, milk, meat cereals, legumes and pulses, oilseeds
vegetables like spinach lettuce.
viii. Copper:
Copper is vital in plants and animals, but its roles are not as well defined as those of iron.
Copper is distributed 100 to 150 mg in the muscles, bones, and liver. Under normal condition,
about 32% of dietary copper can be absorbed. The copper is absorbed in mucosal cells.After Fe
and Zn, copper is the third most dominant element in the human body. Excessive dietary of
either Zn or Mo interferes with the absorption of Cu. After absorption of Cu enter plasma
where it is bound to serum proteins. It is found in a variety of proteins, metalloenzymes, and
naturally occurring colours. Copper is found in cytochrome oxidase, tyrosinase, catalase,
ascorbic acid oxidase, superoxide dismutase, and other enzymes. A lot of invertebrates use
hemocyanin, a copper protein, as an oxygen carrier. Copper is also involved in the colouring of
skin and hair, as well as the creation of a protective layer surrounding nerve fibres. Legumes
and whole grains are the best suppliers of copper.
(a) Copper deficiency
The symptoms of copper deficiency include:
 Copper deficiency turns hair grey and also causes bone disorder and loss in body
weight. Bone demineralisation and blood vessel fragility due to defects in collagen and
elastin formation.
 Anaemia due to defect in iron metabolism.
 Hypercholesterolemia due to increase in ratio of saturated to monosaturated fatty acids

of C18 series.
(b) Impact of Excess of Copper
Excess of copper causes: Fever, high blood pressure, diarrhoea, dizziness, depression, fatigue,
irritability,joint and muscle pain, nausea, premature ageing, vomiting, wrinkling of
skin,headache etc.
Wilson disease illness is brought on by an excessive amount of copper in the body. It's a
genetic disorder. Wilson's disease patients have low quantities of the copper storage protein
ceruloplasmin, which means that even at normal levels, copper is toxic. Wilson disease is

characterised by liver damage, neurological impairment, and brown or green rings on the
cornea. To remove excess copper, many chelating ligands can be utilised, but D-penicillamine
is one of the most effective. This chelating ligands forms a complex with copper ions (Cu + and
Cu2+) that has intense purple colour and the molecular formula of the compound is
[Cu8+Cu62+(penicillamine)12Cl]. The sulfhydryl groups of D-penicillamine effects removal of
copper as Cu+ complex.Chelating therapy with EDTA also eliminates the symptoms.
ix. Cobalt
One of the most important trace metals is cobalt. Itis one the most ancient biocatalysts. Only
around 1.5 mg of cobalt is found in the human body, and the majority of it is in the form of
cobalamin, or vitamin B12. Cobalt is complexed in B12 by a special macrocycle called corrin.a
benzimidazole that is covalently linked to the corrin ring are known as cobalamins. Cobalamins
are cofactors in enzymes that catalyse alkyl transfer reactions and many radical-based
rearrangements. Cobalamin-containing enzymes show strong UV–visible absorption bands;
EPR spectra are observed for Co(II).Vitamin B12 cannot be synthesised by animals or plants. In
reality, only a few microorganisms are capable of producing it. Humans get 100% of their
vitamin B12 from animal sources, particularly meat. Because vitamin B12 is only required in
trace amounts, vitamin B12 deficiency is uncommon. However, vegans who eat no animal
products have been observed to have vitamin B12 deficiency. Vitamin B12 insufficiency is
caused by a failure to absorb the vitamin in the gut, which results in an increase in the excretion
of methyl malonic acid, which the body cannot convert to succinic acid. Pernicious anaemia is
caused by the deficiency.Cobalt is necessary for the action of various enzymes, including
coenzyme A, methyl malonyl oxidoreductase, and others, in addition to being a key component
of vitamin B12. Nausea, vomiting, diarrhoea, and skin rashes are all indications of too much
cobalt in the body.
x. Sulphur
Sulphur is present in the body in its oxidized form sulphate as a part of some proteins. It is also
present along with co-enzyme A and lipoic acid. It is the structural constituent of insulin and
many more proteins. In some enzymes specific sulfhydryl groups are essential for catalytic
activity. It plays an important role in the formulation of acetyl coenzyme A and S-acetyl
lipoate. Protein rich food like meat, fish, egg, milk, etc. provides necessary amount of sulphates
to the body.

xi. Manganese
Manganese is also an essential trace element required by the body and is found concentrated
mainly in the kidneys and liver. It acts as a cofactor or as an activator of many enzymes like
enolase, arginase, isocitrate dehydrogenase, cholinesterase, etc. Manganese and magnesium
may replace one another in case of some of the enzymes. It assist in bone formation and plays a
prominent role in fat and carbohydrate metabolism. Manganese is obtained primarily from
cereals, nuts, whole grains, tea and leafy vegetables.
xii. Fluorine
Fluorine is present in human tooth in trace amounts and helps in tooth development, normal
maintenance and hardening of dental enamel. Fluorine is also required for normal bone
development and increases the retention of calcium and phosphate and prevent old age
osteoporosis. Fluoride is mainly derived in human from drinking water. Other sources of
fluoride include fish, tea, salmon, etc. Destruction of dental enamel and tooth decay are
widespread among both adults and children in areas where drinking water contains less than 0.5
ppm of fluorine. But excess of fluoride in water or diet or its inhalation is harmful and is
considered to be main cause of the fluorosis disease.
xiii. Chlorine
The chloride ion maintain the fluid and electrolytic balance. It also plays a prominent role in
osmatic pressure regulation. In gastric juice, the chloride ion shows importance inthe
production of HCl and thus helps in digestion of food. It is also maintainthe acid base
equilibrium. Significance sources are table salt le maintain the acid base equilibrium.
Significant sources are table salt, processed food and soya sauce. Moderate sources include
meat, egg and milk.
xiv. lodine
The primary function of iodine is its role in thyroid functioning that help to regulate growth
and development. In addition to iodized salt, good dietary sources of iodine are sea food, bread,
dairy products. Deficiency of iodine leads to thyroid hypertrophy.
xv. Chromium
Chromium is widely distributed throughout the body. Chromium plays an important role in
carbohydrate, lipid, and protein metabolism. Chromium enhances the activity of insulinand

helps to maintain insulin levels. When an individual lack chromium, a condition like diabetes
can develop. Good sources of chromium include me/at, brewer's yeast, whole grains,etc.
xvi. Molybdenum
Molybdenum is principally known for its role in biological nitrogen fixation. It is the cofactor
of the enzyme nitrogenase which converts atmospheric N2 into NH3 (Chapter 6 section 5). It
also occurs in several flavoproteins like xanthine oxidase, NADH nitrate reductase, etc.
Molybdenum of all these enzymes participates in internal transfers during oxido-reductions.
Presence of small amounts of molybdenum help in the utilization of copper. On the other hand,
high molybdenum intake produces copper deficiency. Significant sources of Mo are legumes,
cereals and nuts.
xvii. Selenium
Selenium is a trace but an essential metal which is crucial to the heart. It is widely distributed in
the body in all the tissues, high concentrations are found in liver, kidneys and fingernails.
Muscles, bones, blood and adipose tissues show a low concentration of selenium. Selenium is
the prosthetic group of enzyme glutathione peroxidase which is present in cell cystol and
mitochondria and functions to reduce hydroperoxide. It regulate the activity of thyroid gland. It
is also required for normal pancreatic function. The chief sources of selenium ar whole grains,
fruits and vegetables. Deficiency of selenium leads to liver cell necrosis, pancreatic
degeneration infertility, failure of growth and dilation of the heart resulting in cardiac failure.
xviii. Nickel
Nickel plays a crucial role in bacterial enzymes, particularly hydrogenases, where it exploits the
3 and 1 oxidation states, which are uncommon in traditional chemistry. Coenzyme A synthase,
for example, utilises Ni to make CO, which it then combines with CH 3 (supplied by a
cobalamin enzyme) to form a C-C bond in the form of an acetyl ester. Plants also have nickel
as the active site of urease. Urease was the first enzyme to be crystallised (in 1926), but it
wasn't identified to contain Ni until 1976
Table. 3 deficiency symptoms of trace elements
Element Function Main deficiency symptom
Chromium Glucose metabolism Impaired glucose metabolism

Cobalt Vitamin B12 Anemia
Copper Oxidative enzymes Anemia , skeletal defects
Manganese Mucopolysaccharide Growth retardation

metabolism
Molybdenum Purine metabolism, aldehyde Joint pain

oxidation
Zinc Nucleic acid metabolism Poor would healing
3.5 Na+-K+-ATPASE
Jens Skou discovered the Na+K+-ATPase, a transmembrane protein, in 1957. With the
hydrolysis of intracellular ATP, this enzyme is also known as the Na+-K+pump because it
pumps three Na+ out and two K+ into the cell. In animal cells, the Na+-K+ pump keeps the cell's
Na+ concentration low while maintaining a high K+ concentration in the extracellular medium.
The transmembrane electric potential is created by ion transport. E 1 and E2 are the two
conformations of the Na+-K+-ATPase. E has a high affinity Na+ binding site while E2 has a high
affinity K+ binding site.The E1 conformation enzyme binds three Na* from the cell's interior.
The phosphate group is transferred to the aspartic acid residue of the transport protein after E 1,
3Na+ binds ATP, which is hydrolysed. Within the tranet protein, this aspartyl phosphate causes
a conformation change from E to E. The Na+- K+ -ATPase E1 conformation has a low affinity
for Na+ and a high affinity for K+. As a result, the transporter discharges 3Na+ into the
environment and binds 2K+ from the surrounding medium.The phosphate group is hydrolysed,
and the enzyme returns to its E1 shape. Because the E1 conformation of the enzyme has a high
affinity for Na but a low affinity for K+, the transporter releases 2K+ to the cell's interior. A
trans membrane potential of -50 to -70 mV is generated by the migration of 3Na+ ions out of
the cell and 2K+ ions inside the cell. As a result, this ion transport is referred to as electrogenic,
or the creation of electric potential.
3.5 BIOLOGICAL METAL-COORDINATION SITES

Compounds containing metals that are not found in biological systems appear to have a unique
role. Orally given medications are preferred because they prevent the stress and risks associated

with injection. Inorganic substances, such as radioactive technetium, are also utilised to
diagnose sickness or damage. Sequestration of Fe by ligands inspired by or based on
siderophores is used to treat Fe overload. The term "iron overload" refers to a series of major
health problems that impact a huge section of the world's population. It's important to note that,
because of its importance, Fe is a potentially dangerous element, especially because of its
capacity to form damaging radicals when reacting with O 2, and its amounts are usually closely
regulated by regulatory systems. Many people have a breakdown in this control as a result of a
genetic disease. Metal ions coordinate to proteins, nucleic acids, lipids, and a variety of other
molecules. DNA is stabilized by weak coordination of K+ and Mg2+ to its phosphate groups but
destabilized by binding of soft metal ions such as Cu+ to the bases. The binding of Mg2+ to
phospholipid head groups is important for stabilizing membranes.
There are a number of important small ligands, apart from water and free amino acids, which
include sulphide, sulphate, carbonate, cyanide, carbon monoxide, and nitrogen monoxide, as
well as organic acids such as citrate that form reasonably strong polydentate complexes with
Fe3+. from introductory chemistry, a protein is a polymer with a specific sequence of amino
acids linked by peptide bonds. Proteins are synthesized, a process called translation (of the
genetic code carried by DNA), on a special assembly called a ribosome. A protein may be
processed further by post-translational modification, a change made to the protein structure,
which includes the binding of cofactors such as metal ions. Metalloproteins, proteins containing
one or more metal ions, perform a wide range of specific functions. These functions include
oxidation and reduction (for which the most important elements are Fe, Mn, Cu, and Mo),
radical-based rearrangement reactions and methyl-group transfer (Co), hydrolysis (Zn, Fe, Mg,
Mn, and Ni), and DNA processing (Zn). Special proteins are required for transporting and
storing different metal atoms.The action of Ca2+ is to alter the conformation of a protein (its
shape) as a step-in cell signalling (a term used to describe the transfer of information between
and within cells). Such proteins are often known as metal ion-activated proteins.

3.6.1 Metal Complexes in Medicine
i. Cisplatin: Anticancer Drugs
In 1969, B. Rosenberg and co-workers discovered the antitumor activity of simple square
planar Pt(II)complex, cis- diamminedichloroplatinum (II)or cisplatin , [Pt(NH3)2Cl2]. This
compound is used as chemotherapeutic agent to inhibit otherwise rapid division of tumour cells
(i.e. proliferation). Chemotherapy is the use of anticancer drug designed to inhibit growth of
rapid dividing cancer cell in the body. The exact action of this complex is not known. Since the
trans-isomer is inactive, therefore chelation or atleast coordination to Donor atoms at cis-
position is an essential part of activity. The proton NMR studies has suggested that Platinum
binds to N-7 atom of a pair of adjacent quinine bases of a fast-growingtumour with the chloride
ligands first being replaced by water molecules and then by a DNA base.
Cis-platin interact with N atoms of two adjacent guanine bases N-7 on DNA usually within the
same strand(intrastandlinkage) or occasionally between strands (interstrand linking). The N-
7position of guanine base is much more base is much more basic than that of adenine and
provides, therefore, a stronger site for the attack by platinum. Recent X-ray studied on a 12-
base pair fragment of double stranded DNA has suggested that the binding of Pt distorts the
local DNA structure and therefore, inhibits the cells division inherent in the proliferation of
cancer cells. Cisplatin has side effect in kidney and neuro-toxicity. Alternative Platinum
compounds have been developed to avoid these serious side effects. The most important of
these is carboplatin, which replaces the Cis Chloride right ligands with O chelate
cyclobutanedicarboxylate.

Fig. 3.10 Interaction of cis-platin with two guanine bases on a DNA strand
Fig. 3.10: (a) Carboplatin (b)Oxaliplatin (c) Sataraplatin (d)cis-dichloroamine

(cyclohexylamine) Pt(II) (e) trinuclear Pt(II) anticancer drug
ii. Wilson disease
Wilson disease is caused by the overload of copper in the body. It is a genetic disease patients
suffering from Wilson disease have low levels of the copper storage protein ceruloplasmin and
therefore copper can not be tolerated even at normal levels. The Wilson disease is responsible
for liver disease, neurological damage and Brown or green rings in the cornea of the eyes.
Many chelating ligands can be used to remove the excess of copper but one of the best is D-
penicillamine.This Chelating ligand forms a complex with copper ion (Cu + and Cu2+) that has
intense purple colour and the molecular formula of the compound is Cu 8ICu6II
(penicillamine)12Cl]. The sulfhydryl group of D-penicillamine effects removal of copper as
Cu+complex.

Fig. 3.11 Structure of complex of Cu with D-penicillamine chelating ligand
Chelating therapy using EDTA, MoS42-or 2-3 dimercaptopropan-1-ol also causes the
symptoms to disappear.
iii. Anti-Arthritis
Complex of Au(I) have been used most successfully for the treatment of arthritic disorders in
humans. These complexes used to treat Arthritis where painfully administered as
intramuscular injections. These complexes include Na3[Au(S2O3)2] called Sanochrysin, sodium
salt of thiomalate, called Myochrysin.
More recently, the compound auranofin has been developed.It has the advantage that it can be
administered orally and is effective.
iv. Hypercalcemia
Hypercalcemia is a disease which causes the Rapid loss of Calcium from the bones of Cancer
patients. Gallium nitrate Ga(NO3)3 has been found to be most effective for treatment of
hypercalcemia.
v. Siderosis Disease
An excessive intake of iron causes various problems known as siderosis. Chelation therapy is
also used to treat the excess of iron. The patients who suffer from deposit of iron in liver,
kidney and heart lead to failure two of these organs. The excess of iron can be removed by
using chelating ligand such as deferoxamine-B, a polypeptide having a very high affinity of
Fe(III) but not a for other. The concepts of soft and hard metals and ligands can be used for the
process of designing therapeutic chelating agents.
3.7 Magnetic Resonance Imaging (MRI)

Nuclear Magnetic Resonance Spectroscopy can be used to image specific tissues of biological
system because of the differences in relaxation times of water Proton resonance usually brought

about by metal ions which are paramagnetic.The useful metal ions for Magnetic resonance
imaging in humans are Gd(III), Fe(III) and Mn(II) ions. The paramagnetic character of these
ions alters the relaxation of rate of nearby water proton and, therefore, the normal and diseased
tissue can be distinguished an advantage of paramagnetic MRI over radio isotopic imaging
agents is that there is no possibility of radiation damage removal of excess of metal ions from
the body is called chelate therapy.
3.8 TOXICITY OF METALS: Hg,Cd, Pb, As and THE CHELATE

THERAPY
The elimination of toxic metal cations or other excess cations chelating ligands is called chelate
therapy.
i. Lead
Lead is a very poisonous metal it is a cumulative poison sense it keeps on accumulating in the
tissues of human body and plants.The lead compound and organolead compounds such as
Pb(C2H5)4in particular, are highly toxic. Exposure to organoleadcompounds occurs via
inhalation, ingestion and skin contact. Like Hg(II) the heigh toxicity of lead is due to strong
affinity towards sulfhydryl groups SH of cysteine residue of enzyme and proteins.The toxicity
of lead is also due to the ability of lead ions to cause oxidative stress, OH radicals and
peroxides thus generated induced damage of DNA and neurones.
(a) Symptoms of lead poisoning
Symptoms of lead poisoning are anaemia loss of appetite, headache, nervous disorder, brain
damage, liver damage, kidney damage, cholic and skin diseases.
(b) Treatment of lead poisoning
Lead poisoning can be treated by complexing and sequestering the lead by chelating ligands
such as ethylene diamine tetra acetate CaNa2(EDTA) or British anti-lewisite BAL
penicillamine.
Fig. 3.12 Structure of (a) EDTA (b) BAL

ii. Mercury
Hg(II) is the congener of Zn(II) and being the congener of Hg(II) can occupy the Zn(II) binding
sites in many Zn(II) enzymes and proteins. Yet Hg(II) is much larger than Zn(II) and Hg(II)
proteins are formed would not function as well as corresponding Zn(II) proteins or the activity
may be lost altogether. Hg(II) binds specially cysteine residue and hence inhibits enzymes
whose active sites contains cysteine.
The toxicity of Hg(II) is due to the high affinity of Hg(II) and CH 3Hg+ for sulfhydryl group
(SH) of cysteine residue in proteins inhibiting the activity of enzymes and other proteins.
InorganicHg(II) compounds once having entered an organism are biotically converted to
CH3Hg+ (monomethyl mercury) being less ionic and having some affinity toward cell
membrane because of methyl group, can be absorbed relatively easily through membrane.
Mercury having an appreciable vapour pressure is also extremely toxic. Mono methyl Mercury
and dimethylmercury are more dangerous than metallic mercury itself and inorganic
compounds such as HgCl2.These organomercury compounds are more readily absorbed in
gastro-intestinal tract then Hg2+ salts because they have greater ability to penetrate bio
membranes. They concentrate in blood and have immediate and permanent effect on brain and
central nervous system because they bind to the -SH group of cysteine residues in proteins.
(a) Symptoms of Mercury Toxicity
The symptoms of Mercury toxicity are: central nervous disorders, headaches, irritability,
fatigue, inability to make decisions, sleeplessness, diarrhoea etc.
(b) Treatment of Mercury toxicity
More Rapid elimination of cadmium, mercury and lead requires the administration of chelating
and such as 2, 3-dimercaptopropane-1-ol,HSCH2CH(SH)CH2OH and N-acetyl penicillamine.
A very good interesting natural detoxification has been discovered in bacteria resistant to
mercury. Bacteria have developed resistance to heavy metals and detoxifying process is
initiated and controlled by metalloregulator proteins that are able selectively to identify metal
ions. It is a small DNA binding protein that controls transcription of the mer genes.
cadmium is extremely toxic metal becauseCd(II) like Hg(II) being a congerner of Zn(II) can
replace in many Zi(II) enzymes and proteins. Yet Cd(II) is large larger than Zn(II) and Cd(II)
proteins formed would not function as well as the corresponding zinc enzyme or the activity

may lost altogether. Since like Hg(II), Cd(II) is also a soft acid and have strong affinity towards
Cadmium is extremely toxic. It accelerates in the Kidney and liver of human. It has a
strong affinity for the -SH group of cysteine residues in protein and therefore inhibits SH
enzymes.It also inhibits the action of zinc enzyme by displacing zinc.
soft base sulfhydryl (SH) of cysteine residue of protein chain, Cd(II) strongly coordinated to
deprotonated Sulfhydryl groups of cysteine Residue in zinc tablet dependent proteins making
the enzymes and proteins inactive and eventually denaturing the protein and forming insoluble
CdS.
(c) Symptoms of cadmium toxicity
Symptoms of toxicity are:disfunctions of kidney, vomiting, irritation, hypertension, anaemia

and it disease called Hai Itai in which the whole body feels serious pains and the bones and
begins to facture very easily.
iii. Arsenic
Arsenic is a highly toxic elements for most organisms. Once inorganic compounds of arsenic
enterinto the organism, they are metabolized tomethylarsenic compounds, formally derived
from arsenate and arsenite by replacement of upto three OH-/O- functions for methyl groups.
The most commonly occurring Asis arsenate, AsO43-which is readily reduced in organisms
cells by glutathione. Hence, it reduces the availability theantioxidant, glutathione.
The toxicity of arsenic causes: chromel damage, mutagenesis, cancer and oxidative stress.
3.9 SUMMARY
In this Unit you have learnt that:
 Deoxyribonucleic acid (DNA) is a complex molecule that includes genetic instructions

necessary for all known living species to develop and function.
 Watson and Crick discovered the double helical structure of DNA in 1953, making it
one of the most famous discoveries of all time.

 Watson and Crick proposed a probable route for DNA replication while proposing the
double helical model of DNA. DNA replication is the process by which a double-
stranded DNA creates a copy of itself to produce two identical replicas.
 Since then, three distinct DNA replication models have been proposed: conservative,
semi-conservative, and dispersive.
 Essential, Trace, Non-essential, and Toxic elements are divided into four groups.
 At normal quantities, no common element is hazardous, but practically everything can

be dangerous at excessively high amounts.
 The concentration of metal ions in a human's system is tightly controlled.
 Metal ion deficit or excess creates disturbance, which can lead to a variety of disorders.
3.10. SAQS TYPE QUESTIONS

Objective Questions
1. Which of the following is a heme iron protein ?
(a) Ruberodoxin
(b) Transferrin
(c) Hemerythrin
(d) Cytochrome-c
2. In biological systems, the metal ions involved in electron transport are:

(a) Na+ and K+
(b) Zn 2+ and Mg 2+]
(c) a 2+ and Mg2+
(d) Cu 2+ and Fe2+
3. The oxygen free form of myoglobin is a:

(a) 5-coordinate high spin Fe(ll) complex
(b) 6-coordinate low spin Fe(ll) complex
(c) 5-coordinate high spin Fe(lll) complex

(d) 5-coordinate low spin Fe(lll)
4. Iron-sulphur clusters in biological systems are involved in :
(a) proton transfer
(b) atom transfer
(c) group transfer
(d) electron transfer
5. In biological systems, the metal ion involved in the dioxygen transport besides Fe is :
(a) Co
(b) Zn
(c) Mg
(d) Cu
6. The oxidation state of iron in met-hemoglobinis :
(a) Three
(b) Two
(c) Four
(d) Zero
7. The metal ions present in the active site of nitrogenase enzyme cofactor are:
(a) Fe, Mo
(b) Fe, W
(c) Fe, Cu
(d) Fe, Ni
8. In oxyhemoglobin, the iron centre best described by which of the following ?

(a) high spin Fe(ll)
(b) high spin Fe(lll)
(c) low spin Fe(lll)

(d) low spin Fe(ll)
9. The metal atom present in the active site of carboxypeptidase enzyme is :
(a) cobalt (b) zinc
(c) iron (d) magnesium
10. The ligand system present in vitamin B12 is:
(a) porphyrin (b) corrin
(c) phthalocyanine (d) crown ether
Answers:
(d) 2 (d) 3 (a) 4 (d) 5 (d) 6 (a) 7 (a)

1
8 (d) 9 (b) 10 (b)
3.11 LONG ANSWER QUESTION

1. Discuss the role of metal ions in biological systems.
2. What are essential and trace metals? Give the role of iron, manganese and molybdenum
in biological system.
3. Discuss DNA polymerisation in brief. Give the structure and biological significance of
DNA.
4. Write toxic effect of Pb(II). Give reason of its poisonousness. How it can be treated?
3.12 BIBLIOGRAPHY
1. Bio Inorganic Chemistry, Krishna publication,Suchita Tyagi and Vichitra Tyagi
2. Organoetallic and Bioinorganic Chemistry, Aaryush PublicationAjai Kumar.
3. Bioorganic, Bioinorganic Supramolecular Chemistry, New Age Publication, P.S.

Kalsi and J.P. Kalsi

4. Metal ions in Biological Systems, Bioinorganic Chemistry, e-PG Pathshaala, A.K.
Bakshi
5. DNA polymerisation Model systems, Bioinorganic Chemistry, e-PG Pathshaala, A.K.

Bakshi
6. Bioinorganic Chemistry, first south Asian edition,HB Gary, Stephan, J. Lipard and
Joan Silverstone Valentine.
7. Bioinorganic chemistry, First South Asian edition, 1998 by Ivano Bertini, Harry B.
Gary, Stephen J. Lippard and Joan Silverstone Valentine.
8. Bioinorganic chemistry, first edition, 2014 by Dieter Rehder.
9. Biochemisu-y and molecular biology, fourth edition by William H. Elliot and Daphne
C. Elliott. Biochemisüy, sixth edition by Rex Montgomery, Thomas W. Conway,
Arthur A. Spector and David Chappell.
10. Organometallics-l, complexes with transition metals-carbon, c-bonds, Indian edition

by Manfred Bochmann.
11. Inorganic chemistry, Indian edition, 2008 by James E. House.
12. Concise inorganic chemistry, fifth edition by J.D. Lee.
13. Bioinorganic Chemisny--A Survey by EiichiroOchiai.

BLOCK II: BIOORGANIC CHEMISTRY
UNIT 4: INTRODUCTION
CONTENTS:
4.1 Intoduction
4.2 Objective
4.3 Chemical background of biomolecules
4.3.1 Carbohydrates
4.3.2 Lipids
4.3.3 Protein
4.3.4 Nucleic acid
4.4 Proximity effect
4.5 Molecular adaptation
4.6 Summary
4.7 Biobliography
4.8 Suggested readings
4.1 INTODUCTION
Bioorganic chemistry is a chemistry which integrates organic chemistry and biochemistry.
Organic chemistry deals with structure design, synthesis and kinetics. Biochemistry deals
with study of life processes by means of biochemical methodology. Organic chemistry
methods are used to synthesize biological molecules and to examine their structure, to
investigate biochemical reactions. Bioorganic chemistry can be defined as a branch of
chemistry or broadly speaking a branch of science which utilizes the principles, tools and
techniques of organic chemistry to the understanding of biochemical/biophysical process
using chemical methods.

4.2 OBJECTIVE
This unit intended to provide learners with a basic understanding of the chemical nature of
biomolecules. As part of this unit, learners will be introduced to biomolecules such as nucleic
acids, proteins, carbohydrates and lipids. At the end of this unit, learners will be able to:
 Identify and define different types of biomolecules.
 Describe the important functional group and structural features of biomolecules.

 Identify, classify and name carbohydrates, nucleic acids, proteins and lipids on the
basis of their structure and functions.
 Understand the principles of the chemistry behind biochemical molecules and their
biochemical properties.
 Give the composition of nucleic acids and explain the difference between DNA and
RNA.
4.3 CHEMICAL BACKGROUND OF BIOMOLECULES

Biomolecules are molecules that are involved in the maintenance and metabolic processes of
all living organisms. Biomolecules made up of carbon and hydrogen, including large
macromolecules such as proteins, polysaccharides, lipids and nucleic acids. These
biomolecules generally known as the derivatives of hydrocarbons and some of the hydrogen
atoms replaced by various types of functional groups such as hydroxyl, methyl, carbonyl,
carboxyl, amino, phosphate and sulfhydryl to formed different bioorganic molecules or
biological molecules (Table 4.1).
Many biomolecules are polyfunctional, containing two or more functional groups that might
impact each other's reactivity. Basically functional group of biomolecules contain carbon,
hydrogen, oxygen, nitrgen, phosphorus and sulphur besides these biomolecules also contain
several heterocyclic and homocylic ring in their structure for example indol ring in amino
acid tryptophan, phenanthrene ring in steroids. Pyrole is the basic unit of porphyrins found in
several biomolecules hemoglobine, chlorophyll etc. while thiophene ring is the part of
vitamine biotin. The amino acid histadine contain the ring structure of imodazole.
Pyrimidines and purine are the basic constituents of the nucleic acids.

Table 4.1: Important functional group of biomolecule.
Functional group Properties General feature Example of

bimolecule
Name Structure
Hydroxyl Polar, Hydrophilic, Capable Characterized Carbohydrates

of hydrogen bond by presence of
interactions H and O
Carbonyl Polar, Carbonyl group of Characterized Carbohydrates

ketones and aldehydes is by central C and
strongly polar group capable O
of acting as a hydrogen bond Double bond to
acceptor oxygen
increases the
polarity
Carboxyl Ionized to release H+. Since Characterized Fatty acids

carboxyl groups can release by central C
H+ ions into a solution, they bound to O and
are considered acidic. Polar, OH
Capable of hydrogen bond
interactions
Amino Capable of hydrogen bond Characterized Protiens

interactions as a base, by presence of
accepts H+ to form NH3+. N
Since amino groups can
remove H+ from solution,
they are considered basic.
Phosphate Charged, ionizes to release Characterized Nucleic acids

H+. Since phosphate groups by presence of P
can release H+ ions into
solution, they are considered
acidic.
Sulfhydryl Polar Characterized Coenzyme-A

by presence of S
Biomolecules are typically larger than organic molecules. Most biomolecules have molecular
weights in the thousands, millions, or even billions, while small biomolecules have molecular
weights of over 100. Because of their large size, the majority of biomolecules have specific
3-dimensional shapes in which atoms arranged in space in a precise way. The 3-dimensional
shape is maintained by numerous non-covalent bonds between atoms in the molecule. The

structure of a biomolecule is flexible rather than static due to the weak nature of most
noncovalent bonds and interactions between the biomolecule and the solvent. Biomolecules
also exhibit stereochemistry as organic compounds. When the molecule contain stereogenic
(or chiral or asymmetric) carbon then it can exist in two different isomeric enantiomers or
diastereomers forms that have different configurations in space and have different properties.
4.3.1 Carbohydrates
The carbohydrates are an important class of naturally occurring organic compounds. They
occur naturally in plants (where they are produced photosynthetically). During
photosynthesis, plants take up water through their roots and use carbon dioxide from the air
to synthesize glucose and oxygen. Because photosynthesis is the reverse of the process used
by organisms to obtain energy the oxidation of glucose to carbon dioxide and water, plants
require energy to carry out photosynthesis. Carbohydrate originally referred to compounds of
general formula Cn(H2O)n. Where n is the number of carbons in the molecule represents
carbohydrates. In other words, the ratio of carbon to hydrogen to oxygen is 1:2:1 in
carbohydrate molecules. However, only the simple sugars or monosaccharaides fit this
formula exactly. The other types of carbohydrates, oligosaccharides, and polysaccharides, are
based on monosaccharaides units and have slightly different general formula. Carbohydrates
are also called "saccharides" which means sugar in Greek. Carbohydrates are sugars and
starches. They are the major source of energy in many organisms, serve to store energy, and
are structural components in some organisms. Because of their wide distribution,
carbohydrates are the most abundant type of biomolecule.
The carbohydrates are poly functional compounds and contain functional group Alcoholic
hydroxy group (-OH), Aldehyde group (-CHO) and Ketone (>C=O). Thus carbohydrate can
be defined as Polyhydroxyaldehydes such as D-glucose, polyhydroxy ketones such as D-
fructose, and large molecules such as sucrose that produce these compounds on hydrolysis.
We know that Hemiacetal or acetal is formed when a carbonyl molecule (aldehyde or ketone)
reacts with an alcohol (alcoholic -OH). An aldehyde group in carbohydrates reacts with an
alcoholic -OH of the same molecule to create an internal hemiacetal. We shall also see that
bigger carbohydrate molecules are produced by removing H2O molecule from between the
hemiacetal OH groups of two sugar molecules. In view of the foregoing, a better definition of

carbohydrate may be: polyhydoxy molecules with an aldehyde or ketone function, either free
or as hemiacetal or acetal.
4.3.1.1 Classification and nomenclature of carbohydrates
The carbohydrates are divided into three major classes monosaccharides, oligosaccharides,
polysaccharides depending upon whether or not they undergo hydrolysis and if they do, on
the number of products formed. Monosaccharides: The monosaccharides are polyhydroxy
aldehydes (aldose) or polyhydroxy ketones (ketose) which cannot be decomposed by
hydrolysis to give simpler carbohydrates. e.g. Glucose, fructose, Galactose etc. On the basis
of number of carbon in main chain monosaccharides are also classified as trioses, tetroses,
pentoses, hexoses and heptoses (Table 4.2).
Table: 4.2 Clasification of monosacarides

No. of Emprical Aldose Ketose
Carbon Formula
Atoms Types Example Types Example
3 C3H6O3 Aldotriose Gleceraldehyde Ketotriose Dihydroxyacetone
4 C4H8O4 Aldotetrose Erythrose Ketotetrose Erythrulose
5 C5H10O5 Aldopentose Ribose Ketopentose Ribulose
6 C6H12O6 Aldohexose Glucose Ketohexose Fructose
7 C7H14O7 Aldoheptose Glucoheptose Ketoheptose Sedoheptalose
Oligosaccharides (Oligo: few) consist of short chains of monosaccharide units, or residues,

joined by characteristic linkages called glycosidic bonds and upon the hydolysis gives
definite number (2-10) of monosaccharide molecules. The oligosaccharides containing two
monosaccharaides units are called disaccharide and those containing three units are
trisaccharides. The most abundant are the disaccharides, with two monosaccharide units. For
example sucrose (cane sugar), which consists of the six-carbon sugars D-glucose and D-

fructose? Thus sucrose, C12H22O11 is disaccharides because on hydrolysis it gives one
molecule of glucose and one molecule of fructose.
The polysaccharides are sugar polymers containing more than 10 monosaccharide units, and
some have hundreds or thousands of units. Some polysaccharides, such as cellulose, are
linear chains; others, such as glycogen, are branched. Thus carbohydrates which have higher
molecular weight, which yield many monosaccharide molecules on hydrolysis. E.g. Starch,
glycogen, Dextrin, Cellulose etc.
In general monosaccharides and oligosaccharides are crystalline solids, soluble in water and
sweet to taste, they are collectively known as sugars, the polysaccharides on the other hand
are amorphous, insoluble in water and tasteless, they are called non-sugars (Table 4.3).
Table 4.3: Differece between monosaccharides, oligosaccharides and polysaccharides.
Character Monosaccharides Oligosaccharides Polysaccharides
No. of sugar 1 2-10 More than 10

molecules
Glycoside bond Absent Present Present
Molecular weight Low Moderate High
Taste Sweet Minimally sweet taste No teste
Solubility in water Soluble Soluble insoluble
Nature Always reducing May or may not be Always non

sugar reducing sugar
Example Glucose, Fructose, Sucrose, Maltose Starch, Cellulose,

Glactose Glycogen
The carbohydrates may also be classified as either reducing or non reducing sugars. All those
carbohydrates which have ability to reduce Fehiling’s solution and Tollen’s reagents are

referred to as reducing sugars, while others are non reducing sugars. Reducing sugar bear a
free aldehyde (-CHO) or ketonic [RC(=O)R'] group and have the capacity to reduce cupric
ions of benedict’s or Fehling solution to cuprous ion. All monosaccharides and disaccharides
(like lactose, Melibiose, Cellobiose, Gentiobiose) other than sucrose are reducing sugars.
4.3.1.2 Structure and Stereochemistry of Carbohydrates
The chemical structures of carbohydrates are commonly represented by wedge-and-dash

structures or by Fischer projections (Fig. 4.1). Carbohydrates come in a variety of
stereocenters. Glyceraldehyde, for example, contains only one. However, as you get to more
complex carbohydrates, you'll see an increase in stereocenters. There are four chiral carbons
in a molecule like glucose. This indicates that there are a total of 16 stereoisomers in glucose
molecule. The number of stereoisomers is equal to 2n, where “n” is the number of chiral
centres.
Fig. 4.1 Wedge -dash structures and Fischer projections of carbohydrates.
A. Open Chain form of carbohydrates
The stereochemistry of carbohydrates is described using D and L notation, which specifies

the configuration of the last chiral carbon in the chain. It is used to give the name of molecule
by relating it to glyceraldehyde. Fischer's projection is used to distinguish between D and L
carbohydrates. If the hydroxyl group is positioned to the right of the last stereocenter in the
Fischer's projection, the carbohydrates are given the D configuration, and if the hydroxyl
group is placed to the left of the last stereocenter carbon, the carbohydrates are given the L
configuration. It only gives the configuration of the carbohydrates and does not specify
anything relating to the sign of the rotation of the plane-polarized light and the molecules in

which atoms have different structural arrangements are asymmetrical molecules. Absolute
stereochemistry is not designated by the D and L configuration but it can be determined by
the anomeric carbon center by comparing its orientation to the glyceraldehyde. D-L system is
important as it gives the relative configuration of the molecules. D- and L- notation provides
a quick shorthand for designating enantiomers for example D-Glucose is the enantiomer of
L-Glucose (Fig. 4.2).
Fig. 4.2: Open Chain form of carbohydrates and D, L nomenclature.
The presence of asymmetric carbon atoms also confers optical activity on the compound.
When a beam of plane-polarized light is passed through a solution of an optical isomer, it will
be rotated either to the right, dextrorotatory (+); or to the left, levorotatory (-). The direction
of rotation is independent of the stereochemistry of the sugar, so it may be designated D (-),
D (+), L (-), or L (+). For example, the naturally occurring form of fructose is the D (-)
isomer.
B. Cyclic form of carbohydrates
Carbohydrates contain a carbonyl and alcohol functional groups, they can form
intramolecular (cyclic) hemiacetals. A carbohydrate must be at least a tetrose to do that, so
intramolecular cyclic forms don’t exist for smaller carbohydrates. When the -OH on the 4th
carbon is participating in the cyclization, you get a 5-membered ring. When the 5th carbon
provides the -OH, you get a 6-membered ring. Due to the analogy with the common oxygen-
containing heterocyclic compounds furan and pyran, the 5-membered rings are called
furanoses, and 6-membered rings are called pyranoses. Here’s an example of a common
sugar D-galactose forming two different cyclic forms (Fig.4.3).

Fig.4.3 Cyclization of galactose giving two different cyclic products
C. Haworth Projections (Alpha and beta anomers or anomers)
We often use the special type of drawing to depict the cyclic forms of carbohydrates. We call
them Haworth Projections or forms. Basically, a Haworth projection is a cyclic structure
with, traditionally, carbon 1 to the right and the bottom portion of the structure oriented
towards the observer.
C1 in a cyclic carbohydrate is called anomeric carbon. This carbon used to be a carbon of the
C=O in the open-chain structure before the cyclization. Anomeric carbon is special because it
doesn’t have a set stereochemistry and can be in an α-form or a β-form. The α- and the β-
forms are defined as trans or cis isomers of the cyclic carbohydrates where we look at the
anomeric -OH and the C 5 or C6 for furanoses or pyranoses correspondingly.
Cyclization creates an anomeric carbon (the former carbonyl carbon), generating the α and β
configurations of the sugar, for example, α-D-glucopyranose and β-D-glucopryanose. In a
Haworth projection formula of the α-configuration, OH (C1) is trans to the CH2OH (C5)
group while in the β-configuration OH (C1) is cis to the CH2OH (C5) group. Since the α and
β forms are not mirror images, they are referred to as diastereomers. Thus monosaccharaides,
differing only in this configuration around the carbon atom to which the carbonyl group is
attached (the anomeric carbon), are called anomers (Fig.4.4).

Fig.4.4 The α- and the β-forms of Glucopyranoses
D. Chair form of carbohydrates

The planar Haworth projection formulae bear little resemblance to the shape of the six
membered pyranoses that actually adopt a non-planar ring conformation comparable to that
of cyclohexane. The chair form is the most form of the sugar in solution cyclize into rings.
The chair can have two different conformations. The principle effect of the alternative
conformations is to change the axial versus equatorial orientations for the carbon functional
groups. The pyranoses adopt a non-planar ring conformation and the chair form with-the
highest number if equatorial rather than axial hydroxyl groups are favored. It should be noted
that α- D-glucopyranose, in contrast to β -D-glucopyranose has an axial hydroxyl group. In
the α-form hydroxyl group attached to C-1 is below the plane of the ring while in β –form
that it is above the plane of the ring (Fig. 4.5).
E. Glycosidic bonds
 Monosaccharides can be joined to form disaccharides, oligosaccharides and

polysaccharides.
 Important disaccharides include lactose (galactose + glucose), sucrose (glucose +

fructose) and maltose (glucose + glucose).

 The bonds that link sugars are called glycosidic bonds. These are formed by enzymes
known as glycosyltransferases that use nucleotide sugars such as UDP-glucose as
substrates.
Fig. 4.5 Chair form of α and β-D-Glucose

 Glycosidic bonds between sugars are named according to the numbers of the
connected carbons and also with regard to the position of the anomeric hydroxyl
group of the sugar involved in the bond.
 If this anomeric hydroxyl is in the α configuration, the linkage is an α-bond. If it is in

the β configuration, the linkage is a β-bond.
 Lactose, for example, forming a glycosidic bond between carbon 1 of β-galactose and
carbon 4 of glucose. The linkage is, therefore, a β (1→4) glycosidic bond.

G. Epimer
Compounds that have the same chemical formula but have different structures are called
isomers. For example, fructose, glucose, mannose, and galactose are all isomers of each
other, having the same chemical formula, C6H12O6 (Fig. 4.6).
 Carbohydrate isomers that differ in configuration around only one specific carbon
atom are defined as epimers of each other. For example, glucose and galactose are C-
4 epimers-their structures differ only in the position of the -OH group at carbon 4.
[Note: The carbons in sugars are numbered beginning at the end that contains the
carbonyl carbon-that is, the aldehyde or keto group]
 Glucose and mannose are C-2 epimers. However, galactose and mannose are not
epimers-they differ in the position of -OH groups at two carbons (2 and 4) and are,
therefore, defined only as isomers.
Fig. 4.6 Epimers of carbohydrate with chemical formula, C6H12O6

I. Mutarotation
These two possible orientations of the hydroxyl group confer differences in optical properties
when the substance is present in the crystalline anhydrous form and α-D-glucose has a
specific rotation of +113° whereas that of β-D-glucose is +19.7°. However, either form in
aqueous solution gives rise to an equilibrium mixture which has a specific rotation of +52.5°,
with approximately 36% being in the α form and 64% in the β-form, with only a trace present
as the free aldehyde. Because it takes several hours for this equilibrium to be established at
room temperature, any standard glucose solution for use with a specific enzyme assay (e.g.
glucose by the glucose oxidase which is specific for β-D-glucose) should be allowed to
achieve equilibrium before use, so that the proportions of each isomer will be the same in the
standard and test solutions. Mutarotation is defined as the changes in specific optical
rotation by inter conversion of α and β form of D-glucose to an equilibrium mixture.
Enzymes that accelerate the attainment of this equilibrium are called mutarotases and can be
incorporated in assay reagents in order to speed up the equilibrium formation. Mutarotation is
the change in the optical rotation because of the change in the equilibrium between two
anomers, when the corresponding stereocenters interconvert. Cyclic sugars show
mutarotation as α and β anomeric forms interconvert .
 The optical rotation of the solution depends on the optical rotation of each anomer and
their ratio in the solution.

 Mutarotation was discovered by French chemist Sir Dubrunfaut in 1846, when he
noticed that the specific rotation of aqueous sugar solution changes with time. [The
organic fructose molecule was subsequently discovered by Dubrunfaut in 1847].
 Sugars in the ring form can exist in two states, one where the C-1 hydroxy group is
above the plane of the ring (β) and one where it is below (α).
 In aqueous solution there is a constant interchange between the various conformations

via the breaking open of the hemi acetal structure and its subsequent reforming.
4.3.1.2 Functions of Carbohydrates
Carbohydrates have wide range of functions.
 Source of energy for living beings e.g. glucose.

 Storage form of energy e.g. glycogen in animals and starch in plants.
 Serve as structural component e.g. glycosoaminoglycans in humans, cellulose in
plants and chitin in insects.
 Non digestable carbohydrates like cellulose, serve as dietary fibres.
 Constituents of nucleic acid RNA and DNA. E.g. Ribose and deoxyribose sugar.
 Carbohydrates are also involved in detoxification, e.g. glucuronic acids.
4.3.2 Lipids
Lipids are fats and oils. Lipids are family of substances that are soluble in nonpolar solvents
but insoluble in water that can be extracted from cells using organic solvents. Because they
are grouped based on solubility properties, they are chemically more diverse than other
groups of biomolecules. There are several distinct classes of lipids. Most lipids function as
energy storage molecules or as structural components of membranes. Some are also
harmones, vitamins and pigments.
Lipids major roles in human biochemistry are:
 They store energy within fat cells Energy is stored in the form of glycogen for quick
energy when we need it. However, the burning of fats gives more than twice as much
energy as the burning of the equal weight of carbohydrates.
 They are parts of membranes that separate compartments from each other. Most body
constituents, for example carbohydrates and proteins are soluble in water. For

membranes that separate compartments containing aqueous solutions the body needs
insoluble compounds. Lipids provide these membranes.
 They serve as chemical messengers. Primary messengers such as steroid hormones,
deliver signals from one part of the body to another part. Secondary messengers, such
as prostaglandins and thromboxanes, mediate the hormonal response.
4.3.2.1 Classification and Chemistry of lipid
A. Fatty acids
Fatty Acids are carboxylic acids composed of long hydrocarbon chains.The long hydrocarbon
chains can be either saturated or unsaturated. Most naturally occurring fatty acids are
composed of even number of carbon atoms since their biosynthesis requires a concatenation
of C2 units. Most commonly found fatty acids have carbon atoms between 14 and 20. In
higher plants and animals, half of the fatty acids are polyunsaturated. Predominantly found
saturated fatty acids include Palmitic acid (C16), Stearic Acid (C18) and Arachidic acid
(C20). The notation used to represent unsaturated fatty acids is C: n where C represents the
number of carbon atoms and n represents the number of double bonds in the fatty acid.
Commonly found unsaturated fatty acids are Oleic acid (18:1), Linoleic Acid (18:2),
Linolenic acid (18:3) and Arachidonic Acid (20:4).
In the unsaturated fatty acids, the double bonds are always found in the cis configuration.
This cis configuration across the double bonds introduces a rigid 30⁰ bend in its structure
which interferes in its efficient packing due to which unsaturated fatty acids have a lower
melting point than their saturated counterparts.

B. Triacyl glycerols
Triglycerides (triacylglycerols) contain three fatty acids joined by ester bonds to a glycerol
molecule. The three fatty acids may be the same or different. Without the -COOH group, they
are even more non-polar than fatty acids. Triacylglycerols are water insoluble fatty acid esters
of glycerol. They are characterized by the identity of the three fatty acids that are esterified to
the three alcoholic groups of glycerol. They serve as energy reserves in the cell. Their utility
as energy reservoirs in the cell stems from the fact that compared to carbohydrates and
proteins, fats are less oxidized and hence more energy is release on oxidizing the same
amount of fat. Also, since they are nonpolar, they are stored in anhydrous form unlike
carbohydrate polymers like glycogen that bind twice their weight of water.
Triesters formed from glycerol and three molecules of fatty acids

Although some of the molecules contain three identical fatty acids, in most cases two or three
different acids are present (Fig. 4.7).
Fig. 4.7 Different types of fatty acides

C. Glycerophospholipids
Glycerophospholipids contain glycerol, two fatty acids, and a phosphate group at C-3 with a
polar group attached to it. They are derivatives of phosphatidic Acid.
Glycerophospholipids, also known as phosphoglycerides are membrane lipids that are ester
derivatives of L-glycerol-3-phosphate (Fig. 4.8). The C1 and C2 alcoholic groups of glycerol
3- phosphate are esterified to fatty acid residues to generate Phosphatidic acid. This is the
structureal parent fro all glycerophospholipids. The phosphoryl group of phosphatidic acid is
esterified to a highly polar and charged group via a phosphodiester linkage generating a
whole class of glycerophospholipids.

All glycerophospholipids always have a negative charge on the phosphate. The polar group
may have additional charges. Thus they are amphipathic with a polar head (phosphate) and
non-polar tail (fatty acids).
Fig. 4.8 Examples of Glycerophospholipids (phosphoglycerides)

D. Sphingolipids
Sphingolipids are major components of biological membranes. This class of lipids is derived
from the C18 amino alcohol sphingosine and dihydrosphingosine. Figure 4.9 shows the
structures of the sphingosine backbone, the structure ceramide and a typical Sphingolipid In a
sphingolipid, the amino group at the C-2 position of sphingosine is linked via an amide bond
to a fatty acid residue which can be saturated or monounsaturated with 16, 18, 22 or 24
carbon atoms. The C1 position is linked to a polar head group via either a phosphodiester or a
glycosidic bond. Ceramide is the structural parent for this class of lipids.
Fig. 4.9 Example of Sphingolipids

E. Sterols
Sterols are the fourth class of lipids (Fig. 4.10). In contrast to the membrane lipids discussed
above, sterols are structural lipids. Their characteristic structure has a steroid nucleus
composed of four fused rings. Three of these rings have 6 carbons while the fourth ring has
five carbons. It’s a planar ring system and relatively rigid. Any movement around the C-C
bonds in this system is restricted. Cholesterol is weakly amphipathic in nature as it has a
polar hydroxyl group attached to the 3rd carbon atom and a long non polar aliphatic chain
attached to the 17th carbon atom. Cholesterol is the main sterol which serves as not only an
important structural component of biological cell membranes but is also a precursor to major
steroid hormones in the body. Steroids regulate many important physiological processes in
the body as well as carbohydrate metabolism.
Sterols contain the steroid ring system of four fused rings, various side groups, and a
hydroxyl group. Other steroids contain a carbonyl group. They do not contain a fatty acid and
so are non-saponifiable. They are amphipathic with the oxygen group making a polar head
for the molecule. Steroids are non-hydrolyzable lipids. Steroids are compounds containing
four fused carbocyclic rings. Steroids are completely different in structure from the discussed
lipids. They are normally not esters, although some of them are. Steroids are closely related
in structure but are highly diverse in function.
This group of lipids involves:
 Cholesterol
 Sex and adrenocorticoid hormones
 Bile acids
 Vitamins D

Fig. 4.10 Different types of sterols
4.3.3 Protein
A protein is a biologically functional molecule that consists of one or more polypeptides.

Proteins account for more than 50% of the dry mass of most cells.Protein functions include
structural support, storage, transport, cellular communications, movement, and defense
against foreign substances. Protein’s function is highly governed by its stable structure. This
structure has four levels: Primary, secondary, tertiary and quaternary. Protein structure is
stabilized by multiple weak interactions.
Primary structure is the sequence of amino acids joined to each other by peptide bonds.
Secondary structure is the local folding of a part of polypeptide. Next level, the tertiary
structure is mixture of α-helix and β-sheets. While quaternary structure is the subunit
composition of a protein. In this module, we will have deeper insights into the protein
structure and its various levels.

4.3.3.1 Amino acids
Although a wide variety of proteins exist, they all have basically the same structure: They are
chains of amino acids. Amino acid is an organic compound containing an amino group and a
carboxylic group (Fig. 4.11). The 20 amino acids commonly found in proteins are called
alpha amino acids. Except for glycine, which is achiral, all the amino acids in all the proteins
in human body are the L-isomers.
Fig. 4.11 General structure of an amino acid

The most important aspect of the R groups is their polarity. On that basis amino acids are
classified into four groups: nonpolar, polar but neutral, acidic, and basic. The nonpolar
side chains are hydrophobic (they repel water), whereas polar but neutral, acidic, and basic
side chains are hydrophilic (attracted to water).
An amino acid has -COOH and -NH2 groups in the same molecule. Therefore, in water
solution, the -COOH donates a proton to the -NH2 so that an amino acid actually has the
structure of the internal salt. Compounds that have a positive charge on one atom and a
negative charge on another are called zwitterions, from the German word zwitter, meaning
“hybrid.” Amino acids are zwitterions, not only in water solution but also in the solid state
(Fig. 4.12).
Fig. 4.12 Amino acids are zwitterions

4.3.3.2 Peptide Bond
Amino acids are polymerized in living systems by enzymes that form amide linkage from the
amino group of one amino acid to the carboxyl group of another. A molecules formed by
joining amino acids together are called peptides, and amide linkages are called peptide bonds
or peptide linkages. Each amino acid in the peptide is called amino acid residue. Peptides that
contain 2, 3, a few (3-10), or many amino acids are called dipeptides, tripeptides,
oligopeptides and polypeptides, respectively.

Peptides are linear polymers. One end of a polypeptide chain terminates in an amino acid
residue that has a free –NH3+ group; the other terminates in amino acid residues with a free –
CO2- group. These two groups are called the N-terminal and C-terminal residues,
respectively (Fig. 4.13).
Fig.4.13 Formation of a peptide bond by condensation
Linus Pauling and Robert Corey worked on the peptide bond. They deciphered that peptide
bond is planar as an outcome of C-N bond length shorter than in amine. They noticed that
resonance existed between carbonyl carbon and amide nitrogen components of the peptide
bond (OC-NH). The oxygen (O in OC of peptide bond) and hydrogen (H in NH of peptide
bond) lie in trans position. No free rotation was seen around the peptide bond because of
their double bond character. Hence, it is rigid and is planar. Also, the oxygen of carbonyl
carbon has a partial negative charge and the nitrogen has partial positive charge. This forms
the small electric dipole. The only bonds where rotation is possible are N-Cα and Cα-C
bonds. The bond angles upon rotation of Cα-C bond are called the psi angle (ψ) while the
bond angle upon rotation of N-Cα bond is called the phi angle (φ). Psi and phi angles should
be between -180° to +180° (Fig 4.14). The permitted rotations around N-Cα bond (phi angle)
and Cα-C bond (psi angle) were plotted graphically by G.N.Ramachandran (Fig. 4.15).
4.3.3.3 Structure of Protein
The structure of proteins can be described at four different levels (Fig. 4.16). They are:
 Primary Structure: Gives descriptive account of sequence of amino acids and all the
covalent bonds (peptide bonds, disulphide bonds) linking the various amino acids in
the polypeptide chain.

Fig. 4.14 The peptide bond is planar and has a partial double bond character that
imparts rigidity to it. Rotation is allowed around the N-Cα bond (phi angle) and Cα-C
bond (psi angle)

(a) Ramachandran Diagram for Helices. Both (b) Ramachandran Diagram for β
roght and left handed helices lie in regions of strands. The red area shows the sterically
allowed conformations in the Ramachandran allowed conformations of extended, β-
Diagram. However, essentially all α-helices strand-like structure.
in protiens are right-handed.
Fig.4.15 Ramachandran Plot showing permissible phi and psi angles for variety of
structures
 Secondary Structure: Structural patterns made from the arrangements of amino acid
residues. Secondary structure, found in most proteins, consists of coils and folds in the
polypeptide chain.
 Tertiary Structure: Three dimensional folding of the protein and structure is
determined by interactions among various side chains (R groups).

 Quaternary Structure: This takes into account the spatial arrangement of subunits and
structure results when a protein consists of multiple polypeptide chains.
Fig. 4.16 Levels of structure in proteins
A. Primary structure
The primary structure of a protein is its unique sequence of amino acids. Each peptide or
protein has its own unique sequence of amino acids. As with naming of peptides, the
assignment of positions of the amino acids in the sequence starts at the N-terminal end. For
example, In 1953, first amino acid sequence of a protein, bovine insulin ((Insulin is necessary
for proper utilization of carbohydrates, and people with severe diabetes must take insulin

injections) was elucidated by Frederick Sanger. Bovine insulin is 51 amino acids long and is
composed of two polypeptide chains: A (21 amino acids) and B (30 amino acids) joined to
each other by intra- and inter-chain disulphide bonds (Fig. 4.17).
Fig.4.17 Primary Structure of bovine insulin
B. Secondary structure
Structural patterns made from the arrangements of amino acid residues are termed as the
secondary structure of a protein. α-helix and β-sheets are two prominent secondary structures
that occur in proteins (Fig. 4.18). Those protein conformations that do not exhibit a repeated
pattern are called random coils.
i) α-Helix exists in hair protein, keratin. Various conformations can be assumed for a
protein by rotation around single bonds and rigid peptide bonds. The simplest
arrangement of a polypeptide chain is α-helix. The polypeptide coils around an
imaginary axis with side groups protruding out from the helix. The single turn of the
helix is 5.4 Angstrom which is the repeating unit of the α-helix.
Intra-hydrogen bonding stabilizes the α-helix. This bond forms between the first
amino acid and the fourth amino acid. The charge of the side chains can destabilize
the helix. Adjacent Glu, Arg or Lys which are charged at neutral pH impede the
formation of α-helix. Similarly, Pro with ring structure introduces a kink in the helix
and destabilizes it. In the a-helix form, a single protein chain twists in such a manner

that its shape resembles a right-handed coiled spring-that is, a helix. The shape of the
helix is maintained by numerous intramolecular hydrogen bonds that exist between
the backbone –C=O and H-N- groups. Each –N-H points upward and each C=O
points downward, roughly parallel to the axis of the helix. All the amino acid side
chains point outward from the helix.
ii) The β-sheet is zig-zag extended conformation of a polypeptide. The intra molecular
hydrogen bonding is formed between adjacent segments of a polypeptide. The
adjacent segments can be in parallel or antiparallel orientation. Apart from β-sheets,
β-turns are important components of the protein structure that connect the two
adjacent segments of the antiparallel β sheet by hydrogen bonding between first and
fourth amino acids.
Fig. 4.18 Enzyme carboxypeptidase. The β-pleated sheet portions are shown in blue, the
green structures are the a-helix portions, and the orange strings are the random coil
areas

The other important orderly structure in proteins is the β-pleated sheet. In this case,
the orderly alignment of protein chains is maintained by intermolecular or
intramolecular hydrogen bonds. The β-sheet structure can occur between molecules
when polypeptide chains run parallel (all N-terminal ends on one side) or antiparallel
(neighboring N-terminal ends on opposite sides). β-Pleated sheets can also occur
intramolecularly, when the polypeptide chain makes a U-turn, forming a hairpin
structure, and the pleated sheet is antiparallel. The other important orderly structure in
proteins is the β-pleated sheet. In this case, the orderly alignment of protein chains is
maintained by intermolecular or intramolecular hydrogen bonds. In all secondary
structures, the hydrogen bonding is between backbone –C=O and H-N-groups.
C. Tertiary structure
The three dimensional arrangement of all atoms in a protein is termed as the tertiary structure.
When the polypeptides fold in spherical shape, they are called globular proteins while fibrous
proteins have extended conformation (Fig. 4.19)
Fig. 4.19 A fibrous and a globular protein

The tertiary structure of a protein is the 3D-arrangement of every atom in the molecule.
Unlike the secondary structure, it includes interactions of the side chains, and not just the
peptide backbone (Fig. 4.20). In general, tertiary structures are stabilized by five ways.
i) Covalent bond:
The covalent bond most often involved in stabilization of the tertiary structure of
proteins is the disulfide bond.

ii) Hydrogen bonding:
Tertiary structures are stabilized by hydrogen bonding between polar groups on side
chains or between side chains and the peptide backbone.
iii) Salt bridge (electrostatic interaction):
Salt bridges, also called electrostatic attractions, occur between two amino acids with
ionized side chains
iv) Hydrophobic interaction:
The nonpolar groups prefer to interact with each other, excluding water from inward
regions.
v) Metal ion coordination:
Two side chains with the same charge would normally repel each other, but they can
also be linked via a metal ion.
Forces that stabilize the tertiary structures of proteins. The helical structure and the sheet
structure are two kinds of backbone hydrogen bonding. Although the backbone hydrogen
bonding is part of the secondary structure, the conformation of the backbone puts constraints
on the possible arrangement of the side chains.
Fig.4.20 Forces that stabilize the tertiary structures of proteins

D. Quaternary structure
Quaternary structure results when two or more polypeptide chains form one macromolecule.
The highest level of protein organization is the quaternary structure, which applies to proteins
with more than one polypeptide chain. The subunits are packed and held together by
hydrogen bonds, salt bridges, and hydrophobic interactions-the same forces that operate
within tertiary structures.
Hemoglobin in adult humans is made of four chains (called globins): two identical α-chains
of 141 amino acid residues each and two identical β-chains of 146 residues each.
Hemoglobin, the oxygen carrier of blood, has a quaternary structure of 4 polypeptides: 2α
and 2β and hence α2β2. α and β polypeptides resemble the myoglobin structure. In
hemoglobin, each globin chain surrounds an iron-containing heme unit. Proteins that contain
non-amino acid portions are called conjugated proteins. The non-amino acid portion of a
conjugated protein is called a prosthetic group. In hemoglobin, the globins are the amino acid
portions and the heme units are the prosthetic groups (Fig. 4.21).
Fig. 4.21 Quaternary structure of hemoglobin
4.3.3.4. Confirmation and stability of protien
The arrangement of atoms in a protein is called its conformation. A conformational change is

an outcome of rotation around the single bond and without breaking any covalent bonds. The
most stable conformation of a protein under given set of conditions is the conformation that is

thermodynamically most stable, having lowest Gibb’s free energy (G). Multiple ‘most stable
conformations’ exist for a protein defined by the set of conditions such as binding of ligand.
 Native conformation/state of a protein is the one which is most stable form under
given set of conditions.
 Stability of a protein is defined as the tendency to maintain a native
state/conformation.
Different conformations assumed by the protein have different degrees of conformational

entropy. The unfolded state has the maximum entropy. Disulphide bonds and multiple weak
interactions such as hydrophobic interactions, ionic interactions stabilize the native
conformation of a protein. Protein folds in such a way that the hydrophobic residues are
buried inside the core and the polar residues are towards the exterior of the protein. Thus the
protein interior has many such hydrophobic interactions. The aqueous milieu around the
protein forms a solvation shell or layer around it largely defined by the hydrogen bonds that
the protein makes with surrounding water. Largely, the proteins fold burying nearly all the
hydrophobic interactions inside the core while the polar residues tend to stay on the surface
(Fig. 4.22).
Fig. 4.22 Different interactions stabilizing the native conformation of protein

4.3.3.5. Denaturation of Proteins
Denaturation is the loss of the secondary, tertiary, and quaternary structures of a protein by a
chemical or physical agent that leaves the primary structure intact. For example, heat cleaves
hydrogen bonds, so boiling a protein solution destroys the a-helical and β-pleated sheet
structure. In globular proteins heat causes the unfolding of the polypeptide chains; because of
subsequent intermolecular protein–protein interactions, precipitation or coagulation then
takes place. That is what happens when we boil an egg.
Denaturation changes secondary, tertiary, and quaternary structures. It does not affect
primary structures (that is, the sequence of amino acids that make up the chain). If these
changes occur to a small extent, denaturation can be reversed. For example, when we remove
a denatured protein from a urea solution and put it back into water, it often reassumes its
secondary and tertiary structures. This process is called reversible denaturation. In living
cells, some denaturation caused by heat can be reversed by chaperones. These proteins help a
partially heat-denatured protein to regain its native secondary, tertiary, and quaternary
structures. Some denaturation, however, is irreversible. We cannot unboil a hard-boiled egg.
Heavy metal ions (for example, Pb2+, Hg2+, and Cd2+) also denature protein by attacking
the -SH groups. They form salt bridges, as in –S- Hg2+ -S-. This feature is taken in advance

in the antidote for heavy metal poisoning: raw egg whites and milk. The egg and milk
proteins are denatured by the metal ions, forming insoluble precipitates in the stomach. These
must be pumped out or removed by inducing vomiting.
Other chemical agents such as alcohol also denature proteins, coagulating them. This process
is used in sterilizing the skin before injections. At a concentration of 70%, ethanol penetrates
bacteria and kills them by coagulating their proteins, whereas 95% alcohol denatures only
surface proteins.
4.3.4 Nucleic acid
Nucleic acids are biopolymers, macromolecules, essential to all known forms of life. Nucleic
acid, naturally occurring chemical compound that is capable of being broken down to
yield phosphoric acid, sugars, and a mixture of organic bases (purines and pyrimidines).
Nucleic acids are the main information-carrying molecules of the cell, and, by directing the
process of protein synthesis, they determine the inherited characteristics of every living thing.
They are composed of nucleotides, which are the monomers made of three components: a 5-
carbon sugar, a phosphate group and a nitrogenous base. The two main classes of nucleic
acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA is the master
blueprint for life and constitutes the genetic material in all free-living organisms and most
viruses. RNA is the genetic material of certain viruses, but it is also found in all living cells,
where it plays an important role in certain processes such as the making of proteins.
4.3.4.1 Chemistry of nucleic acids:
Nucleic acids are polynucleotides-that is, long chainlike molecules composed of a series of
nearly identical building blocks called nucleotides. Each nucleotide consists of a nitrogen-
containing aromatic base attached to a pentose (five-carbon) sugar, which is in turn attached
to a phosphate group. Each nucleic acid contains four of five possible nitrogen-
containing bases: adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U). A and
G are categorized as purines, and C, T, and U are collectively called pyrimidines. All nucleic
acids contain the bases A, C, and G; T, however, is found only in DNA, while U is found in
RNA. The pentose sugar in DNA (2′-deoxyribose) differs from the sugar in RNA (ribose) by
the absence of a hydroxyl group (―OH) on the 2′ carbon of the sugar ring. Without an
attached phosphate group, the sugar attached to one of the bases is known as a nucleoside.
The phosphate group connects successive sugar residues by bridging the 5′-hydroxyl group

on one sugar to the 3′-hydroxyl group of the next sugar in the chain. These nucleoside
linkages are called phosphodiester bonds and are the same in RNA and DNA.
A. Nitrogenous Bases
Nucleotides are comprised of single-ringed or two-ringed nitrogenous bases. One-ringed

nitrogenous base is called the pyrimidine and the two-ringed nitrogenous base is called the
purine. Basic structure of purine or pyrimidine is shown in (Fig. 4.23). Pyrimidines are six-
membered aromatic rings containing two N-atoms. Purines’ structure is a combination of
pyrimidine ring and a five membered imidazole ring.
Fig. 4.23 Basic structure of purine or pyrimidine

 The Pyrimidines
The pyrimidine bases are of three kinds: Cytosine (C), Uracil (U) and Thymine (T).
Cytosine and Thymine are found in DNA while Cytosine and Uracil are found in
RNA. Their structures are shown in Fig. 4.24.
 The Purines
Two kinds of purine bases exist in nucleotides: Adenine (A) and Guanine (G). Both
are found in DNA as well as RNA.
Fig. 4.24 Chemical structure of purine and pyrimidine bases

B. Pentoses
Pentoses are the five-carbon sugars present in polynucleotides. RNA contains D-Ribose while
DNA contains 2’-deoxy-D-ribose (Fig. 4.25). The hydroxyl group at the 2’-position is not
present in DNA, rather a H atom is present there. We shall see in later sections how this
minor difference has far-reaching consequences on their properties.
Fig.4.25 The structure of pentoses: (a) D-Ribose and (b) 2’-deoxy D-Ribose
C. Nucleoside
Nucleosides are formed by joining a sugar to a nitrogenous base via a β-N-glycosidic linkage.
If the nitrogenous base is a purine, the nitrogenous base is linked to the sugar via its N-9
atom, while if it’s a pyrimidine, it is linked via its N-1 atom. Nucleoside with purine as base
are suffixed with ‘osine’.
For example, adenosine and guanosine. While nucleoside with pyrimidine as bases are
suffixed with ‘idine’. For example, cytidine, uridine, thymidine. If the sugar of the
nucleoside is 2-deoxy ribose, the nucleosides are named as deoxyribonucleosides-
deoxyadenosine, deoxyguanosine etc. Fig. 4.26 shows the structures of all ribonucleosides
and deoxyribonucleosides.
4.3.4.2 Secondary structure of DNA

We learnt about the base composition of nucleic acids in module 5. DNA comprises of
deoxyribonucleotides containing bases adenine, guanine, cytosine and thymine, while RNA
comprises of ribonucleotides with uracil instead of thymine, adenine, cytosine and guanine.
RNA usually exists in a single stranded form while DNA exists in double stranded form. In
double stranded DNA, adenine always pairs with thymine and guanine always pairs with
cytosine. DNA can exists in different forms inside a cell. They are A, B and Z- forms of
DNA defined by different humidity conditions.

A. Deoxyribonucleic acid (DNA)
DNA is a polymer of the four nucleotides A, C, G, and T, which are joined through
a backbone of alternating phosphate and deoxyribose sugar residues. These nitrogen-
containing bases occur in complementary pairs as determined by their ability to form
hydrogen bonds between them. A always pairs with T through two hydrogen bonds, and G
always pairs with C through three hydrogen bonds. The spans of A:T and G:C hydrogen-
bonded pairs are nearly identical, allowing them to bridge the sugar-phosphate chains
uniformly. This structure, along with the molecule’s chemical stability, makes DNA the ideal
genetic material. The bonding between complementary bases also provides a mechanism for
the replication of DNA and the transmission of genetic information.

Fig. 4.26 The structure and nomenclature of ribonucleosides and deoxyribonucleosides
In 1953 James D. Watson and Francis H.C. Crick proposed a three-dimensional structure for
DNA based on low-resolution X-ray crystallographic data and on Erwin Chargaff’s
observation that, in naturally occurring DNA, the amount of T equals the amount of A and
the amount of G equals the amount of C. Watson and Crick, who shared a Nobel Prize in
1962 for their efforts, postulated that two strands of polynucleotides coil around each other,
forming a double helix. The two strands, though identical, run in opposite directions as
determined by the orientation of the 5′ to 3′ phosphodiester bond. The sugar-phosphate chains
run along the outside of the helix, and the bases lie on the inside, where they are linked
to complementary bases on the other strand through hydrogen bonds (Fig. 4.27).

Fig. 4.27 DNA structure
B. Ribonucleic acid (RNA)
RNA is a single-stranded nucleic acid polymer of the four nucleotides A, C, G, and U joined
through a backbone of alternating phosphate and ribose sugar residues (Fig. 4.28). It is the
first intermediate in converting the information from DNA into proteins essential for the
working of a cell. Some RNAs also serve direct roles in cellular metabolism. RNA is made
by copying the base sequence of a section of double-stranded DNA, called a gene, into a
piece of single-stranded nucleic acid.
Whereas DNA provides the genetic information for the cell and is inherently quite stable,
RNA has many roles and is much more reactive chemically. RNA is sensitive to oxidizing
agents such as periodate that lead to opening of the 3′-terminal ribose ring. The 2′-hydroxyl
group on the ribose ring is a major cause of instability in RNA, because the presence
of alkali leads to rapid cleavage of the phosphodiester bond linking ribose and phosphate
groups. In general, this instability is not a significant problem for the cell, because RNA is
constantly being synthesized and degraded.

Fig. 4.28 Camporaive structure of DNA and RNA
In a cell, RNA exists in three predominant forms:
(a) messenger RNA (mRNA)
As the name suggests, this RNA is the messenger that carries the information of the DNA to
protein synthesis machinery. Protein synthesis machinery translates this information into
sequence of polypeptides.
(b) transfer RNA (tRNA)
It is smallest RNA in the cell (~76 amino acids). tRNA serves as a carrier of amino acids
(monomers for protein synthesis). Though it is also single stranded but it forms characteristic
secondary structure by virtue of intrastrand hydrogen bonding.
(c) Ribosomal RNA (rRNA)
rRNA forms the part of ribosomes. Ribosomes, the protein synthesis machinery, comprises
one-third proteins and two-third rRNA. rRNA is the most abundant RNA in the cell. They

participate in the formation of peptide bonds (bonds that link any two amino acids in a
protein chain), hence these are called Ribozymes or RNA enzymes.
4.3.4.3 Chemical properties of DNA and RNA
Two major differences between DNA and RNA are:
(i) Presence of Uracil in RNA versus Thymine in DNA.
(ii) Presence of 2’-deoxy ribose in DNA while RNA contains ribose.
These chemical properties render stability of DNA making it more stable storage form of
genetic information. 2’-OH of ribose in RNA makes it less stable than DNA. The vicinal
hydroxyl group next to 3’-OH (i.e. 2’-OH) is absent in DNA rendering it more resistant to
hydrolysis by alkali. This vicinal hydroxyl group in RNA makes the phosphodiester bond
susceptible to nucleophilic cleavage.
Why does DNA contain thymine instead of uracil?
Cytosine deaminates to uracil in vivo. Cytosine base pairs with G, while U base pairs with A.
Thus this deamination leads to a mutation leading to conversion of CG base pair to UA base
pair. Thus U in DNA is a outcome of domination and is sensed as mutation by the repair. If U
would have been naturally found in the DNA, the repair system of the cell would have been
unable to sense U formed from deamination of C.
4.4 PROXIMITY EFFECT

An enzyme has to form an enzyme-substrate complex with its substrate in order to catalyse a
chemical reaction (Fig. 4.29). The enzyme stabilizes the reaction’s transition state, making it
easier for the bound substrate to form the transition state and convert to product. The
resulting enzyme-product complex then dissociates, releasing free product and regenerating
the free enzyme, which can carry out subsequent rounds of catalysis. The reactive chemical
groups may be aligned and brought together in the ideal orientation and spatial connection for
a reaction to occur through proximity in enzyme-substrate interactions. The enzymatic
reaction acts kinetically like an intramolecular process once the substrates have been fixed in
this manner. According to certain theories, molecules are most reactive when their orbitals
are arranged in a way that reduces the electronic energy of the transition state.

Fig.4.29 Enzymes bind to their substrates to catalyze chemical reactions
The proximity effect, describe the orientation and movement of the substrate molecules
within the active site of the enzyme such that the reactants are much closer together than they
would be in solution. Enhancing the proximity of reactants increases their collision
frequency, thus causing the reaction to proceed at a faster rate. This effect of proximity and
orientation is analogous to an effective increase in concentration of the reagents and endows
the reaction an intramolecular character with a massive rate increase. Intramolecular reactions
between groups that are tied together in a single molecule are faster than the corresponding
intermolecular reactions between two independent molecules.
As an example of the proximity effect in catalysis, consider the rates of the two hypothetical
reactions shown in (Fig.4.30). The reaction at the top relies on the random collision between
the two substrates to bring A and B close enough to react. In contrast, it is much more likely
for A and B to encounter each other in the reaction at the bottom when they are already
tethered together.
Fig.4.30 The proximity effect increase collision frequency

Proximity effect lower the entropic barrier to forming the transition state (ΔS ‡) because they
pre-organize the substrates so that they lose less entropy during the formation of the transition
state than the free substrates would. We can quantify the proximity effect using the effective
concentration of the reactants in the reactions. The effective concentration is defined as the
ratio of the rate constant for the intramolecular reaction (with units of s -1) divided by the rate
constant for the intermolecular reaction (with units of s -1M-1). Effective concentration has
units of molarity (M) and is a measure of the concentration of a reactant you would have to
have in an intermolecular reaction to achieve the same rate as that in the intramolecular
reaction with a substrate concentration of 1M. In the example shown in Figure 4.31, the rate
constant for the intermolecular reaction (top) is k inter = 4 x 10-6 s-1M-1; in contrast, the rate
constant of the intramolecular reaction (bottom) is k intra = 0.8 s-1. The effective concentration
of A and B in the bottom case is kintra/kinter, or 200,000 M. In other words, A and B when
tethered in close proximity and in the proper orientation by an enzyme react at the same rate
as that of the untethered reaction when A or B is an impossibly high 200,000 M.
Fig.4.31 Effective concentration measures the rate enhancement of an intramolecular

reaction relative to a corresponding intermolecular reaction

4.5 MOLECULAR ADAPTATION

The shape of the molecule determines whether like a bioactive molecule it can also be
recognized by a receptor and thus will display the same biological activity. Compounds with
similar structures may compete for the same biological target. This information can be highly
useful in designing new drugs via molecular modification. Examples are presented to explain
these facts.
1. All living systems use nucleic acids (DNA and RNA) to store genetic information.
Thus, any compound which interferes with the synthesis of these vital materials is
toxic to all life forms. These toxic compounds are called antimetabolites and 5-
fluorouracil (5-Fu) which interferes with the synthesis of DNA is one antimetobolite.
This derivative of the natural base uracil inhibits the enzyme which converts uracil to
thymine via methylation. Notice that thymine and uracil differ only by a methyl
group: thymine is 5-methyluracil (Fig. 4.32) one knows that fluorine and hydrogen are
almost similar in size. Thus one can say that 5-fluorouracil being similar in shape and
size with uracil is adapted by the enzyme and subsequently inhibits it, this synthesis
of dTMP is inhibited and this in turn inhibts DNA synthesis.
Fig. 4.32 Chemical structure of natural base and its derivatives

2. A common antibiotic used against bacterial infections is 5-fluorocytosine which is an
analog of natural base cytosine (Fig. 4.33). A new approach is to disguise the drug
chemically so that it can enter the bacterial cell and destroy it. This way the drug will
not harm the tissues of the patient. In the modern approach, to the amino group of the
drug e.g 5-fluorocytosine a small peptide is attached containing D-amino acids (thus
common human enzymes cannot bring about its hydrolysis). This peptide containing
drug thus can sneak into the bacterial cell, where it is metabolized to liberate the drug.

Fig. 4.33 Chemical structure of natural base and its derivatives
4.6 SUMMARY
 Carbohydrates are poly hydroxy aldehydes and ketones.
 When sugar cyclizes and anomeric carbon is created from aldehyde group of an
aldose or a keto group of ketose. This carbon has two configuration α or β, if the
oxygen on anomeric is not attached to any other structure.
 Nucleotides are the monomers that make the polymer DNA or RNA.
 Nucleotides are comprised of a nitrogenous base, a pentose sugar and phosphoric
acid.
 Nitrogenous bases can be two ringed purines or single ringed pyrimidines.
 DNA is double stranded with interstrand hydrogen bonding between adenine and
thymine pair and guanine and cytosine pair that keeps the two strands together.
 Protein structure comprises of four levels: Primary Structure, Secondary structure,
tertiary structure and quaternary structure.
 α-Helix and β-sheets are two predominant secondary structures.
 Tertiary structure is the spatial arrangement of atoms in space while different
polypeptides join to form the quaternary structure of a protein.
4.7 BIOBLIOGRAPHY
 Paula and Bruice, Organic Chemistry, fourth edition.
 Nelson and Cox, Lehninger, Principles of biochemistry, Fourth Edition
 Kalsi P.S., Kalsi J.P.(2006), Bioorganic, bioinorganic and supramoleculer chemistry,

new age international (P) Ltd. Publishers, london, new delhi, nairobi..

 Dr. Sumanta Mondal_B. Pharm V Sem_Applied Biochemistry (PPH 305) _GITAM

University
 e-Pathshala, Bio-organic and Biophysical Chemistry, Paper No. : 16; Module No.: 7;
Protein Structure.
 e-Pathshala, Bioorganic and Biophysical chemistry, Paper No. : 16; Module No.: 5,
Nucleotides and polynucleotides
 NPTEL - Chemistry - Bio-Organic Chemistry, Joint initiative of IITs and IISc -

Funded by MHRD,
 e-Pathshala , Bio-organic and Bio-physical Chemistry, Paper No. : 16; Module No.:
2: Constituents of Cells; Lipids
 e-Pathshala, Bio-organic and Bio-physical Chemistry, Paper No. : 16; Module No.: 6
: Structure of DNA
 e-Pathshala, Bio-organic and Bio-physical Chemistry, Paper No. : 16; Module No.:.3:
Sugars and polysaccharides
 https://projects.iq.harvard.edu/files/lifesciences1abookv1/files/7_dna_structure_chemi
stry_revised_9-24-2018.pdf
4.8 SUGGESTED READINGS

 Nelson and Cox, Lehninger, Principles of Biochemistry, Fourth Edition
 Bioorganic Chemistry; H. Dugas, Springer Verlag, 1999.
 Tyagi V., Tyagi S., (2002), Bioorganic chemistry, Krishna prakashan media meerut,
india,1-302

1. Discuss the different types of protien structure.
2. Write a note on the structure of DNA.
3. Differentiate between anomer and epimer.
4. Write a note on the mutarotation.
5. Wwrite the chemical structure of pyrimidine and purine base.
6. Write a note on the peptide bond and Ramachandran plot.

UNIT 5: ENZYME & MECHANISM OF ENZYME

ACTION
CONTENTS:
5.1 Introduction
5.2 Objective
5.3 Chemical and biological catalysis- transition state theory
5.3.1 How catalysts lower energy of activation-transition state theory
5.3.2 Active site of an enzyme- catalytic power of enzymes
5.4 Remarkable properties of enzymes as catalyst
5.5 Classification and nomenclature
5.6 Extraction and purification of enzymes
5.7 Enzyme mechanism
5.7.1 Fisher's lock & key hypothesis
5.7.2 Koshland's induced fit hypothesis
5.8 Concept and identification of active (enzyme inhibition-reversible and irreversible)
5.9 Kinetics of enzyme action
5.10 Allosteric enzymes
5.11 Enzyme modification by site-directed mutagenesis
5.12 Isoenzymes
5.13 Transition-state theory
5.14 Mechenism of action of enzyme carboxypeptidase a
5.15 lysozyme
5.16 Chymotripsin
5.17 Terminal Questions
5.18 Bibliography

5.1 INTRODUCTION
“Enzymes can be defined as biological polymers that catalyze biochemical reactions.”
Perhaps oldest known to bio-chemical as well as bio-organic phenomenon is fermentation

of juices to alcoholic beverages. Also, this was I st chemical transformation catalysed by
enzymes contained within living Yeast cells. It was discovered in 18th century that
fermentation leads to conversion of Sugars into carbon dioxide and alcohol. The
19thcentury witnessed both the identification of fermentation as physiological act of yeast
cells and introduction of forces review Pasteur’s view that life and fermentation are
inseparable.By now, extraction of enzymes from bio-cells was known and this Discovery
like most scientific discoveries was accidental. In 1897, E. Buchner required a quantity of
purified protein for therapeutic purpose. He grounded yeast and sand, filtered the broken
cells, and add added a large amount of sugar to the filtrate as preservative. Enzymes are
biochemical catalyst (biocatalysts) which are needed in almost all the biochemical reaction
in a living system. Enzymes are proteins in nature, which can be defined as biocatalysts
synthesized by living cell. Kuhne used the word enzyme, which is made up of two words
Greek-en: in and zyme: yeast.Berzelius coined the term catalysis, which means to dissolve
(Greek: to dissolve), in 1836, and was capable of catalysing the fermentation reaction.
5.2 OBJECTIVE
After studying this unit, you shall be able to know
 What is Enzyme? How it behaves as catalyst for biological reactions?
 Biological catalyst (Enzyme) is similar to chemical reactions wherein the rate of

reaction is proportional to the concentration of reacting substrates.
 The rate of an enzyme catalysed reaction or its activity can be measured by a variety
of sensitive assays.
 Classification of catalysts.
 Role of enzyme in biological reaction
 Extraction and purification of enzyme
 Different modules for biological reaction mechanism.

 Role of inhibitor in catalytic reaction.We shall be Understand about Irreversible
Inhibition and Reversible Inhibition.
 Mechanism of different biological catalysts.
1.3 CHEMICAL AND BIOLOGICAL CATALYSIS-

TRANSITION STATE THEORY
A catalyst age an agent which enhance the rate of the chemical reaction and it itself is not
consumed. In the case of biological system, a catalyst e.g. activation energy provides a more
favourable pathway for an organic reaction and acts to lowering of the activation energy and
to stabilize the transition state. The difference in energy between the transition state and
starting materials is termed activation energy. The activation energy determines the rate of
reaction rather than the change in energy between the starting materials and the product.
Fig 5.1 Lowering of activation energy in the presence of the catalyst
5.3.1 How catalysts lower energy of activation-transition state theory
Consider the hydrogenation of 1, 2-dimethylecyclohexene to cis-1,2-dimethylecyclohexane.

This catalyst brings about this transformation and several factors are in operation:
 Catalysts deliver a reaction surface or environment
 Catalysts passage reactants together.
 Catalysts help to position reactants correctly so that transition state configurations,

are attained easily.
 It stabilizes the transition state.

 Catalysts weaken bonds.
 Catalysts may contribute in the mechanism
Fig. 5.2 Catalytic hydrogenation: synaddition with palladium catalyst
Fig. 5.3 Cells and enzymes

These factors are seen in the hydrogenation process of the alkene. We know that the metal
surface (Pd) provide electron to hydrogen gas and form metal-hydrogen bond. Therefore,
hydrogen gas dissociated into atoms. When H2 gas interact with metal surface hydrogen
molecule bonds are weaken.
5.3.2 Active site of an enzyme- catalytic power of enzymes
We know that enzymes display their remarkable feat of catalysis by presenting their three-
dimensional environment (made out of L-amino acids) to the substrates. The active sites of
an enzyme i.e., the functional portion of an enzyme occupies only a very small portion of
the enzyme molecule. The active sides site of enzyme may be shown in an overall fashion
by either of the representations. When a substrate binds to an enzyme, the enzyme changes
the substrate's structure, causing it to move toward the transition state. An enzyme's active
site usually has a form that closely resembles the transition state rather than the
substrate.For this region transition state analogues are potent inhibitors.
 In the tertiary structure of protein enzyme (folding of enzyme), the active sites
(amino acid residues) are relatively nearer, whereas in the primary structure the
active sites are far from each other. These amino acid residues are sown by the
letters A, B, and C.
 Catalysis takes place in the active site of every enzyme molecule. The binding site is
located within the active site, and it is here that the amino acid residues (R-groups)
bind the substrate in the correct location for reaction. In an intramolecular reaction,
this is analogous to the appropriate location of the reactive group. Hydrogen
bonding, electrostatic interactions, and other factors all play a role in this binding.
These favourable interactions with amino acid residues in the active site stabilise the
transition state, lowering the active activation energy of the process and increasing
the rate of reaction.
 There is also a pocket (P) in the active site which e.g., in the case of
carboxypeptidase can accept the side chain of terminal amino acid when this enzyme
splits the terminal amino acid from a peptide chain.
 Cofactors are non-protein molecules that many enzymes require for the reaction to
take place. These cofactors are either can metal ion, small organic molecule called

coenzyme (NAD+, NADP+). Most of the coenzyme are bound by ionic bond or other
non-covalent bonding interactions however, others are bound covalently and are
termed prosthetic groups.In carboxypeptidase A zinc ion is essential for enzymatic
activity. Zinc Ion located in a groove near the surface of the enzyme and held in
position via coordinate to two histidine side chains, a glutamate side chain, and
water molecule.
 After the substrate is properly bound in the active site of the enzyme, the R group of
other amino acids brings about the catalytic activity.
5.4 REMARKABLE PROPERTIES OF ENZYMES AS CATALYST

 Enzymes are highly effective catalysts and enhance the reaction rate by a factor of
105-1017.
 Then substrate is tightly bound in the active site to form an ES complex.
 Like other catalysts enzyme lowering the activation energy for a reaction and
enhances the reaction rate. The enzyme has no effect on the reaction's equilibrium.
 The energy used for enzymatic rate enhancement is largely derived from weak
interaction like hydrogen bonding, ionic interactions and hydrophobic interactions.
The active sites provide these interactions and these stabilize the transition state.
 The general acid bas4e catalysis, covalent catalysis and metal ions catalysis help to
provide a lower energy path.
 The binding energy helps to lower the entropy of the substrate and can bring about a
conformational change in the enzyme to bring induced fit.
 Binding energy also justify the enzyme behaves as a catalyst.
5.5 CLASSIFICATION AND NOMENCLATURE

Some enzymes were given names at random, such as trypsin (hydrolases protein) and
pepsins (hydrolyse peptides), but the majority of enzymes are given names by adding the
suffix 'ase' to either the function they perform or 'substrate' on which they work.For
example, transferase, isomerase, hydrase, dehydrogenase, oxidase belongs to the first group

and esterase, protease, urease, and amylase belong to second group. Thus, there are two
ways to classify and name the enzyme:
(A) Based upon the function they perform
(B) Based on substrate they act upon
(I) Classification into hydrolytic and oxidative enzymes:
Enzymes were earlier classified into two groups:
(a) Hydrolytic enzymes
(b) Oxidative enzymes
(a) Hydrolytic enzyme
They catalyse hydrolysis.
Sub-class of this class are:
(1) Proteases
(2) Carbohydrases
(3) Esterases
(4) Amidases
(1) Proteases
These are proteinases and peptidases. Some enzymes of sub-class are:
(i) Trypsin: this enzyme occurs in pancreas, hydrolyses, proteins and polypeptides into
amino acid.
(ii) Pepsin: This occurs in cells and hydrolyses synthetic peptides into proteins and
peptones.
(iii) Rennin: Rennin is found in gastric juice. It is responsible for the clotting of milk and
clots ten-million times of its weight o0f milk and converts casein of milk to paracasein.
(iv) Papain: Papain is found in Carica papaya. It is strong proteolytic action and milk
clotting power.
(2) Carbohydrases:These enzymes act upon carbohydrates. Some of them are following:

(i) Maltase: It is occurring in yeast and hydrolyses maltose into glucose.
(ii) Invertase:It is also found in yeast and hydrolyses sucrose into a mixture of glucose
and fructose.
(iii) Amylase:Amylase found in animals and plants, particularly in saliva and pancreatic
juices. It hydrolyses amylum (starch) into maltose.
(iv) Lactase: Lactase is found in yeast and gastric juices of young animals and hydrolyse
lactose into a mixture of glucose and galactose.
(v) Inulase: This enzyme found in invertebrates and hydrolyse insulin into fructose.
(3) Esterase: it hydrolyses esters. Their examples are following:
(i) Lipases: This enzyme found in pants and pancreas. These hydrolyses fats to fatty
acids and glycerol.
(ii) Phosphatases: This enzyme occurs in animal and plant tissues, it hydrolyses
phosphate ester.
(4) Amidases: These enzymes act on amides.
(i) Ureases: It is found in jack beans, liver and few other seeds. It hydrolyses urea into
other ammonia and carbon dioxide.
(ii) Arginase: It is found in liver cells and hydrolyses arginine into urea and ornithine.
(b) Oxidative enzyme
These enzymes bring about oxidation and reduction in biosystems.
(1) Dehydrogenase
(i) Alcohol dehydrogenase:It is found in the East and oxidizes alcohol to study height
acetaldehyde.
(ii) Glutamic dehydrogenase: It is the source of beef liver. It converts glutamic acid to
Alpha ketoglutaric acid.
(2) Oxidases:
(i) Catalase: It is also found in beef extract and oxidises hydrogen peroxide to oxygen.

(ii) Ascorbic acid oxidase: Plants are its sources and it oxidizes ascorbic acid to
dehydroascorbic acid.
(II) INTERNATIONAL UNION OF BIOCHEMISTS (I.U.B.) CLASSIFICATION
According to the International Union of Biochemists (IUB), enzymes are classified into six
functional classes based on the sort of reaction they catalyse. The six categories of enzymes
are hydrolases, oxidoreductases, lyases, transferases, ligases, and isomerases.
ENZYME
Hydrolases Oxidoreductases Lyases Isomerases Transferases Ligases
Table 5.1: Classification of enzymes and their biochemical property.
S.N. Types Biochemical property
1 Hydrolases Hydrolases are hydrolytic enzymes that catalyse the

hydrolysis reaction by cleaving and hydrolyzing bonds in
the presence of water.
2 Oxidoreductases The enzyme Oxidoreductase catalyses the oxidation

reaction in which electrons tend to move from one form of
a molecule to the other.
3 Lyases Adds water, carbon dioxide or ammonia across double

bonds or eliminate these to create double bonds.
4 Transferases Transferases enzymes aid in the translocation of functional

groups between acceptors and donors.
5 Ligases The Ligases enzymes are known to charge the catalysis of

a ligation process.
6 Isomerases The Isomerases enzymes catalyze the structural shifts

present in a molecule, thus causing the change in the shape
of the molecule.

(1) Hydrolases: These enzymes are hydrolyse C-O, C-N, C-C and some other bonds,
including phosphoric anhydride bonds.
(2) Oxidoreductases: These enzymes are catalyze oxidation and reduction reactions, e.g.
catalysing the oxidation of pyruvate to acetyl coenzyme-A, pyruvate dehydrogenase. The
substrate that is oxidized is regarded as hydrogen donor. The systematic name is based
on donor:acceptor oxidoreductase. The common name will be dehydrogenase, wherever
this is possible; as an alternative, reductase can be used. Oxidase is only used in cases
where O2 is the acceptor.
(i) Oxidases: It passed hydrogen directly to oxygen. In oxidases flavin moiety of

coenzyme FAD acts as hydrogen carrier:
(ii) Dehydrogenases: It catalyse removal of hydrogen from one substrate and transfer it
to other.
Here A and B stand for substrate and E for an enzyme.
Different apoenzymes have different apoenzyme but same co-enzymes.
For instance, Nicotinamide adenine dinucleotide (NAD+)or Nicotinamide adenine

dinucleotide phosphate (NADP+). Nicotinamide acts as hydrogen carrier or oxidising
agent.
(3) Lysases: It is catalysed addition of group to double bond or elimination of groups to

create double bond without oxidation, reduction or hydrolysis.

(4) Transferases: The Enzyme which catalyse to the transfer of a group of atoms, such
as amine, carboxyl, carbonyl, methyl, acyl, glycosyl, and phosphoryl from a donor substrate
to an acceptor compound.
5.6 EXTRACTION AND PURIFICATION OF ENZYMES

(A) Extraction
In the extraction and purification of enzymes, material availability is critical. A single

enzyme's concentration can change between tissues. It is crucial to choose a tissue with a
high enzyme content. As a result, yeast, bacteria, and fungi provide certain advantages as
source materials. The benefit is that these cells can be cultivated in a favourable
environment. However, there is one disadvantage: obtaining large quantities of microbial
cells other than yeast is difficult.
After selecting the starting material, a number of techniques can be used to extract and
isolate the enzyme. The following are some specific examples of various methods:
1. Sedimentation: Many mitochondrial & other particle cell bodies remain constant when
liver tissue is homogenised using the Potter-Elvenhjem apparatus rather than usual blending
devices. They easily sediment out of solution, taking with them a slew of enzymes. Only in
the early stages of separation is physical separation by e sedimentation useful.
2. Extraction: Previously, enzymes were divided into two categories: soluble or

lyoenzyme, and bound or desmoenzyme. Desmonzymes are likely enzymes for which
suitable techniques have yet to be developed, hence this is a bad classification.
Because of its fat-free nature, acetone-powder (from which enzymes can be extracted using
buffer) is often the easiest substance from which to extract enzymes. The initial stage, in
any case, is a fine-grinding. Autolysis, lysozyme digestion, grinding, freezing and thawing,
sonic disintegration, shaking with solvents, shaking with fine-glass beads, and ultimately,
explosion by quick release of pressure is some of the methods for eliminating enzymes from
microorganisms.
3. Salt Fractionation: In enzyme fractionation, ammonium sulphate is the most helpful

salt. Its benefits include high water solubility (760 g/lt) and a nearly neutral response (pH 5
to 6) in concentrated solution. Dixon devised a monogram (chart) for preparing ammonium
sulphate solutions, while Kunits devised an equation for estimating the amount of

ammonium sulphate to add to a solution in order to get the appropriate final concentration.
One downside of using ammonium sulphate in a somewhat alkaline solution is that
50 percent of the ammonium ions are transformed to ammonia, even at pH 9.3. A buffer
should be used to keep the pH of the ammonium sulphate solution under control. However,
sodium sulphate has been widely employed to crystallise glutamic dehydrogenase from cow
liver.
4. Solvent Fractionation: The isolation of enzymes is aided by water-soluble solvents such

as acetone, ethanol, methanol, and dioxane.
When extracting acetone, start below 0 oC and work your way up to higher temperatures.
Fractionation at the maximum temperature will not result in significant yield loss. Because
acetone absorbs strongly in the ultraviolet area, it must be entirely eliminated by dialysis or
distillation at a low pressure before spectral measurement.
The use of ethanol in enzyme isolation is becoming more common. It was used to extract
crystalline lactic dehydrogenase from the liver of rats.
5. Solvent-Metal ion fractionation: Combining metal ions and solvents, notably Zn2+ and
ethanol, is an important approach for separating blood proteins. Protein zinc salts are often
more soluble than sodium and potassium salts and separate out of solutions more quickly.
Metal ions can be extracted from these by using citrate, ethylene diamine tetraacetate, or
ion-exchange resin.
6. Adsorption: Protein adsorbents have been made from a number of materials, with
hydrated aluminum oxide being one of the first. The use of a calcium phosphate gel and
bentonite in the isolation of lysozyme has also proven to be beneficial.
7. Adsorption chromatography: For the separation of proteins and thus enzymes, column
chromatography on adsorbents is particularly successful. A calcium phosphate gel was
developed by Anger. Swingle & Tiselius tested the same adsorbent for generic protein
chromatography. Zechmeister has written a review on the topic of enzyme chromatography
in general. Using a biochemically specific adsorbent, another method isolates enzymes
based on their catalytic specialisation rather than their overall features as proteins. Various
adsorbents containing p-azophenol and similar groups were produced from aromatic ethers
of cellulose, for example, in the isolation of mushroom tyrosinase.

7. Ion-exchange chromatography: The isolation of small molecular weight molecules

like amino acids has proven to be a huge success for this approach.
Cytochrome C was isolated in 1950 using an amberite IRC-50 column and the game
method, which was useful in separating cytochrome from Ustilago. In a similar way,
ribonuclease and lysozyme have been purified. Ion-exchange chromatography is essentially
an electrophoretic separation in which the resin serves as one electrode and gravity as the
other. It may yet prove to be a useful technique as the chemical industry continues to
provide novel resins to enzymologists.
9. Complex Formation: Protamine was used as a complexing agent in this method to a

lower level. To get rid of unwanted proteins, basic lead acetate has been used.
(B) Purification of Enzymes
There are various methods for isolating enzymes from tissues. Acetone powders of tissues
or cells are made by blending the tissue (1 vol) with acetone (5-10 vol) at 0o C in one of the
techniques. To remove room moisture and lipids, the smooth slurry is filtered and rinsed
with acetone many times. It is then dried after being rinsed with ether. A powdery residue is
left behind, which could be a mixture of enzymes. The powdered substance is separated,
and the different fractions are evaluated for catalytic activity in vitro. The fraction with the
required activity is chosen for fractional crystallisation, resulting in the pure state of the
desired enzyme. Chemical or physical fractionation processes are used in the purifying
process.
The goal is to keep the majority of the targeted enzyme while freeing it from other proteins,
nucleic acids, and other contaminants. To remove denatured protein, heat guide the cell free
extract to 500C for 5 minutes. This can be accomplished by precipitating ammonium
sulphate and then using ion exchange chromatographic methods, gel filtering, and so on.
Final purification of enzymes, whether in the laboratory or in commercial operations, is a

time-consuming process that depends mainly on chromatographic technologies. Gel
permeation chromatography & affinity chromatography hold very high promise for
simplifying the purification of enzymes. The latter method entails connecting a crude
enzyme preparation with a solid support to which a reversible inhibitor or other compounds
that will selectively and reversibly bind to the enzyme of interest are attached.The support-

inhibitor-enzyme complex is isolated from the initial crude feed after the enzymes have
been attached to immobilised inhibitors, and the purified enzyme is eluted from the support-
inhibitor part. Because of the lower cost of enzyme purification as well as the bigger
quantity and variety of enzymes, methods such as gel filtering and affinity chromatography,
as well as other chromatographic methods, are appropriate. However, there is more work to
be done in terms of scaling up these technologies. Anaphylactic or precipitation reactions
are also effective in purification. The antigen-antibody reaction is carried out in a gel, such
as agar, in this approach. The chemicals are evident in the gel as a precipitated zone.
5.7 ENZYME MECHANISM
5.7.1 Fisher's lock & key hypothesis
Enzymes are highly specific; therefore, the reasonable question is what is their mechanism
of action. According to Arrhenius, enzymes catalyse the reaction through the formation of
unstable intermediate. Simplest model to explain enzymatic action is Lock & Key model
proposed by Fischer. This model assumes that enzyme is rigid three-dimensional body, the
surface of which has active-sites which have slots for fitting definite substrates just as a key
fit in a particular lock (Fig 5.4).
Fig 5.4 Key lock model reaction
An enzyme molecule is very large (consisting of 100 to 200 amino acid residues); but
active-sites, which combine with substrate have definite shape in which substrate can fix,
are comparatively small (with few amino acid residues). Amino acids of active sites are
located at different places in the chain, whereas, other amino acids, which are not part of

active-sites, are located in a definite sequence. This is because of the fact that this sequence
allows the whole enzyme molecule to fold in exactly required manner.
An hypothetical example of mechanism of enzyme-action is given in above fig 5.4. This is

referred to as a "Lock and Key Mechanism."
An enzyme-substrate complex is formed when the enzyme reacts chemically with the
substrate (Michaelis-Mention hypothesis).
The enzyme-substrate complex then breaks down to give the products of reaction. The
enzyme is released & can be used over and over again.
E + Product
The Lock and Key model explain the action of many enzymes. But of other enzymes, there
is evidence that this model is too restrictive. Enzyme molecules are in dynamic state, not in
static one. There are constant motions within them, so that the active site has some
flexibility.
5.7.2 Koshland's induced fit hypothesis
This model was given by Koshland the Fischer model (1966). In the Fishcher model, i.e.,
Lock and Key model, the active-site is presumed to be preshaped to fit the substrate. In the
induce -fit theory, the substrate induces a conformational change in the enzyme. This aligns
amino acid residue or the other groups on the enzyme in the correct spatial orientation for
substance binding & catalysis both. At the same time, the other amino acid residues may get
buried in the interior of the enzyme. This is shown in the following Fig 5.5.
Fig. 5.5 Representation of an induced fit by conformational enzymes

In fig 5.5 in the absence of substrate, the catalytic and the substrate-binding groups are
several bond distances removed from one another. When the substrate approaches there
occur a conformational change in the enzyme protein, aligning the groups correctly for
binding and for catalysis. At the same time there also occurs a change in the spatial
orientation of the other regions.
Fig. 5.6 Hypothetical representation of alternate pathways of substrate induced

conformational changes.
The main evidence in favour of induced fit model comes from demonstration of
conformational changes during substrate binding & catalysis with creatine kinase,
phosphoglucomutase, and several other enzymes.Upto this time, the exact sequence of
events in a substrate induced conformational change has not been established. There may be
several possibilities as shown in Fig. 5.6. Evenif one knows the complete primary structure
of enzyme, it is not very easy to decide exactly which residues exactlyconstitute

5.8 CONCEPT AND IDENTIFICATION OF ACTIVE (ENZYME

INHIBITION-REVERSIBLE AND IRREVERSIBLE)
The substrate is attached to a specific cavity or site in an enzyme. The cleft has an active
core, which is where the amino acids are clustered together to allow them to mix with
substrate. The reactive amino acids may be separated by a long distance in the polypeptide
chain. The chain, on the other hand, folds in such a way that the reactive amino acids are
brought together in the active site. When the substrate molecule connects to the active site,
it is thought that the Parts are held together in such a way that chemical bonds are distorted,
i.e. the bonds are weakened. The reactivity of the substrate or chemical bonds is increased
as a result of this distortion, which speeds up the reaction rate.The strain model of catalysis
describes how the reaction products are liberated because they are less tightly bound.
Inhibitors are chemicals that slow down the rate of a process catalysed by an enzyme. The
following is a diagram of inhibition:
Fig. 5.7 Inhibition
Enzyme inhibitors are molecular agents, which interferes catalysis, to show or eliminate
enzymatic activity. These compounds have to the ability to combine with certain enzyme
but do not serve as substrate: instead these after or even block enzymatic catalysis. Poison
act upon living bodies by inhibiting enzymes. For instance, Carbon monoxide poisoning by
combining with haemoglobin thus making it useless fort performing its usual role as carrier
of' oxygen. Cyanide poisoning due to its combination with natural substances particularly
with metallic centre of Cytochrome. Poisonous effect of arsenate is due to its blocking of
enzyme sites in place of phosphates. A wide range of naturally occurring and synthetic

compounds has the ability to bind reversible or irreversible to specific enzymes. These
substances fall into two categories:
A) Irreversible Inhibition
B) Reversible Inhibition.
(A) Irreversible Inhibition: These substances are bind tightly to the active of the enzyme
molecule alternating them, so that they permanently lose their catalytic properties. These
inhibitors bind with or destroy the functional groups of the enzyme molecules that are
necessary for their catalytic activities. For example, the compound
diisopropylfluorophosphate (DFP) is irreversible inhibitor, which inhibits the enzyme
acetylcholinesterase, important in the transmission of nerve impulses.
Diisopropylfluorophosphate is a very reactive and combines with the hydroxyl group of an
essential serine residue at the active site of the enzyme to form a catalytically inactive
derivative. Once this derivative formed, the enzyme inhibitor is iodoacetamide, which can
react with sulfhydryl (-SH) groups of essential cysteine residues or with the imidazole group
of essential histidine residues.
(B) Reversible Inhibition
These substances bind less tightly to enzymes, and their inhibiting effect can be reversed.
These are of three main types:
Fig. 5.8 Competitive and Non-competitive inhibitors

(i) Competitive inhibitors
(ii) Uncompetitive inhibitors
(iii) Non-competitive inhibitors
Competitive inhibition: These types of inhibitors compete with the substrate for binding to
the active site of the enzyme, but once bound, cannot be transformed by the enzyme. Their
effect can be reversed or relieved simply by increasing the substrate concentration.
These inhibitors generally resemble the normal substrate in three-dimensional structure. As

a result, the ability of the substrate to bind to the enzyme is reduced. Because of
resemblance, the competitive inhibitor tricks the enzyme into binding to the active site.
However, the inhibitor molecules can not be attacked by the enzyme molecules and since
their active site is occupied.
However, if substrate concentration is increased, the inhibitor molecules are displaced from
the enzyme and enzyme becomes functional. Therefore, a higher concentration than normal
is needed to achieve the same rate of reaction. These types of inhibitors compete with the
substrate for binding to the active site of enzyme, but once bound, cannot be transformed by
the enzyme. Their effect can be reversed or relieved simply by increasing the substrate
concentration.
Fig 5.9 Competitive inhibition transformation
The typical example of competitive inhibition is the inhibition of succinate dehydrogenase

by the malonate anion. Succinate dehydrogenase is involved in citric acid cycle, which
catalyses the removal of two hydrogen atoms from succinate one from each of the two
methylene groups.

Malonate resembles succinate in having two

ionised carboxyl groups at pH7.0, but differs in
having only three carbon atoms. However,
malonate is not dehydrogenated by succinate
dehydrogenase; it simply combines with the
active site and prevents it from acting on its
normal substrate Malonate inhibition is reversible
in nature, as the increase in the succinate
Another common example of this typeconcentration reduces
of inhibition is the extent
the inhibition of inhibition
of ribulose by
biphosphate
a given
carboxylase by oxygen molecule. Carbon concentration
dioxide of malonate.
(CO2) is the normal substrate for ribulose
'biphosphate carboxylase, which is important enzyme during photosynthesis. This enzyme is
competitively inhibited by O2 and even relatively low O2 concentrations reduce the rate at
which CO2 is incorporated into sugars. Competitive inhibitors are not always structure
analogues of substrates.
Competitive inhibitors change Km but not Vmax of enzyme-catalysed reactions because

number of active sites remains unaltered. However, larger the concentration of substrate is
required for the maximum utilization of active sites, this is why Km is increased.
Uncompetitive inhibition: These types of enzyme inhibitors are not very specific and they
bind at a site on the enzyme other than the active site. This binding alters the conformation
of the enzyme molecule so that reversible inactivation of the active site occurs. These
inhibitors bind reversibly to both the free enzyme and the enzyme substrate complex to
form the inactive complexes.

Fig 5.10 Uncompetitive inhibition reaction
The uncompetitive inhibitors are the compounds, which reversibly combine with only
enzyme substrate complex but not with the free enzyme, and this type of inhibition is not
overcome by high concentration of the substrate. The increase of substrate concentration
increases the degree of inhibition, instead of releasing the inhibition. Uncompetitive
inhibitor binds with already formed enzyme-substrate complex and has equal effects both
on Km, and Vmax. This type of inhibition is rare in one-substrate reaction, but causes a type
of product-inhibition in reactions with multiple substrates and products.
Fig 5.11 Un-competitive inhibition
Non-competitive inhibition: These inhibitors are reversible type which is not bind to the
active sites of an enzyme. These are not competed with the substrate for the active site and
do not prevent S from binding to the enzyme. Non-competitive inhibitors bind to the
enzyme and cause a conformational change in it. The enzyme thus becomes inactive. The
inhibitors reduce Vmax for the reaction, but do not change Km.

5.9 KINETICS OF ENZYME ACTION

Enzyme kinetics can be studied in two parts:
(A) Energy of activation
(B) Steady state enzyme kinetics
(A) Energy of activation: Enzymes catalyze the rate of reaction via an alternate pathway
using low energy of activation, enabling reactions to take place under extreme conditions at
suitable temperatures. The energy required to bring the reactants to a transition state in
which new bonds are partially created and old bonds are partially broken. This is the state
with the highest energy during the reaction, therefore it is very unstable and breaks down to
generate lower energy products. Only a small fraction of the reactant molecules have
enough energy to react, i.e. the threshold energy.As the temperature rises, molecules gain
energy equal to the threshold energy. However, in living systems, reactions take happen in a
nearly isothermal environment with very little temperature change. By activating the
reactant molecules, infect enzymes aid in the development of enzyme-substrate complexes.
The enzyme-substrate combination reaches high energy and creates strained bonds in the
reactants, causing them to react quickly. Furthermore, the reactant molecules in the enzyme-
substrate complex are arranged in such a way that their reaction becomes a certainty rather
than a matter of chance, which is why the reaction rate is thousands of times faster.
Fig. 5.12 Effect of temperature increase on fraction with threshold energy.
Alternatively, the energy of activation to reach the transition state (T.S.) is reduced in the
presence of enzyme (Fig 5.12). However, given typical parameters of temperature and

pressure (∆GO) of reaction, the total free energy change remains constant.The enzyme
catalyses both forward and reverse equilibrium reactions to the same amount. Because
(∆GO) has not changed, the equilibrium constant has not changed either, but equilibrium is
rapidly attained. The proportions of reactant and product in catalysed and uncatalyzed
reactions are the same.
Fig 5.13 Lowering of activation energy
(B) Steady state enzyme kinetics: This theory, proposed by Michaelis and Menten in 1913,
is based upon following assumption:
(i) Enzyme-substrate complex (ES) is in equilibrium and substrate
(ii) Product-formation is possible only through enzyme-substrate complex:
Based on those assumptions Michaelis-Menten equation can givenbelow:
Let us consider formation of enzyme substrate complex ES.

d [ES]
 K1[ E ][ S ]..........(1)
dt
It is clear from assumptions (i) and (ii) that clear equilibrium is not in the fast process, as
Enzyme-substrate complex [ESJ is constantly removed in the slow process. Concentration
of enzyme is very concentration of substrates. Therefore, [E]<< [S]. Hence, [ES]<<[S]. the
rate of reaction is given as:
d [S]  d [P]
r   K 2 [ES].............(2)
dt dt
Using steady state approximation for the formation of ES:
d [ES]
 K1[ E ][ S ]  K 1[ES]  K 2 [ES]  0..........(3)
dt
Concentration of free enzymes [E] is not measurable in living process. But total enzyme
concentration [E]0 is measurable and can be given by Equation (4).
[E]0  [E]  [ES]............(4)
Here [ES] is bound enzyme concentration, therefore [E] can be given as
[E]  [E]0  [ES]............(5)
Putting this value in Eq. (3), we get
d [ES]
 K1{[E]0  [ES]}[ S ]  K 1[ES]  K 2 [ES]  0..........(6)
dt
Upon simplification of above equation & grouping the constants
K1[E]0[S ]  {K1  K2  K1[S]}[ES]............. 7 
K1[E]0 [ S ]
[ES]  .......................(8)
K 1  K 2  K1[S]
Upon puttting this value of [ES] in Eq. (2)

K1 K 2 [E]0 [ S ]
r ............(9)
K 1  K 2  K1[S]
K 2 [E]0 [ S ] K 2 [E]0 [ S ]
r  ...................................(10) [upon dividing both numerator and
(K 1  K 2 ) K m  [S]
denominator pf eq.(9) by K1]
K1  [S]
This equation correlates thocomponontg of cnzyrnc reaction, [S]&[E], to initial &

maximum velocity through rate Constant (Km):
(K1  K 2 )
Km 
K1
This equation correlates the components of enzyme reaction, [S] &[E], initial and maximum
velocity through rate constant Km:
Rate of breakdown of ES
Km 
Rate of formation ES
(C) Michaelis-Menten & Lineweaver-Burk Plots
V or (Vmax.) representsmaximumvelocity of enzyme reaction, whereas, Michaelis constant

(Km) is the substrate concentration at which enzyme demonstrates 50% of its maximum
velocity. Michaelis-Mentenequation shows the relation:
K 2 [E]0 [ S ]
r ........(11)
K m  [S]
This equation can be further simplified. When all the enzymes have reacted with substrate
the reaction shows maximum velocity (Vmax). As at that stage no free-enzyme is left [E]0 =
[ES]. Therefore, Equation (2) becomes
rmax  Vmax  K 2 [E]0 .........(12)
Hence, Michaelis-Menten equation (ll) can be written as:
Vmax [S]
r .........(13)
K m  [S]
Now, here are two cases:

(i) If Km >> (S). Then [Sl can be neglected from the denominator. Then
Vmax [S]
r  K ' [S] ..........(14)
Km
This is a first-order reaction,
(ii) If [S] Km. Then, Km can be neglected from denominator;
r  Vmax [S]  Constant .....(15)
This reaction rate follows zero-order kinetics.
1
If K m  [S]; r  Vmax
2
As already stated, Michaelis Constant is equal to concentration of S_at which rate of

formation of product is half of maximum rate. K2of equation (12) is known as turnover-
number of enzyme. It is the number of molecules converted in unit-time by one molecule
of enzyme. It’s value is in between 100-1000 per second. However, sometimes it may be as
large as 105 to 106 per second.
Upon increase in concentration of substrate these active sites get occupied and cause rate
enhancement.However, at high substrate concentration reaction rates become constant as all
the active-sites remain occupied all the time.Michaelis -Menten plot for the kinetics of
enzyme catalysed reaction is given in fig 5.14.
Fig. 5.14 Plot of kinetics of Enzyme-catalysed reaction

Fig. 5.15 Rate of Reaction in presence of inhibitors in Enzyme-catalysed reaction.
Thus, Km is substrate concentration at which half of the active-sites of enzymeare involved

in the formation of substrate enzyme complex. From Michaelis-Menten equation it is
possible to calculate the rate of reaction at any substrate concentration if K m&V are known.
Kinetics of enzyme-action is helpful in understanding metabolic pathways. Determination
of Vmax& also Km directly from the plot of r against [S] is rather difficult. However,
Michaelis-Menten equation can be modified to get plots from which V max can be easily
determined. Two such plots are Lineweaver- Burk plot and Eadic-llofstee plot.
Lineweaver-Burk Equation & plot: Michaelis-Monten equation is
Vmax [S]
r .........(16)
K m  [S]
Taking reciprocal of both sides
1 K m  [S]
 .........(17)
r Vmax [S]
Upon arranging it
1 Km [S]
 + .........(18)
r Vmax [S] Vmax [S]

1 Km 1
 + .........(19)
r Vmax [S] Vmax
Equation (19) is Lineweaver-Burk equation. A plot of 1/r against 1/[S:
Note:
The Lineweaver -Burk plot of enzyme reaction rates is very useful to distinguish between
some types of enzymatic reaction mechanisms and to study enzyme inhibition.
5.10 ALLOSTERIC ENZYMES

When initial velocity of enzyme catalysed reaction (V o) is plotted as a function of
concentration of substrate [S]; curve is not hyperbolic but is sigmoidal. Rate of reaction
given [S] is increased or decreased by the addition of specific substance, i.e., activators or
inhibitors (modulators). Such enzymes are known as allosteric enzyme. Besides substrate
active sites, these enzymes possess other sites in which activators & inhibitors may bind and
affect catalysis through induced conformational change; in the structure of enzyme.
Fig 5.16 Allosteric Inhibition and activation

5.11 ENZYME MODIFICATION BY SITE-DIRECTED

MUTAGENESIS
An important piece of informant between the structure and its function can be derived from
mutagenesis. In this method one amino acid of a protein is replaced with another. When Asp
102 of chymotrypsin is replaced with Asp 102, although the enzyme, ability to bind the
substrate remains unchanged, but its ability to catalyse the reaction decreases to less
than0.05% of its value with the native enzyme. This shows that Asp 102 is involved in the
catalytic process; its negative charge stabilises histidine positive charge.
5.12 ISOENZYMES
Isoenzymes (also known as isozymes) are oligomeric enzymes which catalyze same reaction
but differ in their subunit composition. These differences modify the rate at which molecular
species transform the substrate. Isozymes may be primary or secondary. Primary isozymes
are produced by multiple gene loci which code for distinct protein molecules or are produced
by multiple alleles at a single gene locus. These are also called alloenzymes. Secondary
isozymes are product of post-translational modifications including glycosylation. On account
of their different amino acid compositions primary isozymes may be identified on the basis of
their different electrophoretic mobility. When enzymevariations are within game species they
are known as intra-specific variants. But enzyme variation from different species is called
interspecific or phylogeneticvariant.
Isoenzymes: Lactate dehydrogenase is an oligomeric enzyme in which each sub-unit

performs same function. It catalyses following reaction:

Enzyme assay based on determination of catalytic activity can not distinguish between
isoenzymes. Activity measured will be sum of contributions of active forms of enzymes
being assayed. Even if only one coenzyme is present, its, molar activity, for example, in a
homogenate is not the same as in pure form.
Plasma upon electrophoresis (e.g. on cellulose acetate strips) at pil of 8.6 separates all the
five isoenzymes of LDH. All those isoenzymes may be located by specific stain, e.g. a
mixture of lactate, NAD+ and a chromogen which will give coloured product where LDH is
present to catalyse a step of the reaction An abnormal pattern helps in diagnosis. Separation
may also be achieved by ion-exchange chromatography (for instance, on QAE-Sephadex).
Relative proportions of isoenzyme in plasma can be assessed with their separation on the
basis of their properties. If total LDH activity is higher than normal it can be determined if it
is due to excess of (as in case of heart disease, haematologieal disorder or renal disease) or
duo to excess of M4 (as in case of skeletal muscle of liver disease).
Fig. 5.17 Separation of Isoenzymes of LDII by electrophoresis at a pH of 8.6.

5.13TRANSITION-STATE THEORY
 During enzymatic reactions all reaction groups are brought together at the active site
in the proper position for reaction.
 Some of the amino acid residues serve as catalytic groups, these side chains are in the
proper position relative to the substrate to act as catalysts. This is analogous to the rate
enhancements observed for intermolecular catalysis.
 The conformational change that an enzyme undergoes after binding a substrate can
introduce strain into the substrate, to make it more reactive.
 Groups on the enzyme can stabilize an intermediate and, therefore, the transition state
leading to the intermediate, by van der Waals and electrostatic interactions and by
hydrogen bonding. As a direct result of stabilization of the transition state the
activation energy of the reaction is lowered.
5.13.1 Catalysis
(A) Introduction when the substrate is bound to an enzyme, the catalytic functional groups
which are properly positioned around the substrate in the active site lead to the cleavage and
formation of bonds involving several mechanisms some of these are :
 Metal ion catalysis
 General acid-base catalysis
 Covalent catalysis
Enzymes catalyze reactions using general acid-base catalysis since at physiological pH (pH
= 7.3) only a very small concentration of H+ for specific-acid catalysis or OH- for specific
base catalysis is available.
A. Metal Ion Catalysis: Many enzymes require metal ions for maximum activity. An enzyme
is called a metalloenzyme, if it binds the metal very tightly or requires the metal ion to
maintain its stable, native state. Enzymes which bind metal ions weakly are referred to as
metal activated enzymes. One role for metals in metal activated enzymes and
metalloenzymes is to act as electrophilic catalysts, stabilizing the increased electron density
or negative charge that can develop during reactions.

The ionic interactions between a metal bound to the enzyme and a substrate can favourably
orient the substrate for reaction. These metal ions can also stabilize the charged transition
states.
 Metal ions complex with water and loss of a proton from such a complex gives a
metal bound hydroxide ion which is a better nucleophile compared to water.
 A leaving group can become a weaker base (thus a better leaving group) in the
presence of a metal ion.
 A catalyst must increase the rate of a slow step, since increasing the rate of a fast step
will not increase the rate of the overall reaction
Another potential function of metal ions is to provide a powerful nucleophile at neutral pH.
Coordination to a metal ion can increase the acidity of a nucleophile with an ionizable proton:
The reactivity of the coordinated, de-protonated nucleophile is typically intermediate between

that of the un-ionized and ionized forms of the nucleophile. Carboxypeptidase enzyme
contains an active site Zn2+, which facilitates deprotonation of a water molecule in this
manner.
Liver alcohol dehydrogenase: Enzyme Liver alcohol dehydrogenase catalyses the transfer
of a hydride ion from NADH to acetaldehyde (CH3CH0), forming ethanol (CH3CH2OH). An
active-site zinc ion stabilizes negative charge development on the oxygen atom of
acetaldehyde, leading to an induced partial positive charge on the carbonyl C atom. Transfer
of the negatively charged hydride ion to this carbon forms ethanol.
A. Acid-Base Catalysis
Almost all enzyme catalysed reactions involve some degree of acid or base catalysis. Acid-
base catalysis is two types of:
 The proton must be fully transferred to the reactant before the slow step can
occur in a specific-acid catalysis. H+ or OH- catalysis is a type of acid-base catalysis
in which the reaction is accelerated by H+ or OH-. As a result, a specific acid catalyst
must be a strong acid in order to protonate the reactant before the slow step begins.

 The proton is transported to the reactant during the slow step of the reaction in
general-acid catalysis. The use of an acid or base other than H+ or OH- to accelerate
a process is known as general acid-base catalysis. A general acid is any material that
is weakly ionizable; hence, a general-acid catalyst is a weaker acid since it only
partially transfers a proton in the slow step transition state.
General acid or general base catalysis may increase reaction rates 10-100 times.Specific
and general acid catalysts increase the rate of the reaction in the same way, by donating
a proton to make bond formation and bond breaking easier. These differ as to the extent to
which the proton is transferred in the transition state of the slow step of the reaction. In
general acid catalysed reaction, the transition state has a partially transferred proton while in
specific-acid catalysis the proton must be fully transferred to the reactant before the
beginning of the slow step.
5.14 MECHENISM OF ACTION OF ENZYME

CARBOXYPEPTIDASE A
Carboxypeptidase A is a metalloenzyme in which Zn2+ ion is acting as cofactor for the
hydrolytic cleavage of peptide bonds. A tightly boundZn2+ ion is located in a groove near
the surface of the enzyme and held in its position through coordination with a molecule of
water, Glu72, His69 and His196. The Zn2+ ion increases the reactivity of the bound water
during the reaction as water does not react readily with peptide bonds at neutral pH in the
absence of protease. But in the presence Zn2+ ion water behaves like OH- ion. Many groups

at the active site of the enzyme carboxypeptidase-A bind the substrate in the optimum
position for the hydrolytic cleavage reaction.
1 2
3 4
Arg145 forms two hydrogen bonds with the C-terminal carboxyl group of the substrate. Tyr128
is also involved in hydrogen bonding with the substrate. The non-polar side chain of the
substrate is bound in the hydrophobic pocket lined with non-polar amino acids. The
hydrolytic reaction involves general acid-base mechanism. Glu270acts as a general base
catalyst along with Arg127 and Zn2+.
The first step of the reaction involves the positioning of the phenyl group of the substrate in
the hydrophobic pocket and binding of the C-terminal carboxylic group with Arg145 via
electrostatic interaction as well as hydrogen bonding (1).
The binding of the substrate is made further made stronger by hydrogen bonding of the C-
terminal carboxylic group with Tyr128. Zn2+ ion also partially complexes with amidic

carbonyl group on binding of the substrate in the above manner. Zn2+ ion complexes with
water to increase its acidity and thus water behave as hydroxides. Glu 270 acts as a general
base andwithdraws proton from water which attacks as hydroxide ion on amidic carbonyl
group to give a tetrahedral intermediate which acquires negative charge (2).
This negatively charged tetrahedral intermediate is stabilized by Zn2+ion as well as Arg127.

The tetrahedral intermediate undergoes collapse with Glu 270acting as a general acid catalyst,
the leaving ability of the amino group is increased; the reaction is completed with formation
of a protein with one less amino acid residue (3).
The unfavourable electrostatic interactions between the negatively charged carbonyl group of
the residual peptide product and the negatively charged carboxyl of residue helps in the exit
of the product from the active site of the enzyme (4).
5.15 LYSOZYME
Lysozyme is an enzyme which hydrolyses polysaccharide chains. It breaks certain bacterial
cells by cleaving the polysaccharide chains that build up their cell wall. The most thoroughly
studied form of lysozyme is from hen egg whites. The bacteriolytic properties of hen egg
white lysozyme were first described in 1909 by the Russian scientist P. Laschtchenko. In 1922
Alexander Fleming gave the name lysozyme to the agent in mucus and tears that destroyed
certain bacteria, because it was an enzyme that caused bacterial lysis.

(A) Hydrolysis by lysozyme
The bacterialcellsare surrounded by a rigid, strong wall of peptidoglycan,a copolymer of

twosugar unit. N-acetylmuramic acid (NAM)andN-acetylglucosamine (NAG) and in bacterial
cell wall polysaccharides, they are joined in ß(1-4) glycosidic linkages. Lysozymehydrolyzes
glycosidic bond between C-1 of NAM and C-4 of NAG but does not act on the ß(1-4)
linkages betweenNAG and NAM.
(i) Carbocation mechanism
Glu35 is in a nonpolar or hydrophobic region of the protein, whereas Asp52 is located in a

much more polar environment. Glu35 is protonated, but is ionized (1). In the first step, Glu35
act as a general acid, donating a proton to the oxygen atom of the glycosidic bond and
accelerating the reaction. This protonation makes the leaving group a weaker base and hence,
a better leaving group (2).


A carbocation is formed on displacement of an alcohol. Asp 52, on the other hand, probably
stabilizes the carbonium ion generated at the site upon bond cleavage (3). Following bond cleavage,
the first product formed diffuses away, and the carbonium ion intermediate can then react with H20
from the solution. can now act as a general base, accepting a proton from the attacking water (4)
and formation of the second product (5).
(ii) SN2 mechanism
In 2001, an alternative mechanism involving two consecutive direct bimolecular

nucleophilic displacement (SN2) steps, with inversion of configuration on each
displacement step at C-1 carbon of NAM residue, leading to the formation of product with
net retention of configurationhas been suggested.

5.16 CHYMOTRIPSIN
Chymotrypsin hydrolysis to peptide bonds at body temperature and physiological pH.In
Chymotrypsin is a protease (series protease since serin is at active site). The overall reaction
shown below:
(Lehninger Principles of Biochemistry Fourth Edition)

(Lehninger Principles of Biochemistry Fourth Edition)
The following points confirm the mechanism of hydrolysis of a peptide bond (when
adjacent to aromatic amino acids) by the enzyme chymotrisin. Chymotrypsin can hydrolyse

other acetate functional group for example esters amides, and anhydrides, p-niotro phenyl
acetate, an ester containing an aromatic portion are react with chymotrypsin
p-Nitrophenol and acetic acid are formed at with different speed, while p-nitrophenol forms
very rapidly, while acetic acid form slowly. The results indicate a two-step reaction sequence
commonly observed in enzyme catalysis. In between of reactant and product the enzyme-
substrate complex is found.
5.17 ----
5.17 SUMMARY
This unit covered following points:
 Enzyme is the biological catalyst which increases the rate of reaction and decrease
the activation energy of biological reaction.
 Enzyme is classified on different basis.
 The role of inhibitor in different site of substrate. It influences the rate of reaction in
the presence of enzyme.
 Learnt about Transition State theory
 Mechanism action of Carboxypeptidase A, Lysozyme and chymotrypsin enzyme in

different reactions.

1. What are the forces involved in binding substrate and intermediates to the active sites of
enzyme?
2. What is the difference between specific and general acid catalyst? Explain with suitable
examples.

3. Chymotrypsinis a serine protease which specifically hydrolyses the peptide bond
adjacent to aromatic amino acid residue. Explain.
4. Why lysozyme distorts one of the rings of the bacterial cell wall from the chair to half
chair form?
5. Enzymes are multifunctional catalyst. Explain with example.
6. Is there a difference between the initial and the final energy levels in catalyzed and non-
catalyzed reactions?
7. What are the main theoretical models that try to explain the formation of the enzyme-
substrate complex?
8. What is the chemical basis of enzyme specificity?
9. What is the Michaelis-Menten equation and its Lineweaver-Burk form?
10. How does the Michaelis-Menten equation explain why the rate of an enzyme-catalyzed
reaction is proportional to the amount of enzyme? How does the Michaelis-Menten
equation explain why the rate of an enzyme catalyzed reaction reaches a maximum value
at high substrate?
11. How does the formation of an E.S complex explain the reaching of a maximal velocity in
the Vo vs So graph?
12. What is antibody and what is hapten.
13. Define the following terms: (a) Enzyme model and (b) Biomimetic Synthesis.
14. Is there a difference between the initial and the final energy levels in catalyzed and non-
catalyzed reactions?
15. What are the main theoretical models that try to explain the formation of the enzyme-
substrate complex?
5.18 BIBLIOGRAPHY
1. Outline of Enzyme Chemistry, J.B. Neilands and Paul K. Stumpf, John-Wiley & Sons,
In.anism
2. Bio-organic Chemistry: A chemical Approach to Enzyme Action; Herman Dugas & C.

Penny, Springer-Verleg.

3. Enzyme Mechanism, Ed. M.I. Page and Williums, Royal Society of Chemistry.
4. Enzyme Structure and Mechanism; A. Fershi, W.H. Freeman.
5. Immobilisation Enzyme: An introduction & application in Biotechnology, Michael D.

Trevan, John Wiley.
6. Essential of Bio-Organic Chemistry; Vinay Prabha Sharma, Pragati Prakashan.
7. Bioorganic, Bioinorganic and Supramolecular Chemistry, P.S. Kalsi & J.P. Kalsi;New
Age Publication
8. Bioorganic Chemistry; H.C. Chopra, Narosa publication.
9. Dagmar Heinová, ZuzanaKostecká,TomášCsank, Separation of turkey lactate

dehydrogenase isoenzymes using isoelectric focusing technique; Wiley Analytical
Science
10. Lehninger Principles of Biochemistry Fourth Edition

UNIT 6: KINDS OF REACTIONS CATALYSED BY

ENZYMES
CONTENTS:
6.1 Introduction
6.2 Objective
6.3 Nucleophilic displacement on phosphorus atom
6.3.1 Important in living things is the chemical ATP
6.3.2 Phosphorylation of glucose
6.4 Multiple displacement reactions of phosphorus
6.5 Coupling of ATP cleavage to endergonic processes
6.5 Transfer of sulphate
6.7 Addition reaction
6.8 Elimination reaction
6.9 Enolic intermediates in isomerization reactions
6.10 Cleavage and condensation
6.10.1 Aldol cleavage
6.10.2 Claisen Condensation
6.10.3 Decarboxylation of β-keto acids
6.11 Enzyme catalyzed carboxylation and decarboxylation
6.12 Summary
6.13 SAQs type question
6.14 Glossary
6.15 References

6.1 INTRODUCTION
Enzymes are biological catalysts (biocatalysts) necessary for practically all biochemical
processes in a living system. Natural enzymes are proteins that may be thought of as bio-
catalysts since they are created by living cells. Recently, however, it has also been shown that
a small number of RNA molecules possess some catalytic activity. In a lab setting, it takes a
few days for a protein to be hydrolyzed by a strong acid at pH 100. However, the digestive
enzyme digests the same protein over a few hours at a considerably lower temperature (37 C,
body temperature). Many laboratory reactions call for greater temperatures, as well as a
number of solvents and high reagent concentrations. A live cell can't access these
circumstances, but they won't kill it either. Enzymes allow the body's reactions to occur
quickly and effectively at body temperature with just modest reagent concentrations in water,
the cell's solvent. Kuhne used the word enzyme (Greek: in yeast) to refer to the catalysis that
takes place in biological systems before Berzelius's 1836 use of the phrase catalyst (Greek: to
dissolve). In 1883, Duclaux first used the word "substance." The enzyme was found in yeast
(Greek: en =, and zyme = yeast) and was able to catalyse the fermentation processes. In 1883,
Buchner isolated an enzyme system from a yeast extract devoid of yeast cells. Zymase was
the name of the active ingredient, which was able to turn sugar into alcohol.
6.2 OBJECTIVE
You will be able to learn after this unit:
 Understand nucleophilic displacement on the phosphorus atom; numerous displacement

reactions; and the connection between ATP cleavage and endergonic processes after
finishing this unit.
 Sulfate transfer, addition and elimination reactions, enolic intermediates in isomerization

events, cleavage and condensation, certain isomerization and rearrangement reactions,
and Enzyme-catalyzed carboxylation and decarboxylation are all covered in this section.
6.3 NUCLEOPHILIC DISPLACEMENT ON PHOSPHORUS

ATOM
6.3.1 Important in living things is the chemical ATP.
In most biological tissues, phosphoric esters make up more than 3% of the organic

components. They participate in practically all facets of cellular activity. Phosphorylation,
which is the process by which a phosphoryl group is transferred from one group to another, is
one of their most significant processes. The reaction, which involves a nucleophilic
substitution on phosphorus (Fig. 6.1), results in the same kind of structural modification
as that seen in acyl transfer reactions involving carboxylic acid derivatives.
Fig 6.1 Nucleophilic substitution on phosphorus
As a "high-energy" phosphate, adenosine triphosphate (ATP) is frequently mentioned. In

biological terms, this indicates that energy is released when ATP phosphorylates an acceptor
(nucleophile) and transfers a phosphate group to that acceptor. The breaking of phosphate
bonds is crucial for the transmission of energy in a biological system.
The type of nucleophile used in the phosphorylation affects how much energy is released.
With water serving as the reference (Fig. 6.2), ATP is hydrolyzed to produce adenosine
diphosphate (ADP), which releases energy at a rate of around 7 kcal/mol (30 kJ/mol).
Fig. 6.2 ATP is hydrolyzed to produce adenosine diphosphate (ADP)

On a chemical level, the significant shift in free energy that occurs during ATP hydrolysis is
explicable. First, the product ADP exhibits a weaker than expected electrostatic repulsion
between the four negative charges in ATP (Fig.6.3). Resonance serves to stabilise the
inorganic phosphate (Pi), the other byproduct of hydrolysis.
Fig.6.3 Electrostatic repulsion between the four negative charges in ATP
Due to its two anhydride connections, adenosine triphosphate (ATP) is an energetically dense
molecule. The free energy required to hydrolyze ATP into the corresponding monoanhydride
(ADP) is -7.3 kcal/mol (fig. 6.2). This significant decrease in free energy demonstrates how
favourable the reaction is. With Go =-7.3 kcal/mol, one may conclude that ATP hydrolysis is
a strongly exergonic process.
6.3.2 Phosphorylation of glucose
The ATP hydrolysis-induced favourable free energy change is employed to shift the
equilibrium of unfavourable biological reactions in the desired direction. Think about how
glycolysis, which is derived from the Greek words for "sweet" and "splitting," releases
energy from glucose in biological systems. In contrast to glycolysis, which begins with the
phosphorylation of glucose with ATP to produce glucose-6-phosphate, the breakdown of
glucose involves a sequence of enzyme-catalyzed processes to produce two molecules of
pyruvate (Fig. 6.4). If one considers the reaction of D-glocose with hydrogen phosphate
(Fig.6.5), the formation of a phosphate ester.

Fig. 6.4 Enzyme-catalyzed processes
Glucose 6-phosphate will be produced at the main hydroxy group. The free energy of this
reaction, however, is +3.3 kcal/mol (13.8 kJ/mol), making it an endergonic reaction (fig.6.6).
Due to the equilibrium favouring the reactions, there is a positive free energy. Thus, this
phase in the metabolism of glucose is undesirable.
Fig.6.5 Reaction of glucose with hydrogen phosphate-an unfavourable reaction
However, glucose and ATP easily combine to form glucose 6-phosphate and ADP in a
straightforward one-step nucleophilic substitution process (Fig. 6.6). Without the need for an
intermediary, a phosphoanhydride bond is broken when the 6-OH group of glucose engages
the terminal phosphate of ATP as a nucleophile. In order to phosphorylate glucose, ATP is
needed because the 6-OH group of D-glucose acts as a nucleophile and displaces weakly
basic ADP. Without ATP, the 6-OH of glucose would have to displace a very basic OH group
(Fig 6.5).Instead of cleaving the link, glucose's nucleophilic attack on ATP cleaves a

phosphoanhydride bond. This is comparable to a carbonyl group being attacked by a
nucleophile when the bond is the first bond to break.
Fig. 6.6 Nucleophilic substitution process
The free energy change of this reaction is computed by combining the free energies for the
phosphorylation of glucose and the hydrolysis of ATP (Fig. 6.6), where the species present
on either side of the reaction arrow cancel. This reaction has a free energy change of -4.0
kcal/mol. Coupled reactions are two reactions in which the energy of one reaction is used to
drive the other, such as the favourable hydrolysis of ATP and the unfavourable
phosphorylation of glucose (with hydrogen phosphate).
A phosphoanhydride bond is broken during the beneficial reaction of phosphorylation, which

transfers the electrophilic phosphate group from one nucleophile to another. The primary role
of ATP is phosphorylation because it offers good leaving for reactions that would not
otherwise take place due to insufficient leaving groups.

Fig. 6.7 Total enegry change from ATP to ADP conversion process
6.4 MULTIPLE DISPLACEMENT REACTIONS OF

PHOSPHORUS
In biological systems, ATP reacts with a variety of nucleophiles. These are oxygen from an
alcohol or carboxylate, nitrogen from creatine, oxygen from the side chain of arginine, or

oxygen from the side chain of histidine. Depending on the enzyme initiating the reaction,
each of the three phosphates of ATPα-, β-, orγ-is susceptible to nucleophilic displacement
and gives a unique kind of product.
When a pyrophosphate is hydrolyzed, it yields two equivalents of phosphate. So, when a

pyrophosphate is produced as a result of nucleophilic displacement on phosphorus, its
subsequent hydrolysis moves the reaction to the right, guaranteeing its irreversibility. When
such irreversibility is necessary, biochemical processes take place by nucleophilic
displacement on α or β phosphorus of ATP.
6.5 COUPLING OF ATP CLEAVAGE TO ENDERGONIC

PROCESSES
As long as the overall path is exergonic, high energy compound exergonic reactions can be
connected to endergonic processes to drive them to completion. The exergonic hydrolysis of
ATP to produce ADP and Pi supplies free energy for a number of bodily functions. Instead of
using the conventional chemistry terms "exothermic" and "endothermic," exergonic and
endergonic are used to indicate that a process is accompanied by a loss or gain of free energy
in any form, not just heat.A connected exergonic-endergonic reaction system with an overall
net change that is exergonic is the only way that an endergonic reaction can exist
independently. Exergonic processes refer to catabolism, the disintegration of fuel molecules.
Molecules are assembled synthetically during anabolism. Catabolic and anabolic processes
both occur during metabolism, and a number of organophosphates, including ATP, ADP, and
phosphoenolpyruvate, are important for energy storage and transfer.
This ATP cleavage is frequently connected to an endergonic metabolic process that is

thermodynamically forced upward. Such reactions require the sequential displacement of two
different types of reactions. The sequential displacement of an atom of phosphorus followed
by an atom of carbon, or vice versa, is used to couple two processes. The initial stage in the
coupling process involves the displacement of one of the three phosphorus atoms on which

the phospho, pyrophospho, and adenyly groups from the ATP molecule are transferred to a
nucleophile. A second nucleophile attacks a carbon atom in the following step, dislodging the
transferred group.The creation of acetyl CoA is a typical illustration of a multiple
displacement reaction. In bacteria, acetyl-CoA is produced from acetate in two separate
processes that are each mediated by an S-acetyl transferase and an acetate kinase. The oxygen
atom in the carboxylate group acts as a nucleophile in the first step, dislodging the
phosphorus in ATP to create acetyl phosphate. In the second reaction, Pi is displaced by the S
atom of the coenzyme A SH group, acting as a second nucleophile and attacking the carbon
atom of acetyl phosphate.
Fig. 6.8 Synthesis of acetyl-CoA in bacteria
The cleavage of ATP provides the energy needed for the synthesis of acetyl-CoA, which has
a high group transfer potential. In this manner, acetyl-CoA production and the energy of ATP
hydrolysis are linked.

Acetate + Coenzyme A → Acetyl-CoA ∆G = + 35.1 kJ/mol
ATP + H2O → ADP + Pi ∆G = − 34 .5 kJ/mol
The two processes required for the synthesis of acetyl-CoA from acetate are catalysed by the
same enzyme, acetyl-CoA synthetase, also referred to as acetate thiokinase, in the majority of
eukaryotic cells. The displacement of the adenylyl group from the ATP's phosphorus atom by
the nucleophilic attack of the carboxylate's O atom to produce acetyl adenylate, followed by
the displacement of the AMP group from the carbon atom, is how two processes are coupled.
Fatty acyl CoA synthetase is an enzyme that catalyses a process similar to acetyl CoA
synthetase. With the help of the cleavage of ATP into AMP + PPi and the production of fatty
acyl-CoA, this enzyme catalyses the activation of fatty acids. Two stages are required to
complete the reaction:
1. To produce a fatty acyl-adenylate and PPi, which are then quickly hydrolyzed into two
molecules of Pi, the oxygen atom of the carboxylate group of the fatty acid displaces the
adenylyl group on the phosphate atom of ATP.
2. To generate the thioester fatty acyl-CoA, the coenzyme A thiol group displaces AMP and
causes the second carbon atom to move.

6.6 TRANSFER OF SULPHATE

Natural product sulfation is a common occurrence. The enzyme ATP sulphurylase, which has
been investigated using kinetic and stereochemical techniques, is responsible for bringing
inorganic sulphate to cells and activating it there. It has been demonstrated that the enzyme
catalyses the direct "in line" displacement of inorganic pyrophosphate by inorganic sulphate
from P of ATP. The resulting adenosine 5'-phosphosulphate is then phosphorylated by APS
kinase at the 3' site to produce 3'-phosphoadenosine 5'-phosphosulphate, the most prevalent
sulphating species in biology.
Fig. 6.9 Activation of sulphate
Fourier Transform Infrared Spectroscopy has been used to build a method for the
stereochemical analysis of chiral [16O17O18O]-sulphate esters. A generic strategy for their
synthesis has also been proposed. It has been demonstrated that an Aspergillus oryzae aryl
sulphotransferase's stereochemical course proceeds with the retention of configuration at
sulphur, supporting a ping-pong-type mechanism with a sulpho-enzyme intermediate on the
reaction pathway.During enzymatic reactions mediated by sulfotransferases, nucleophilic
displacements of the sulphur atom also take place. These enzymes transfer the sulphate group
from 3′-phospho-adenosine-5′-phospho sulphate (PAPS) to the oxygen and nitrogen atoms of

an acceptor molecule. PAPS, also known as activated sulphate, is created when the enzymes
ATP sulfurylase and APS kinase work together to create sulphate.
Sulfatides (15 percent of white matter lipids in the brain) are formed, for example, by
transferring the sulphate group from PAPS to the C3-OH group of galactose in cerebroside (a
component of brain lipids).
6.7 ADDITION REACTIONS
In an addition reaction, a nucleophile and a proton are added to a polarised double bond, such as
C=O or C=N. If the C=C bond has been polarised by conjugation with C=O or C=N, the
nucleophile may also attack it. Alcohols, amines, and thiols are the most common nucleophiles
used because they quickly attack the electrophilic carbon atom of the carbonyl group. The water
molecule can add to the carbonyl group by acting as a nucleophile. The transformation of CO2
into bicarbonate ion, which is catalysed by the enzyme carbonic anhydrase, is an illustration of
an addition process.
The Zn2+ ion that is coupled to the protein is tetrahedrally connected to three histidine residues,
and a water molecule occupies the fourth co-ordination site. As a base, His 64 pulls a proton
from the water molecule and binds it to Zn2+ to create an OH ion. However, the distance
between His 64 and the water-bound Zn2+ prevents it from directly abstracting a proton; as a
result, a hydrogen bonding network between these two molecules connects them. The
hydrogen-bonded network functions as a proton shuttle. As a result, the Zn2+-bound OH attacks
the CO2 substrate to change it into HCO3- by acting as a nucleophile. The production of imine
intermediates is a common step in many enzymatic processes. These imine intermediates are
called Schiff base. The formation of amine intermediates involves the amine engaging in a
nucleophilic assault on the carbonyl group, followed by the removal of the OH ion.

Fig 6.10 Mechanism of action of carbonic anhydrase
At its active site, the lysine side chain of the enzyme aldolase forms Schiff bases with the
substrate's ketonic group. Fructose-1,6-bisphosphate is transformed into glyceraldehyde-3-
phosphate and dihydroxyacetone phosphate by the enzyme aldolase.

The protonated Schiff base, an iminium cation, is created when the substrate fructose 1,6-
bisphosphate binds to the lysine residue in the aldolase enzyme's active site. The release of
glyceraldehyde-3-phosphate, the initial reaction product, causes the cleavage of the C—C
link, which causes the creation of the intermediate enamine. Dihydroxyacetone phosphate is
the second result of the reaction with the regeneration of free enzyme after protonation of the
enamine to iminium cation and hydrolysis.
There are other metabolic processes in which the nucleophile is also added to the C=C bond
if the C=C bond is conjugated with the C=O bond and the polarisation from the C=O is
transferred to the C=C bond.
At the carbon of the carbonyl group, the nucleophile adds to the carbon double bond. By
dispersing negative charge, the added product stabilises itself. The reaction is finished by
adding a proton to the enolate anion.
A typical example of this reaction is the addition of water molecule to α,β-unsaturated CoA
derivative catalyzed by the enzyme enoyl-CoA hydrase.

6.8 ELIMINATION REACTIONS
Small molecules like H2O, NH3, and other similar molecules are eliminated in elimination
reactions by creating a double bond between the carbon atoms from which the atoms or groups
are separated.
Reaction proceeds buy any of tree mechanism
1. By concerted reaction
2. By the formation of a carbocation resulted by the breaking of C-O bond
3. By the formation of a carbanion resulted by the breaking of C-H bond
However, enzymes either use one of two mechanisms to catalyse the dehydration reaction:
(i) Acid catalysis, or protonation of the OH group by an acidic group.
(ii) Proton extraction by a basic group, also known as base catalysis.
The dehydration of 2-phosphoglycerate to phosphoenol pyruvate, which is catalysed by the

enzyme enolase, is a classic illustration of an elimination reaction.

Fig.6.11 Mechanism of action of the enzyme enolase
The two bound Mg2+ ions in the enzyme's active site interact strongly with the substrate 2-
phosphoglycerate, increasing the acidity of the C—2 proton. Before the substrate binds to the
enzyme, it forms a combination with Mg2+. In the first phase of the reaction, the amino acid
Lys-345 functions as a base to remove a proton from the 2-phosphoglycerate's C—2, and in the

second step, the amino acid Glu-211 functions as an acid to provide a proton to the leaving
group (—OH). As a result, this is a straightforward instance of acid-base catalysis.
6.9 ENOLIC INTERMEDIATES IN ISOMERIZATION

REACTIONS
Isomerization reactions are those in which an H-atom is moved intramolecularly to alter the
location of a double bond. A proton is moved from one C-atom to another in these reactions.
The process proceeds through an intermediate called enediol.
Fig.6.12 Formation of enediol intermediate
The two ionizable groups on the enzyme are X1 and X2. In the first step, X1 provides a proton
and X2 donates an electron to allow the production of a C=C bond and the intermediate enediol.
The second phase involves the donation of a proton by X1 and the abstraction of a proton by
X2, which allows the C=O bond to form. The C—H bond is created as a result of the
displacement of an electron pair from the C=C bond. The enzyme triose phosphate isomerase
catalyses the conversion of dihydroxy acetone phosphate to glyceraldehyde-3-phosphate, which
is an illustration of an isomerization reaction. The enediol intermediate is formed as the reaction
progresses.

The catalytic mechanism of the enzyme triose phosphate isomerase involves the abstraction of a
proton from the substrate by glutamate residue and the donation of a proton by lysine residue,
resulting in the creation of the intermediate enediol. Glyceraldehyde-3-phosphate is produced in
the second stage by proton donation by glutamate and proton abstraction by lysine.
The transformation of glucose-6-phosphate into fructose-6-phosphate, which is catalysed by the

enzyme phosphoglucose isomerase, is another illustration of an isomerization reaction. In order
to create a cis-enediol intermediate, a base from the imidazole component of the His-Glu dyad
abstracts a proton from the C-2 atom. In a whole proton transfer, a proton is transferred onto the
C-1 atom.
Fig. 6.13 Mechanism oF action of triose phosphate isomerase

Fig. 6.14 Conversion of glucose-6-phosphateto fructose-6-phosphate
Isopentenyl pyrophosphate isomerase, an enzyme, catalyses the conversion of isopentenyl

pyrophosphate to dimethyl allyl pyrophosphate in the cholesterol biosynthesis process, which is
another example of an isomerization reaction. An intermediate carbocation is created when the
enzyme's Cys residue acts as a proton donor by donating a proton to the substrate. As the
product is formed, the Glu-residue simultaneously removes a proton from the intermediate
carbocation.
6.10 CLEAVASE AND CONDENSATION
The processes that form or dissolve carbon-carbon (C—C) bonds are a part of the catabolic and
anabolic pathways of metabolism. Organic molecules' tightly bonded carbon skeleton makes it
difficult to create or break them. A nucleophilic carbanion must be joined to an electrophilic
carbon atom in order to form a C—C bond. Due to its ability to extract electrons, the carbonyl
atom in aldehydes, ketone esters, and CO2 is the most frequently electrophilic carbon atom in
these processes.
To add to an electrophilic centre, one needs a stabilised nucleophilic carbanion. A resonance

stabilised carbanion, or enolate, is produced by the carbon atom of the carboxylate group on an
adjacent carbon. This enolate anion acts as a nucleophile.. The cleavage and production of the
C—C bond are thus made easier by the carbonyl group.

Aldol cleavage, Claisen condensation, and decarboxylation of β-keto acids are examples of
processes of this sort. These reactions involve the formation and breaking of bonds between α
and β carbon atoms of a carbonyl compound and will be referred to as β-condensation and β-
cleavage.
6.10.1 Aldol Cleavage
A typical example of aldol cleavage, which is a common reaction of C—C bond formation, is
the reaction that fructose bisphosphate aldolase catalyses during glycolysis. By stabilising its
enolate intermediate, which functions as a nucleophile, aldol cleavage is catalysed. Aldolases
come in type I and type II varieties. The intermediate enolate ion is stabilised by type I
aldolases, which are found in both plants and mammals. They achieve this by converting the
carbonyl group into a protonated Schiff base. The enolate ion is stabilised by the type-II
aldolases, which are found in fungi, algae, and some bacteria. They achieve this by coordinating
the enolate ion with a metal ion, usually Zn 2+ or Fe 2+.
A type I aldolase called fructose bisphosphate aldolase is responsible for converting fructose-
1,6 bisphosphate into glyceraldehyde-3-phosphate and dihydroxyacetone phosphate during
glycolysis.

6.10.2 Claisen Condensation
One instance of claisen ester condensation in which the carbanion is stabilised by the carboxyl
of a neighbouring thioester is the reaction catalysed by citrate synthesis during the citric acid
cycle.
Acetyl CoA and oxaloacetate are condensed in the first phase of the citric acid cycle, which is
catalysed by citrate synthase (ketone). Due to the fact that the substrate (acetyl CoA) is a
thioester, the reaction is known as Claisen ester condensation. There are three steps involved in
the reaction those citrate synthase catalyses.
 An enolate of acetyl CoA is produced when the enzyme Asp-375 removes a proton from
the methyl group. The H bonds to His 274 stabilise the enolate intermediate.
 Citryl-CoA is created when the acetyl CoA enolate nucleophilically attacks the carbonyl
carbon of oxaloacetate by donating a proton from His-320 to the carboxyl group of
oxaloacetate.
 After that, citryl-CoA is hydrolyzed to produce citrate and coenzyme A.
The formation of acetoacetyl CoA, a precursor to cholesterol, from the condensation of two
acetyl CoA molecules by thiolase is sometimes referred to as a claisen condensation.

Fig. 6.15 Claisen Condensation

6.10.3 Decarboxylation of β-keto acids
A resonance stabilised carbanion is produced after the decarboxylation of β-keto acids by the
removal of CO2. An illustration of this kind of reaction is the acetoacetate decarboxylase
catalysed process.
6.11 ENZYME CATALYZED CARBOXYLATION AND

DECARBOXYLATION
The most important C—C bond building and bond breaking reactions in biological processes
result in the gain or loss of one carbon, in the form of CO 2. Carboxylation refers to the addition
of a CO2 unit to a substrate molecule, whereas decarboxylation refers to the removal of carbon
in the form of CO2. The bulk of carboxylation reactions that occur in metabolic pathways are
catalysed by RuBisCO and biotin-dependent carboxylases. The enzyme ribulose-1, 5-
bisphosphate carboxylase oxygenase (RuBisCO) catalyses the synthesis of 3-phosphoglycerate
from ribulose-1, 5-bisphosphate (RuBP).
The most prevalent protein on the planet, this enzyme contains up to 50% of leaf proteins.
The 500–560 KD protein known as RuBisCO from higher plants is made up of eight large (L)
subunits that are encoded by chloroplast DNA and eight tiny (S) subunits that are encoded by
a series of nuclear genes. At the top and bottom of the protein, eight small subunits are

grouped as two caps (tetramer), while eight big subunits are present in the space between the
two caps. The L-subunit contains the catalytic site of the enzyme. The removal of a proton
from RuBP's C-3, which results in the production of an enediolate, initiates the carboxylation
process of RuBisCO.The intermediate enediolate then engages in nucleophilic attack on CO2
to produce a β-keto acid. This β-keto acid then reacts with water to produce an adduct, which
splits to produce a molecule of 3-phosphoglycerate and an intermediate carbanion. In order to
create a second molecule of 3-phosphoglycerate, the carbanion is protonated. The finding that
the homolog of the -keto acid intermediate, 2-carboxyarabinitol-1-phosphate (CA1P), binds
closely to the active site of the enzyme from spinach supports this enzyme's mechanism.
(A) Production of an enediolate intermediate that attacks CO 2 nucleophilically to produce a

keto acid.

(B) Water and β-keto acid combine to generate an adduct that breaks into 3-phosphoglycerate
and carbanion.
Fig. 6.16 The carboxylation reaction catalyzed by RuBisCO

Bacteria use phosphoenol pyruvate (PEP) carboxylase for biosynthetic purposes, and C4 plants
like sugarcane and corn use it as part of a CO2 concentrating mechanism. In the leaves of C4
plants, mesophyll cells encircle a single layer of bundle sheath cells in the leaves. The PEP
carboxylase but not the RuBisCO enzyme is present in the mesophyll cells. In the mesophyll
cells, the PEP carboxylase combines HCO3-with PEP to produce oxaloacetate. Malate, which is
transferred to the bundle sheath cells, is created by the reduction of oxaloacetate.In animals, the
pyruvate carboxylase catalyses the carboxylation of pyruvate with ATP cleavage to produce
oxaloacetate (OAA). A biotin prosthetic group on pyruvate carboxylase serves as a CO 2 carrier
during carboxylation. Two stages make up the pyruvate carboxylase reaction:
(i) An enzyme-catalyzed transfer of a phosphoryl group from ATP to bicarbonate results in

the formation of a carboxyphosphate. Biotin is carboxylated when its N1 atom hits the
C-atomof carboxy phosphate.
(ii) Oxaloacetate is produced when the activated carboxyl group from carboxybiotin is
transferred to the pyruvate's carbon.
Acetyl-CoA carboxylase, which also facilitated the first committed step of fatty acid
biosynthesis, catalyses a similar process. Two stages are needed to complete the reaction.
The enolate ions generated during the carboxylation reaction are used in carbon-carbon (C—C)
bond formation. Decarboxylation in metabolism can be either non-oxidative or oxidative. The
reaction catalysed by PEP carboxykinase is a good example of non-oxidative decarboxylation.

Phosphoenol pyruvate (PEP), a metabolic intermediate, is the phosphate ester of pyruvate which
is formed by GTP-driven decarboxylation of oxaloacetate (OAA). The reaction is catalysed by
PEP carboxykinase (PEPCK). The decarboxylation of OAA generates an enolate anion whose
oxygen atom attacks the phosphate group of GTP, forming PEP.
Fig. 6.17 The catalytic mechanism of reaction catalyzed by PEP carboxykinase
A β-hydroxy acid is converted to a β-keto acid through the process of oxidative

decarboxylation, which is followed by its decarboxylation. Isocitrate dehydrogenase, malic
enzyme, and -ketoglutarate dehydrogenase all catalyse this kind of reaction.
6.12 SUMMARY
The present chapter is summarised as follows:
 "Metabolic reactions catalysed by enzymes are categorised into 5 classes: substitution,

addition, elimination, isomerization, and rearrangement reactions.
 In a substitution reaction, one atom or group is swapped out for another. In an electrophilic
substitution reaction, the atom or group is replaced by an electrophile, and in a nucleophilic
substitution reaction, a nucleophile.
 On saturated C-atoms, carbonyl C-atoms, phosphorus atoms, and sulphur atoms, a

nucleophilic substitution process can occur. The endergonic process and ATP hydrolysis
are coupled through a number of displacement processes.

 The reactions known as addition reactions occur when the attacking reagent adds to the
substrate. Small molecules are removed during the product generation process in
elimination reactions.
 The intramolecular displacement of the H atom to modify the location of the double bond
occurs during the isomerization reaction.
 The production of the intermediate enediol is the first step in isomerization reactions.
 In rearrangement processes, substituents are moved around within the molecule to create
the molecule's structural isomer.
 Condensation and -cleavage are the terms used to describe the reactions that result in the
production and dissolution of a C—C bond between a carbonyl group's carbon atoms,
respectively. While CO2 is removed during a decarboxylation reaction, it is added to the
substrate during a carboxylation reaction.
6.13 SAQs TYPE QUESTIONS
A. Multiple choice questions
1. The conversion of 2-phosphoglycerate into phosphoenol pyruvate:
(a) Enolase (b) Aldolase
(d) Mutase. (c) Isomerase
2. The following reactions transform geranyldiphosphate into geraniol:
(a) Isomerization reaction (b) Elimination reaction
(c) Addition reaction (d) Nucleophilic displacement
3. From dimethyl allyl pyrophosphate produced isopentenyl pyrophosphate is through the

following reactions:
(a) Schiff base (b)Tertiary carbocation
(c) Enediol intermediate (d) Carbanion intermediate
4. Triose phosphate isomerase catalyzes the conversion of dihydroxy acetone phosphate

toglyceraldehyde-3-phosphate via the formation of:
(a) Enediol intermediate.(b) Carbocation intermediate

(c)Carbanion intermediate (d) Schiff base
5. Which of the following enzyme can catalyze the thiol disulfide exchange reaction?
(a) RNase A (b) PEP carboxylase
(c) RuBisCO(d) Thiol transferase
6. The enzyme that converts fructose-1, 6-bisphosphate into dihydroxy acetone phosphate and
glyceraldehyde-3-phosphate:
(a) Enolase (b) Isomerase
(c) Mutase (d) Aldolase
7. When dihydroxyacetone phosphate is converted to glyceraldehyde 3-phosphate, the reaction

is catalyzed by?
(a) Pyruvate kinase (b) Phosphoglycerate mutase
(c) Triose phosphate isomerase (d) Enolase
8. Conversion of glucose-6-phosphate to fructose-6-phosphate is catalyzed by the enzyme:
(a) Aldolase (b) Phosphoglucose isomerase
(c)Enolase(d) Triose phosphate isomerase.
9. What is the name for the following reaction intermediate? H 2N+=CH2
(a) Nucleophilic (b) Carbanion
(c) Schiff base (d) Carbocation
10. The reaction catalyzed by acety-CoA synthetase is an example of:
(a) Carboxylation reaction.(b) Multiple displacement reaction
(c) Elimination reaction(d)Addition reaction
11. Mg2+is an inorganic activator for the enzyme:
(a) Aldolase. (b) Carbonic anhydrase
(b) Chymotrypsin (d) Enolase
12. The reaction catalyzed by RuBisCO is:
(a) Decarboxylation of RuBP (b) Carboxylation of RuBP

(c) Carboxylation of PEP(d) Decarboxylation of β-keto acid.
13. The cleavage of fructose-1, 6-bisphosphate to glyceraldehyde-3-phosphate and dihy-
droxyacetone phosphate is an example of:
(a) Aldol condensation(b) Claisen condensation
(c) Aldol cleavage (d) None of the above.
14. The enzyme which incorporates HCO3 − into phosphoenol pyruvate to form oxaloacetate:
(a) PEP carboxylase(b) Aldolase
(c)RuBisCO (d) None of the above.
15. Zn2+ is an inorganic activator for enzyme :
(a) Chymotrypsin (b) Carbonic anhydrase
(c) Phosphate (d) Mutase
16. In nucelophilic displacement reaction on sulfur atom, which of the following act as a
nucleophile?
(a) Enolate anion (b) Carboxyl oxygen
(c) Hydroxyl group(d) Thiolate anion
17. When a nucleophile attacks the α-phosphorus atom of ATP, what kind of transfer occurs?
(a) Pyrophosphoryl transfer (b) Phosphoryl transfer
(c)Adenosine transfer(d) Adenylyl transfer
18. Which of the following protein can reduce the disulfide bridges in proteins?
(a) Reduced form of thioredoxin (b) Oxidized form of thioredoxin
(c) Glutathione reductase (d) All of the above
19. Which of the following has a thioester bond?
(a) ATP(b) Phosphoenolpyruvate
(c) Phosphocreatine (d) Acetyl-CoA.
20. Which of the following is not a nucleophile that commonly participates in biochemical

reactions?
(a) Imidazole(b) Hydroxide ion
(c) Phosphorus of a phosphate group (d) Carbanion
B. Fill in the blank
(i) The exergonic hydrolysis of ATP to produce ADP and Pi supplies………. for a number of
bodily functions.
(ii) A phosphoanhydride bond is broken during the beneficial reaction of phosphorylation,

which transfers the …………. group from one nucleophile to another.
(i) Isomerization reactions are those in which an H-atom is moved …………….to alter the
location of a double bond.
(ii) First, the product ADP exhibits a weaker than expected electrostatic repulsion between the
….………. charges in ATP.
(iii) A resonance stabilised carbanion is produced after the decarboxylation of β-keto acids by
the removal of ……………..
C. True/ False
1. A nucleophilic substitution process occurs when one nucleophile is replaced by another on a

saturated carbon atom. True/False
2. The disulfide exchange reaction is catalyzed in its oxidized state by the protein disulfide
isomerase. True/False
3. Dihydroxyacetone phosphate is transformed into glyceraldehyde-3-phosphate by an

isomerization reaction catalyzed by the triose phosphate isomerase. True/False
4. The phosphorus atom of ATP nucleophilically attacks the carboxylate oxygen of acetate in
the reaction mediated by acetyl-CoA synthetase. True/False
5. One instance of a multiple displacement reaction is the production of fatty acyl CoA from
fatty acid and coenzyme A. True/False
6. The Claisen ester condensation reaction is the condensing of acetyl CoA with oxaloacetate.

True/False
A. Berzelius's i.Zn2+
B. Carbonic anhydrase ii.Schiff base
C. imine intermediates iii.Sugar
D. Glucose iv. Enzyme
Answer key
A. 1 a 2d 3b 4a 5d 6d 7 c 8 b 9 c 10 b
11 d 12 b 13 c 14 15 d 16 d 17 d 18 a 19 d 20 c
B. i free energy, ii electrophilic phosphate, iii intramolecularly, iv four
negative, v CO2
C. 1 True 2 False 3 True 4 False 5 True 6 False
D. A vi B i C ii D iii
6.14 GLOSSARY
PEP = Phosphoenol pyruvate
RNA = Ribonucleic acid
DNA = Deoxyribonucleic acid
ATP = Adenosine triphosphate
ADP = Adenosine diphosphate
AMP = Adenosine diphosphate
GDP = Guanosine diphosphate
GTP = Guanosine diphosphate
RuBP = RIbulose-1, 5-bisphosphate

6.15 REFERENCES
1. Kohl, Issaku E.;Asatryan, Rubik;Bao, Huiming (1991), No oxygen isotope exchange

betweenwater and APS-sulfate at surface temperature: Evidence from quantumchemical
modeling and triple-oxygen isotope experiments, Philosophical transactions : Biological
science, 332, 141-141.
2. Kalsi P.S., kalsi J.P.(2006), Bioorganic, bioinorganic and supramoleculer chemistry, new
age international (P) Ltd. Publishers, london, new delhi, nairobi.
3. Tyagi V., Tyagi S., (2002), Bioorganic chemistry, Krishna prakashan media meerut,
india,1-302.
6.16 FURTHER READING
1. Crueger W, Crueger A (2017), Cruegers Biotechnology: A Textbook of Industrial

Microbiology, (3rd Ed.). Medtech publication. ISBN: 978-9385998638
2. Singhania RR, Patel AK, Pandey A (2010). The Industrial Production of Enzyme. In:
Industrial Biotechnology Sustainable Growth and Economic Success, Soetaert W and
Vandamme EJ (Ed.). Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. pp: 207-
225.
3. Mohamad NR, Marzuki NH, Buang NA, Huyop F, Wahab RA (2015) An overview of
technologies for immobilization of enzymes and surface analysis techniques for immobilized
enzymes, Biotechnol Biotechnol Equip. 29(2):205-220.
4. Cacicedo ML, Manzo RM, Municoy S, Bonazza HL, Islan GA, Desimone M et al. (2019),
Immobilized Enzymes and Their Applications. Singh RS, Singhania RR, Pandey A, Larroche
C (Ed.) In Biomass, Biofuels, Biochemicals, Advances in Enzyme Technology, Elsevier pp.
169-200. ISBN 9780444641144. https://doi.org/10.1016/B978-0-444-64114-4.00007-8.
5. Raveendran S, Parameswaran B, Ummalyma SB, Abraham A, Mathew AK, Madhavan A,

Rebello S, Pandey A., (2018), Applications of Microbial Enzymes in Food Industry, Food
Technol Biotechnol. 56(1):16-30.

1. What are processes involving nucleophilic displacement? Describe their mechanism using
an appropriate example.
2. Describe the addition reaction's mechanism using an appropriate example.
3. The transformation of 2-phosphoglycerate into phosphoenol pyruvate is catalysed by the
enzyme enolase. Describe the reaction's mechanics.
4. The enzyme triose phosphate isomerase catalyses the transformation of dihydroxy acetone
phosphate into glyceraldehyde 3-phosphate.
5. Using an appropriate substrate, how would you explain the enolic intermediate in bio-
organic isomerization? or Through enolic intermediates, discuss the involvement of
enzymes in the isomerization step.
6. Describe how SN 1 and SN 2 reactions work.
7. Talk about the phosphorus atom's nucleophilic displacement process. Explain the
sulfotransferase-catalyzed sulphate group transfer mechanism.

UNIT 7: CO-ENZYME CHEMISTRY

CONTENTS:
7.1. Introduction
7.2. Objective
7.3. Co-Factors
7.4. Coenzymes
7.5. Coenzyme A
7.6. NAD+ and NADP+
7.7. FMN and FAD
7.8. Thiamine pyrophosphate
7.9. Pyridoxal phosphate
7.10. Lipoic acid
7.11. Vitamin B12
7.12. Summary
7.13 SAQs
7.14 Terminal Questions
7.15 References
7.1 INTRODUCTION
Enzyme is the most efficient catalyst known in nature. They have the ability to enhance
reaction rate by lowering the activation energy of reaction and by stabilizing the reacting
molecules at their activated complex states. Enzyme catalyse a wide variety of chemical
reactions, but to bring about some biochemical reaction, the amino acid side chain of the
protein is not sufficient e.g they are less suitable for catalyzing oxidation reduction reaction
and many type of group transfer reaction. Although, enzyme catalyze these reaction, in
association with some other non-enzymatic substance. Such substance are called co-factor.

In the presence of these co-factors the catalytic activity of enzyme is greatly enhanced.
Enzymes are the biological catalyst as well as act as receptor by acting with the substrate.
7.2 OBJECTIVE
After going through this unit you will be able to:
 Define Coenzyme
 Know the important Co-enzyme i.e Co-enzyme A, Nicotinamide nucleotide, Flavin

nucleotides and lipoic acid
 Know the mechanism of oxido-reduction by NAD+/ NADP+
 Understand NADH and NADPH dependent reaction
 Learn about the metabolic role of Co-enzyme
7.3 CO-FACTORS
Co-factors assist the enzymes in their catalytic action. A catalytically active enzyme co-factor
complex is called haloenzyme. The enzymatically inactive protein resulting from the removal
of co-factor is called an apoenzyme.
Apoenzyme + Co-factor → Haloenzyme
Co-factor may be metal ions, such as Zn+2 required for the catalytic activity of
carboxypeptidase-A. Such metal ion bound enzyme is called metallo enzymes. If the co-
factors are organic molecules, these are called coenzyme. Some factor like NAD+ is
transiently associated with a given enzyme. Other co-factors, which are permanently
associated with the protein by covalent linkage are called prosthetic group e.g., heme, the
prosthetic group of haemoglobin is tightly bound to its protein through covalent bond along
with extensive hydrogen bonding and hydrophobic interaction.
7.4 CO-ENZYME
Coenzyme as derived from vitamins are organic molecules required by many enzymes for
catalytic activity.Co-enzymes are transiently associated with a given enzyme and function as
co-substrates,e.g.,an enzyme alcohol dehydrogenase utilize NAD+ as coenzyme in the
catalytic oxidation of primary or secondary alcohols.Coenzyme plays vital roles in

biochemical reactions,e.g.,they transfer atoms or groups from substrate to other
molecules,they participate in redox reactions and thus help in electron transfer,they function
as hydrogen acceptors like NAD+ or NADP,and they provide nucleophilic sites for the
biochemical reactions.
7.5 COENZYME A
Coenzyme A has a complex structure consisting of an adenosine triphosphate, a pantothenic
acid which is a vitamin and cysteamine. The coenzyme is involved in transfer of acyl-groups.
The sulfhydryl (-SH) group of cysteamine moiety of this coenzyme forms a thioester with the
carboxyl (-COOH) group of the acyl-compound, such as acetic acid to produce acetyl-CoA
which is one of the most important CoA derivatives. The thioester bond is energy-rich and
can easily transfer the acetyl-group to an acceptor.Coenzyme A is an acyl activating enzyme
derived from vitamin pantothenic acid. Panthotheric acid is pentoic acid and β alanine joined
together in a amide linkageCoenzyme A can bedivided into two components, adenosine 3, 5
diphosphate and pantotheine, which is formed the combination of pantothenic acid and
βmercaptoethylamine.
The SH group of thioethanol amine moeity is an active group acting as carrier and coenzyme
A is abbreviated as CoA or CoASH. The rest of molecule provides enzyme binding site. In
acylated derivatives, such as acetyl-coenzyme A, the acyl group is linked to thiol group of
form an energy rich thio ester.
CoA reacts with many compounds to form important derivatives such as:
 Acetyl CoA
 Succinyl CoA

 HMG CoA
 Fatty acyl CoA
Acetyl Co-A and succinyl Co-A are important intermediates at cross roads of many metabolic
pathways. Acyl Co-A is formed as an intermediate both in fatty acid biosynthesis and
oxidation. HMG Co-A is an important intermediate involved in ketogenesis as well
ascholestrol biosynthesis we designate the acylated forms of coenzyme A as
or acyl Co-A, and unacylated as CoA-SH. While Co-A wasdiscovered as the "acetylation
coenzyme”. It has far more general function. It is required in the form of acetyl Co-A, to
catalyze the synthesis of citrate in citric acid cycle.
It is essential to the β oxidation of fatty acids and carries propionyl and other acyl groups in a
great variety of other metabolic reactions. The acyl group [such as acetyl or aceto acetyl
group] is attached to the Co-A through a thio ester linkage to the β- mercaptoethylamine
moiety. Acyl groups are covalently linked to thiol group, forming thioesters. Because of their
relatively high standard free energies of hydrolysis, thioesters have high acyl group transfer
potential and cars donate their acyl group of variety of acceptor molecules. The acyl group
attached to coenzymeA may thus be thought of as "activated" for group transfer.
The vitamin precursorof coenzyme A is pantothenate. Panthotheric acid is pentoic acid and β-
alanine joined together in a amide linkage.
Pyruvate is oxidized to acetyl CoA by pyruvate dehydrogenase complex by an oxidative

decarboxylation reaction.

The pyruvate dehydrogenase complex is a group of three enzymes responsible for the
conversionof pyruvate to acetyl-CoA, viz. pyruvate dehydrogenase, dihydrolipoyl
transacetylase, and dihydrolipoyl dehydrogenase. The pyruvate dehydrogenase complex also
requires TPP along with four other coenzymes (lipoate, coenzymeA, FAD and NAD +).
The first enzyme pyruvate dehydrogenase, a TPP requiring enzyme, decarboxylates pyruvate,
with the intermediate formation of hydroxyethyl thiamine pyrophosphate. This reaction is
same as catalyzed by pyruvate decarboxylase, however, unlike pyruvate decarboxylase,
pyruvate dehydrogenase does not convert the intermediate hydroxyethyl thiamine
pyrophosphate into TPP and acetaldehyde but transfer the intermediate to the second enzyme
dihydrolipoyl transterase . Second enzyme requires lipoate, a coenzyme that is attached to its
enzyme by an amide linkage to a lysine residue.
The hydroxyethyl thiamine pyrophosphate carbanion attacks the disulphide linkage of lipoate
followed by the eliminationof TPP carbanionfrom the intermediate adduct to form acetyl-
dihydrolipoamide and regenerate active pyruvate dehydrogenase. Dihydrolipoyl
transacetylasecatalyzesthe transfer of the acetyl group to CoA forming acetyl CoA and
dihydrolipoamide-dihydrolipoyl transacetylase. The third enzyme dihydrolipoyl
dehydrogenasealso calledlipoamidedehydrogenasereoxidizesdihydrolipoamide utilizing the
coenzyme FAD. Oxidation of dihydrolipoate by FAD forms enzyme bound FADH2. NAD+
then oxidizes FADH2 back to FAD.
7.6 NAD+ AND NADP+

The dinucleotide coenzymes, nicotinamide adenine dicucleotide (NAD +) and nicotinamide
adenine dinucleotide phosphate (NADP+) play a vital role in biochemical reaction. These are
derived from the vitamin niacin (nicotinic acid). These are also known as pyridine nucleotide
due to the presence of pyridine ring.
Nicotinamideadenine dinucleotide NAD+ is two-electron oxidizing agent, and is reduced to

NADH. NADH is in turna two-electron reducing agent and is oxidized to NAD+.
NAD in derived from vitamin commonly called niacin and niacinamide is the equivalent
form of the vitamin. NAD+ is made of two nucleotideunits joined to one another via their
phosphategroups. The heterocycliccomponentof the nucleotidesof NAD + is nicotinamide, and
the heterocyclic component of the other is adenine. This explains the coenzyme's name

(nicotinamideadenine dinucleotide). The positive charge in the NAD abbreviation refers to
the positivelycharged nitrogen of thepyridine ring.
The only difference between NADP+and NAD+ is the phosphate group bonded to the 2’-OH
group of the ribose of the adenine nucleotide; this explains the addition of "P’’ to its name.
NAD+ and NADH are generally used as coenzymes in catabolic reactions and the
phosphorylated derivatives, NADP+ and NADPH, are generally used as coenzymes in
anabolic reaction.

An enzyme alcohol dehydrogenase (ADH) involves NAD+ as coenzyme in the catalytic
oxidation of primary or secondary alcohol to aldehyde or ketone respectivly.
The coenzyme NAD+ binds to the Alcohol dehydrogenase enzyme at the specific site and
accepts a hydride anion from the substrate at C-4 position ring to form the carbonyl
compound. The enzyme alcohol dehydrogenase in the presence of NAD + has the ability to
differentiate between enantiotopic hydrogen. For example, Alcohol dehydogenase remove
only the pro R hydrogen of ethanol.
The mechanism for the reduction of NADH is the reverse of the mechanism for oxidation of
NAD+. If a substrate is to be reduced, a hydride ion from the 4-position of the
dihydropyridine ring is donated to the substrate. An acidic group of the enzyme aids the
reaction by donating a proton t the substrate.
Another enzyme which uses NAD+ and NADP+ as coenzyme are as follows:
Malate dehydrogenase, Lactate dehydogenase, Homoserine dehydrogenase, Glyceraldehyde

3-phosphate dehydrogenase.
7.7 FLAVIN ADENINE DINUCLEOTIDE (FAD) AND FLAVIN

MONONUCLEOTIDE (FMN)
Flavin nucleotides are derived from riboflavin (vit. B2). Vit B2 deficiency causes inflamation
of the skin. Like nicotinamide nucleotide, Flavin Adenine Mononucleotide (FMN) and Flavin
Adenine Dinucleotide (FAD), also participate in oxidation- reduction reaction.
Flavin nucleotides are derived from vitamin B12 [Riboflavin] and are actively involved in
hydrogen transfer reactions.Riboflavin is 6, 7 dimethyl-iso-alloxan bound to ribitol [alcohol

of ribose]. The intense yellow color is due to iso-alloxan ring. Active co-enzyme forms are
FMN and FAD.
FMN [Flavin Mono-Nucleotide] and FAD [Flavin Adenine Dinucleotide] are the active or
coenzyme form of riboflavin.FMN is formed by phosphorylation of riboflavin by kinase
using ATP in intestine. Flavin nucleotides are derived from vitamin B12 [riboflavin] and are
actively involved in hydrogen transfer reactions.
FAD and FMN are co-enzymes of proteins known as flavoproteins.Those coenzymes which
are the flavin nucleotides are derived from vitamin riboflavin. As the names indicate FAD is
a dinucleotidein which one of the heterocycliccomponentsis flavin and the other is adenine.
FMN contains flavin but not adenine and therefore, it is a mononucleotide. Riboflavin
(vitamin B2) is thus composed of flavin and ribitol. FAD and FMN participate in several
types of enzyme-catalyzedoxidation/reduction reactions.Oxidationof a carbon-carbonsingle
bond in the hydrocarbon chain to a carbon-carbondouble bond is brought about by FAD or
FMN. As seen from balanced half-reactions, the two-electron oxidation of the hydrocarbon
chain is coupled with the two-electron reduction of FAD.
After oxidation of the substrate the coenzyme is reduced to (FADH 2) or (FMNH2).

Significantlyall the oxidation/reduction reactionsoccur on the flavin ring. An example is the
dehydrogenation of succinic acid into fumaric acid by succinate dehydrogenase. The active

site of the enzyme consists of FAD covalently bonded to the enzyme. Suchtightlybound
coenzymes are correctly called prosthetic groups. In the conversion of succinic acid
intofumaric acid not a trace of the other geometrical isomer (maleic acid) is formed, thus the
reaction is like stereoselective trans-elimination. The mechanism of conversion of -CH2CH2-
to-CH=CH- with these co-enzymes may be presented as:
The reaction involves a transfer of a hydride ion from the carbon of the hydrocarbon chain (a
substrate)to FAD (or FMN). A basic group on the surface of the enzyme removes a hydrogen
from the adjacent carbon and ultimately a new basic group is created on the surface of the
enzyme.
Thus when e.g., FAD is convertedto FADH2, one of the hydrogen atoms comes as a hydride
from the hydrocarbon chain, while the other comes as a proton from an acidic group on the
surface of the enzyme catalyzing this oxidation. Moreover, one may note that one group of
the enzyme functions as a protonacceptorwhile the other acts as a protondonor.
7.7.1 Metabolic role as Co-enzyme
Riboflavin containing proteins are called flavoproteins. Many oxido-reductase enzymes are
flavoproteins containing FMN and FAD as prosthetic group. Example L-amino oxidase
[FMN dependent], succinate dehydrogenase in citric acid cycle is FAD + dependent.
These enzymes participate in reversible oxidation reduction reactions. N-atoms at position 1

and 10 in isoalloxan ring of riboflavin undergo oxidation reduction. Example of enzymes
requiring FMN or FAD as a coenzyme and reaction where they are involved.

7.7.2 FMN dependent enzymes
L- amino acid oxidases
They catalyze oxidative deamination of amino acid producing H 2O2 which is split by catalase.
NADH dehydrogenase complex
This enzyme is a part of electron transport chain and contains FMN. The electrons are
transferred from NADHto FMN and then to CoQ in the electron transport chain.
7.8 THIAMINE PHOSPHATE (TPP)

TPP is a coenzyme which has a thiazole ring containing an acidic hydrogen ( the hydrogen
attached to imine carbon) and is thus capable of giving a carbanion. Thus carbanion act as a
nucleophilic towards carbonyl groups.
Thiamine was the first of B vitamins to be identified. It is also called as vitamin B 1. The
vitamin is structurally complex, made up of 2,5 dimethyl 6-amino pyrimidine joined to 4
methyl, 5 hydroxythyl thiazole by a methylene linkage. But its conversion to the coenzyme
form, thiamine pyrophosphate, or TPP involves simply and ATP dependent
pyrophosphorylation.Thiazole ring of TPP is the functional part of coenzyme.
7.8.1 Role of thiamine phosphate as a Coenzyme
Thiamine pyrophosphate participate as a coenzyme in following reactions:
Decarboxylation of an α-keto acid (pyruvate to acetaldehyde)
Pyruvate decarboxylase is the enzyme which needs the coenzyme TPP to catalyze the
decarboxylation of pyruvate. Thiamine pyrophosphate is the coenzyme for all
decarboxylations of α-keto acids. The mechanism shown below for pyruvate decarboxylation
is involved in all of these reactions. Note that TPP contains two heterocyclic rings a

substituted pyrimidine and a thiazole. Recent NMR studies have shown that both rings
participate in the formation of a reactive carbanion at C-2 of the thiazole ring-the carbon
atom between the nitrogen and the sulfur. As shown in the following diagram, a glutamate
carboxyl group in the enzyme attracts a proton. Linked to N-1 of the pyrimidine, which in
turn increases the basicity of the amino group, facilitating the deprotonation of C-2 of the
thiazole ring.
This carbon forms a carbanionwhich in turn can attack the carbonyl carbon of α-keto acids,
such as pyruvate, giving an addition compound. The addition compound undergoes
nonoxidative decarboxylation with the thiazole ring acting as an elect stabilized eneamine.
Protonationgive hydride or more accurately, hydroxyethyl-TPP.
7.8.2 Thiamine pyrophosphate in the pyruvate dehydrogenase reaction
Thiamine pyrohphosphate (TPP) is the coenzyme for the pyruvate dehydrogenase reaction
and other nonoxidative de-carboxylations of α-keto acids. The key reaction is attack by the
carbanion of TPP on the carbonyl carbon of pyruvate and is followed by nonoxidative
decarboxylation of the coenzyme - bound pyruvate the electron pair remains with the ring
nitrogen. In next stepthe two-carbon fragment bound to TPP extracpts a proton from
pyruvatedecarboxylase, generating a hydroxyethyl group. This fragments remains at the

aldehyde oxidation level. The pyridoxal phosphate is a phosphate ester of aldehyde form of
vitamin B6. Pyridoxal phosphate [or PLP]. Vitamin B6, which is also known as anti-dermatitis
factor includes three closely related forms, pyridoxanine, pyridoxal and pyridoxine. Active
forms are phosphate ester such as pyridoxal phosphate and pyridoxamine phosphate or
coenzyme form are formed by phosphorylation formed by enzyme pyridoxal kinase using
ATP. Pyridoxal phosphate is required by many enzymes catalysing reactions of amino acid
and amines.
The reactions are numerous and pyridoxal phosphate is surely on of nature's most versatile
catalyst. The story begins with biochemical transamination. In 1937, Alexander Braunstein
and Maria Kritzmann, in Moscow, described the transamination reaction by which amino
groups can be transferred from one carbon skeleton to another. The transamination reaction is
a widespread process of importance in many aspects of nitrogen metabolism of organisms.
For large number of transaminases glutamate is one of the reactant.In 1944, Esmond snell

reported the non-enzymatic conversion of pyridoxal into pyridoxamine by heating with
glutamate. He recognized that this was also trannsamination and proposed that pyridoxal
might be a part of coenzyme needed for amino transterases. The hypothesis was soon verified
and coenzyme was identified as pyridoxal 5’-phosphate or pyridoxamine 5’- phosphate.
7.9. PYRIDOXAL PHOSPHATE

Pyridoxal PhosphatePuridoxal phosphate (PLP) is derived from pyridoxine (vit. B 6).
Deficiency of vit. B6 causes anemia. This coenyzme is required by the enzymes that catalyze
certain transformations of amino acids. It possessreactivealdehydegroup throughwhich it can
bind itself to a number of enzymes.
Pyridoxin (Vit. B6) Pyridoxal-5’phosphate (PLP)
PLP is covalently attached to the enzyme via a Schift base (imine) linkage by the
condensation of its transimination reaction, the substrate (amino acid) reacts with the
enzymePLP Schiff base, forming a tetrahedral intermediate. A new imine is
formedbetweenPLP and the amino acid by the expulsion of lysine residue. PLP catalyzes
several different amino acid transformations as given below.
A. Decarboxylation
If the PLP catalyzes the decarboxylation reaction, the carboxyl group of the α-carbon of the
amino acid is removed. Decarboxylatedintermediateattains its aromaticity from lysine residue
or some other acid group. Transimination with a lysine side chain regenerate enzyme bound
PLP by releasing the decarboxylated substrate.

B. Mechanism of amino acid metabolism/ transformation- transamination
The enzymes that catalyze transamination reaction are called aminotransterase

(transaminase). In the first step of the reaction, a proton is removed from the α-carbon of the
amino acid bound PLP.
Protonation of the α-carbon attached to the pyridine ring followed by hydrolysis of imine,
forms the α-keto acid and pyridoxamine. In the second step, pyridoxamine foms an imine
with α-ketoglutarate, the second substrate of the reaction. Removal of the proton from the

carbon attached to the pyridine ring and donation of a proton to the α-carbon of the sulosuate
forms an imine, which on transimination with lysine residue releases glutamate and
regenerate the enzyme bound PLP-Schiff base.
C. Racemization
The first step of racemization is same as that of transamination of amino acid i.e., removal of
proton from the carbon of the amino acid bound PLP. In the second step,
reprotonationoccurs. The proton can attack the sp hybridized carbon from either side of the
plane forming the racemic mixture of both D- and L-amino acid.
Mechanism for Racemisation of L-amino acid catalyzed by PLP
7.10. LIPOIC ACID

Lipoic acid (often called lipoic acid) is a naturally occuring compounds that is also
synthesized by human.
Lipoic acid
Lipoic acid acts in the transfer of hydrogen during oxidative decarboxylation reactions. In the
structure of lipoamide, where lipoic acid is bound in the amide linkage to the ε amino group
of lysine residue of dehydrolipoamide acyl transferases. The complex reactions of the
carbohydrate metabolism catalyzed by pyruvate dehydrogenase system and α-ketoglutarate

dehydrogenase require participation of lipoic acid. It acts as a carrier and undergoes inter
conversion between reduced and oxidized form as. The two thiol groups that can undergo
reversible oxidation to a disulfide (-s-s-), similar to that between two Cys residues in a
protein. Because of its capacity to undergo oxidation- reduction reactions, lipoate can serve
both as an electron hydrogen carrier and as an acyl carrier.
Lipoic acid is also known as α-Lipoic acid or ALA and thioctic acid. Lipoic acid is cofactor
for atleast five enzymes systems, two of these are in the citric acid cycle through which many
organisms turn nutrients into energy e.g. The pyruvate dehydrogenase complex and the α-
ketoglutarate dehydrogenase.
7.11. VITAMINE B12

Coenzyme B12 as derived from vit. B12 catalyzes certain rearrangement reactions. In the
structure of B12 the cobalt metal is co-ordinated with a tetra pyrole ring system, called a
corrin ring which is as that porphyrin ring of heme compounds. The metal ion is linked to the
four nitrogen atoms of the ring by an covalent and three co-ordinate bonds but the four bonds
are almost equivalent due to resonance. The fifth which is as that the ring by one orientation
site on the cobalt is filled by a nitrogen atom from an imidazole ring [dimethyl benzimidazole
(DMB)]. The sixth co-ordination site is occupied by OH group or cyano group. In coenzyme
B12, the cyano group is replaced by a 5-deoxyadenosyl group. Vit. B12 is the only vitamin
which contain a metal ion (the metal ion has an oxidation state of +3). Deficiency of vit. B12
causes pernicious anemia.
The coenzyme form of vit. B12 is obtained after activation of NADH linked reducing systems.
Reaction with ATP, mediated by an adenosyltransferase, resultin the formation of 5’-
deoxyadenosyl cobalamin coenzyme. Coenzyme B12 is the co-factor form of vitamin B12. This
vitamin is unique among all the vitamins in that is not only consist complex organic molecule
but an essential trace element Cobalt.
Methyl malonyl Co-A mutase convert methyl malonyl CoA to succinyl-CoA utilizing
coenzyme B12. The cleavage of C-Cobond forms 5-deoxyadenosyl radical and cobalamin in
its +2 oxidation state. Removal of a proton from methyl malonyl CoA by the deoxyadenosyl
radical generates method Co-Aradical. Rearrangement of the intermediate form succinyl CoA
radical. Abstraction of a succinyl Co-A radical from 5-deoxyadenosine form succinyl Co-A

and again generates the 5-deoxuad radical 5-deoxyadenosyl radical and Co(II) combinesto
generate the coenzyme.
Coenzyme B12
Methyl cobalamin is known to participate during the transfer of methyl groups e.g., in the
regeneration of methionine from homocysteine in mammals. In this reaction, the methyl
group of 5-methyl tetrahydrofolate is passed to a reactive, reduced form of cobalamin to form
methylcobalamin, which can transfer the methyl group to the thiol side chain of
homocysteine.

7.11.1. Co-enzyme form of B12
Two active forms of vitamin B12 are involved in metabolism. These are (a) methyl cobalamin
and (b) 5’- deoxy adenosyl cobalamine also called cobamide coenzyme only two reactions
use vitaminB12 as coenzymes. Conversion of homo cysteine to Methionine, Methyl
cobalamine is the coenzyme in this reaction. Conversion of methyl malonyl CoA to succinyl
CoA and 5’- deoxy adenosyl cobalamine is used as coenzyme.
Function of 5’-deoxyadenosylcobalamin involves isomerizations involving exchange of

carbon bound hydrogen with another carbon bound functional group as methyl malonyl Co-A
mutase catalyzes reaction of this type given.
7.12 SUMMARY
 Four enzymes which are involved in transfer of group are Vitamin B12, Biotin, TPP, and
Coenzyme A.
 Biotin participates in the transfer of Carboxylic groups.
 Thiamin pyrophosphate or thiamine TDP is the coenzyme responsible for the transfer of
aldehyde and glyoxal group, and it is derived from vitamin B, by phosphorylation.
 Coenzyme A is derived from vitamin pantothenic acid. This is abbreviated as CoA. It is

also called as acetylation coenzyme
 Coenzyme B12 is the cofactor form of vit B12 and involved in isomerisation reaction and
methyl group transfer.
7.13 SAQs
Multiple Choice Questions
1. When metal ions are loosely attached with the enzymes, they become
(a) Catalysts
(b) Coenzymes
(c) Cofactors
(d) Substrates

2. Which of the following participates in enzyme-catalysed reactions
(a) Cofactors
(b) Coenzymes
(c) Meatl ions
(d) None
3. In which form coenzymes transfer energy from one enzyme to another
(a) Oxygen atoms
(b) Nitrogen atoms
(c) Carbon atoms
(d) Hydrogen atoms
4. Nicotinamide adenine dinucleotide (NAD) is made from which vitamin
(a) Vitamin A
(b) Vitamin B
(c) Vitamin C
(d) Vitamin D
5. The non-protein organic molecules are
(a) Coenzymes
(b) Enzymes
(c) Cofactors
(d) All the above

1. Expain the β-oxidation of fatty acids.
2. What are the co-enzymes.
3. Why are co-enzymes necessary. Explain
4. Explain NAD+ and NADP+

5. What are the cofactors? Discuss role of cofactors in coenzyme chemistry.
7.15 REFRENCES
1. V.P Sharma, Eseentials of Bioorganic Chemistry,A Pragati Edition
2. H.K.Chopra, A.Parmar and P.S.Panesar, Bioorganic Chemistry, Narosa Pub.
3. D.V.Vranken and G.A.Weiss, Introduction of Bioorganic Chemistry.
Answers of MCQ
1. c 2. b 3. d 4. b 5. a

UNIT 8: BIOTECHNOLOGICAL APPLICATIONS OF

ENZYMES
CONTENTS:
8.1 Introduction
8.2 Objective
8.3 Large-scale production and purification of enzymes
8.3.1 Upstream process
8.3.2 Downstream process
8.4 Techniques and methods of immobilization of enzymes
8.4.1 Advantages of enzyme immobilization
8.4.2 Methods of Immobilization
8.5 Use of enzymes in food and drink industry-brewing and cheese-making
8.5.1 Enzymes in food industry
8.5.2 Enzymes in drink and brewing industry
8.5.3 Enzymes in cheese making
8.6 Syrups from corn starch
8.6.1 Production of corn syrups
8.6.2 Applications of corn starch
8.7 Enzymes as targets for drug design
8.7.1 Mechanism of inhibition of enzymes by drugs
8.7.2 Applications
8.8 Clinical uses of enzymes
8.8.1 In medical device cleaning
8.8.2 In medicine

8.8.3 In treatment of disorders
8.8.4 Used to assist metabolism
8.8.5 To assist drug delivery
8.8.6 To diagnose disorders
8.8.7 In preparation of toothpaste
8.9 Enzyme therapy
8.9.1 How the enzyme therapy works?
8.9.2 Benefits behind enzyme therapy
8.9.3 Limitations of enzyme therapy
8.10 Enzymes and recombinant DNA technology
8.11 Summary
8.12 SAQs type questions
8.13 Glossary
8.14 References
8.15 Suggested Readings
8.1 INTRODUCTION
Enzymes are high molecular weight proteins that act as biological catalysts. Enzymes have
the tendency to participate in reaction and accelerate it via lowering the activation energy.
Due to such properties enzymes have wide applications industrial sectors such as paper and
food industry, starch industry, textile industry, baking and brewing industry etc. For industrial
applications enzymes can be isolated from various sources including animals, plants and
microorganisms. Enzymes play an essential role in manufacturing of various industrial food
products also. Cheese, beer, wine and vinegar are such few examples. Enzymes are helpful in
controlling various processes such as process time, enrich flavour, improve texture, increase
shelf life etc.

In this unit, we find the commercial methods for production of enzymes, different
immobilization techniques of enzymes, its applications in different sectors i.e. food and drink
industries, cheese making etc., enzymes used as target for drug designing, enzyme therapy
and recombinant DNA technology for developing recombinant enzymes. Enzyme
immobilization is a widespread technology to achieve more stable, active and reusable
enzymes. In this technique, the confinement of enzyme molecules is done onto/within a
support or matrix via physically or chemically or both for retaining full activity. Enzymes not
only energies the industrial sectors but also clinical sectors. Currently, various kind of
enzyme associated diseases are there which is cured via the enzyme therapy or enzyme
targeted drugs. Such diseases or disorders may include lysosomal storage disorders, cancer,
Alzheimer’s disease, irritable bowel syndrome, exocrine pancreatic insufficiency, and
hyperuricemia etc. Enzymes may act as markers in various disease such as myocardial
infarction, jaundice, pancreatitis, cancer, neurodegenerative disorders, etc. Thus, enzymes can
provide insight into the disease process by diagnosis, prognosis and assessment of response
therapy.
8.2 OBJECTIVES
In this unit you will be able to learn the
 The commercial methods for enzyme production including the downstream and
upstream processes.
 Techniques and methods of immobilization of enzymes
 Enzyme applications in clinical and industrial sectors i.e. food and drink industry,
brewing and cheese making industry etc.
 Enzyme therapy and recombinant DNA technology for developing recombinant
enzymes
8.3 LARGE-SCALE PRODUCTION AND PURIFICATION OF

ENZYMES
Enzymes are specific, versatile, and efficient biocatalysts which participate in reaction and
cause lowering of activation energy. Activation energy is the minimal amount of extra energy
required by a reacting molecule to convert into product. The sources of various industrial
enzymes are based on animals (pepsin, trypsin, pancratin, chimosin), plants (papain,

bromelain, ficin), and microorganisms (amylase, proteases, isomerases, glucose oxidases,
pectinases, lactase, cellulase, xylanase, lipase, phytase, invertase, catalase, etc.). Industrial
fermentation is one of the important step in enzyme production in which the substrates are
converted into products. The factors influence this process may depend on temperature,
substrate, pH, aeration and agitation, inhibitors etc.
The essential steps for industrial enzyme production can be categories into two parts
including (1) upstream and (2) downstream processes.
8.3.1 Upstream process
Upstream process refers to all those activities which needed to gather the materials required
to create a particular desired industrial product including inoculum development, media
preparation, cell culture, cell Separation and harvest. When the cells have reached the desired
density, they are harvested and moved to the downstream section of the bioprocess.
8.3.1.1 Sources
Enzymes can be obtained from three different sources including plants, animals and
microorganisms. Some commercial enzymes such as papain, bromelain, ficin, and malt
diastase are obtained from plants. While, pepsin, trypsin, alpha-chymotrypsin, lipase,
catalase, rennin, and pancreatic enzymes are derived from animal sources. The
microorganisms i.e. fungi, bacteria and yeast are chiefly used for the production of industrial
enzymes. A brief list of industrial enzymes, their source and applications are mentioned in
Table 8.1 & 8.2.
Table 8.1 Commercial enzymes from plant sources and their applications
Enzyme Plant source Industrial applications
β-Amylase Barley, Soybean Baking, preparation of maltose

syrup
Bromelain From Bromeliaceae family Pharmaceuticals industry

members i.e. pineapple
Esterase Wheat Ester hydrolysis
Ficin Fig Meat tenderiser

Malt diastase Malted barley Food supplements
Papain Papaya In baking industries, meat

tenderiser, tanning
Peroxidase Horse Radish Diagnostic purposes
Urease Jack Bean Diagnostic purposes
Table 8.2 Industrial microbial enzymes, source and their applications
Enzyme Microbial source Industrial applications
Amylase Bacteria (Bacillusamyloliquifaciens, B. Starch industry, paper and food

licheniformis, B. coagulans, Geobacillus industry, in preparation of
sp.); Fungi (Aspergillusoryzae, A.niger, glucose and maltose syrups,
Rhizopus sp.) high fructose corn syrups,
clarified fruit juices etc.
Catalase Aspergillus sp. Used in food industry and also

in egg processing, in textile
industry removing hydrogen
peroxide from fabrics
Cellulose Fungi (Trichodermareesei, T. viride, Textile industry, pulp and

Penicillium sp., Humicolagrisea, paper industry, and food
Aspergillus sp., industry, as well as an additive
Chrysosporiumlucknowense, in detergents and in
Acremonium sp.) improvement of digestibility of
animal feeds
Dextrinase Gluconobacteroxydans Used in preparation of corn

syrup in starch and syrup
making
Glucose oxidase Aspergillusniger, Penicillin sp. Bakery and food industry
Hemicellulase Fungi (A. niger, T. reesei, Penicillium Food, feed, bioethanol, pulp
sp.) and paper, chemical, and
beverage producing industries
as well as in biorefineries and
environmental biotechnology
Invertase Saccharomyces spp. Food industry, baking and

brewing industries

Keratinase Bacteria (Bacillussubtilis), Fungi Textile industry, detergent

(Aspergillusstelliformis) preparation, leather industry,
Animal feed production
Lactase Yeast (Kluceromyces) Dairy and food industries
Laccases and Aspergillusnidulans, Aspergillus sp., Textile and biofuel production

peroxidase Basidiomycetes
Lipase and A. oryzae,A. terreus, Pseudomonas sp., In dairy, baking, fruit juice,
proteinase Alcaligena spp., Staphylococcus sp., beer and wine industries
Candidaalbicans, Rhizopus sp., Mucor
sp.
Pectinases Aspergillusniger, Penicillin sp. In food industry useful for fruit

juice extraction and wine
clarification; tea, cocoa, and
coffee concentration and
fermentation
Phytase Aspergillus sp., A. ficuum, Paper and pulp industries

P. funiculosum, Bacillus sp.,
Pseudomonas, Xanthomonasoryzae
Protease Bacteria (Bacillusamyloliquifaciens, Leather processing, food

Pesudomonas, Clostridium); Fungi industry, pharmaceuticals,
(Aspergillusoryzae, A. niger, Washing powders
Penicilliumchrysosporium,
Rhizopusoligosporus, Actinomycetes sp.)
Xylanase Fungi (Myeciliophthorathermophila, Wood pulp bioleaching,

Bacillus sp., A. oryzae, Trichoderma sp.) papermaking, the manufacture
of food and beverages, animal
nutrition, and bioethanol
production
8.3.1.2 Screening of microorganisms
The microbial screening is theprocedure of isolation, detection and separation of

microorganisms of interest from a particular population. In term of enzyme production,
screening procedure helps to detect the potential microorganisms with higher production or
yield of certain enzymes. For examples, fungi (Trichodermareesei, T. viride, Penicillium sp.,
Humicolagrisea, Aspergillus sp., Chrysosporiumlucknowense, Acremonium sp.) yeast
(Saccharomycescerevisiae), bacteria (Bacillussubtilis, Pseudomonas, Xanthomonasoryzae,

Clostridium) are few examples of potential microorganisms used in various industrial
production including leather processing, food industry, pharmaceuticals etc.
Fig. 8.1 Steps of Industrial enzyme production
For commercial production the selected microorganisms should fulfil the following criteria.
1. The microorganisms should be capable to grow on an inexpensive medium at a rapid

and constant rate.
2. The microorganisms should produce the enzyme with high yields at a higher
concentration.
3. The selected microorganisms should be genetically stable during the entire production
process.

4. The microorganisms should be able to grow on a concentrated medium for
better/higher enzyme yield.
5. Undesirable enzyme contaminants and the content of metabolites in the fermentation

broth should be minimal.
6. Recovery of the enzyme should be technically feasible and inexpensive.
7. In terms of biohazards, the production process and its product must be safe to the
personnel involved and to the consumer.
8.3.1.3 Raw materials, media formulation and processing
Microorganisms constitute the major source of enzymes, but several enzymes are obtained
from animals and plants sources. The traditional enzyme production relied on the natural
hosts as raw materials. However, selection of wild or genetically engineered microorganisms
may yield sufficiently higher quantities of enzymes. The formulation of culture media is an
essential step. It should provide all nutrients supporting for enzyme production in high
amount but not for good microbial growth. For this, an ideal medium must have a cheap
source of carbon, nitrogen, amino acids, growth promoters, trace elements and little amount
of salts. Some parameters should be regulated including pH, temperature etc. of the culture
media. In media preparation, the source of carbohydrates including molasses, barley, corn,
wheat and starch hydrolysate and for proteins meals of soybean, cotton seed, peanut and
whey, corn steep liquor and yeast hydrolysate are generally used.
8.3.1.4 Industrial scale fermentation process
Medium is sterilized batch-wise in a large size fermenter. For this purpose, continuous
sterilization method is generally used. After medium sterilization, sufficient amount of
inoculum is inoculated to start fermentation process. In traditional method, enzymes
production are done by surface culture technique where inoculum remains on upper surface
of broth. This technique is useful for production of some of the fungal enzymes i.e. amylase
(from Aspergillus sp.), protease (from Mucor sp. and Aspergillus sp.) and pectinase (from
Penicillium sp. and Aspergillus sp.) (table 8.2). Currently, submerged culture method is
widely used due to less chance for contamination and higher yield of enzymes. Growth
conditions e.g. pH, temperature and oxygen are maintained in fermenter at optimum level.
These factors may vary with particular group of microorganisms. Antifoaming agents may

add to fermenter to control foaming during fermentation process. After 30-150 h incubation,
extracellular enzymes are produced by the inoculated microbe in culture medium. Most of
enzymes are produced when exponential phase of growth completes but in a few cases, they
are produced during exponential phase. Besides extracellular enzymes, other metabolites (10-
15 %) are also produced in the fermented broth. These metabolites are removed after enzyme
purification. When fermentation is over broth is kept at 5°C to avoid contamination.
8.3.2 Downstream process
8.3.2.1 Recovery of enzymes
Enzyme purification is a complex process. Recovery of enzymes from the fermented broth
(fluid) of bacteria is quite difficult in comparison to filamentous fungi. Fungal broth is
directly filtered or centrifuged after pH adjustment. Therefore, the bacterial broth is treated
with calcium salts to precipitate calcium phosphate which help in separation of bacterial cells
and colloids. Then the liquid is filtered and centrifuged to remove cell debris.
Thus, the essential steps of purification are mentioned below:
(i) Preparation of concentrated solution by vacuum evaporation at low temperature or by

ultrafiltration
(ii) Clarification of concentrated enzyme by a polishing filtration to remove other

microbes
(iii)Addition of preservatives or stabilizers i.e. calcium salts, proteins, starch, sugar,

alcohols, sodium chloride (18-20 %), sodium benzoate etc.
(iv) Precipitation of enzymes with acetone, alcohols or organic salts, e.g. ammonium
sulfate or sodium sulfate,
(v) Drying the precipitate by free drying, vacuum drying or spray drying, and Packaging
for commercial supply.
8.4 TECHNIQUES AND METHODS OF IMMOBILIZATION OF

ENZYMES
Enzyme immobilization is a widespread technology to achieve more stable, active and
reusable enzymes. Enzyme immobilization can be defined as the confinement of enzyme

molecules onto/within a support or matrix via physically or chemically or both for retaining
full activity. The properties of an ideal support or matrix used should be inert, physically
strong and stable, cost effective, regenerable, biocompatibility, ease of derivatization, mean
particle diameter and swelling behaviour, reduction in product inhibition, enhance specificity
of enzymes, and made up of insoluble material e.g. calcium alginate. The first immobilized
enzyme was amino acylase isolated from Aspergillusoryzae for the production of L-amino
acids in Japan.
8.4.1 Advantages of enzyme immobilization
1. Protection from degradation and deactivation of enzymes
2. Retention of enzymes, enzyme-free products
3. Recycling or repetitive use of enzymes
4. Cost efficiency
5. Enhanced stability and efficiency of enzymes
6. Use as controlled release agents
7. Ability to stop the reaction rapidly by removing the enzyme from the reaction
8. Development of multi-enzyme system
9. Minimum reaction rate and high enzyme substrate ratio
10. Less chance of contamination of products
8.4.2 Methods of Immobilization
Based on support or matrix and types of bonds involved, there are six major types of
principal techniques for immobilization of enzymes mentioned below.
1. Adsorption
2. Covalent binding
3. Entrapment
4. Cross-linking or copolymerization
5. Metal linked Immobilization
6. Encapsulation

8.4.2.1 Adsorption
Adsorption is the simplest, oldest and reversible method. In this method, enzyme is adsorbed
to external surface of the support. The support may be different types (1) mineral support
(e.g. aluminium oxide, clay), (2) organic support (e.g. starch), and (3) modified sepharose and
ion exchange resins. No parmanent bond formation happens between the carrier and enzyme
in this method. Adsorption of enzymes onto the carriers can proceed via different types of
interactions i.e. hydrogen bonds, hydrophobic bonds and vander Waals forces.
Fig. 8.2 Diagrammatic representation of Adsorption method of enzyme immobilization
This method depend on following four processes.
A) Static process
Immobilization to carrier by allowing the solution containing enzyme to contact the carrier
without stirring. It usually uses saturated salt solutions kept inside desiccators with still air.
B) Dynamic batch process
This method uses air with a low flow velocity and this low velocity air will reduce the
equilibrium time. The carrier is placed in the enzyme solution and mixed by stirring or
agitation.
C) Reactor loading process
The carrier is placed in the reactor and the enzyme solution is transferred to the reactor with
continuous agitation.
D) Electrode position process
The carrier is place near to an electrode in an enzyme bath and then the current is put on.
Under the electric field the enzyme migrates to the carrier and deposited on its surface.

Advantages of Adsorption
1. Easy to perform and cheap
2. No reagents are required
3. Enzymes can be mobilized in native states, no activation is required
4. Less disruption of enzyme than chemical methods, and
5. Wide applicability and capable of high enzymes loading.
Disadvantages
1. Desorption of the enzyme resulting from changes in pH, temperature and ionic
strength
2. Slow process, and
3. Low efficiency.
8.4.2.2 Covalent binding
Covalent binding is a classical method. It involves direct attachment of the enzyme onto the
support matrix through the covalent linkage. It provides a strong and stable attachments as
these are formed through reactions between functional group in the support matrix and the
enzyme surface that contains the amino acid residues. It can be used on unmodified proteins
since they relay only on naturally present functional group. Use of spacer arm may provide
greater degree of the mobility to the enzymes to show higher activity. Common support or
carriers are polyacrylamide porous glass, cellulose, collagen, gelatin, DEAE cellulose, and
porous silica.
Fig. 8.3 Diagrammatic representation of Covalent bonding method of enzyme

immobilization

Advantages
1. Tight binding of enzyme to support matrix
2. Comparatively simple and mild method
3. Minimized enzyme leaching in aqueous media
4. Wide applicability.
Disadvantages
1. Enzymes may be chemically modified, can be denatured.
2. Only small amounts can be immobilised (about 0.02 g/ gram of Matrix).
8.4.2.3 Entrapment
In this method, enzymes are physically entrapped inside a porous matrix. This method is the
best immobilization strategy to avoid any negative influence on enzyme structure. Enzymes
are immobilized by occlusion in the synthetic or natural polymeric networks. Entrapment can
be achieved through gel or fibre entrapping and micro-encapsulation. Sol-gels widely used or
highly porous silica materials, extensively used for protein immobilization particularly for the
development of the biosensors.
Fig. 8.4 Diagrammatic representation of Entrapment method
Entrapment can be further classified into lattice type and microcapsule types.
A) Lattice-type entrapment involves entrapping enzymes within the interstitial spaces of a

cross-linked water insoluble polymer.

B) Microcapsuletype
It involves enclosing the enzymes within semipermeable polymer membranes.
Advantages
1. Fast cheap and mild conditions required for reaction process.
2. Easy to practice at small scale.
3. Large surface area between polymer and enzyme.
4. Loss of enzyme activity upon immobilization is minimized.
5. Used in sensing application.
6. Very less chance for conformational changes in enzyme.
Disadvantages
1. Limitation in mass transfer.
2. Enzyme inactivation during encapsulation.
3. Leakage of enzyme.
4. Pore diffusion limitation.
5. Chance of microbial contamination.
8.4.2.4 Cross linking or copolymerization
This method is an irreversible carrier-free enzyme immobilization. The enzyme acts as its
own carrier.Formation of intermolecular cross linkage between the enzyme molecules by
meaning of bi or multifunctional reagents i.e. glutaraldehyde. Nanodiametric supports such as
electrospun.
Cross-linked enzyme aggregates is prepared by first aggregating the enzymes in precipitance

such as acetone, ammonium sulphate, ethanol or 1,2-dimethoxyethane followed by addition
of a cross-linker like glutaraldehyde.
Advantages
1. Very little enzyme desorption.
2. Best used in conjunction with other methods.

Disadvantages
Cross-linking may cause significant changes in the enzyme’s active site.
Fig. 8.5 Diagrammatic representation of Cross linking method
8.4.2.5 Metal linked Immobilization
In this method, transition metal salts or hydroxides are precipitated onto the support by
heating or neutralization. The deposited matrix cannot occupy all coordination sites of metal,
some are free to bind with enzymes. The metals i.e. titanium and zirconium salts and for
supports i.e. cellulose, chitin, algenic acid, and silica – based carriers are used. The
immobilized metal – ion affinity (IMA) absorbents - Chelator ligands viz. EDTA can
immobilized first onto the solid supports by means of a stable covalent bonds, metal irons are
then bound by coordination.
Advantages
Simple and the immobilized specific activities can be obtained with enzymes.
Disadvantages
Operational stabilities are highly variable and the results are not easily reproducible.
8.4.2.6 Encapsulation
This type of immobilization is done by enclosing the enzymes in a membrane capsule. The
capsule made up of semipermeable membrane like nitrocelulose or nylon. In this method, the
effectiveness depends upon the stability of enzymes inside the capsule.

Advantages
1. This method is cheap and simple method.
2. Large quantity of enzymes can be immobilized by encapsulation.
Fig. 8.6 Diagrammatic representation of encapsulation method
Disadvantages
1. Pore size limitation.
2. Only small substrate molecule is able to cross the membrane.
8.5 USE OF ENZYMES IN FOOD AND DRINK INDUSTRY-

BREWING AND CHEESE-MAKING
Enzymes play an essential role in manufacturing of various industrial food products. Cheese,
beer, wine and vinegar are such few examples. Enzymes are helpful in controlling various
processes such as process time, enrich flavour, improve texture, increase shelf life etc.
Enzymes breakdown complex molecules into smaller units such as carbohydrates into sugar.
They are natural substances involved in all biochemical reactions. Enzymes are produced by
all living cells and act as catalysts for specific chemical reactions. Enzymes are highly
efficient which help in cut off effective cost of its respective processes. The application of
enzymes as used in various sectors of food, drink, brewing and other industries are mentioned
below.

8.5.1 Enzymes in food industry
Enzymes are very essential component of life processes such as digestion, respiration and
metabolism in organisms. Due to its impressive catalytic efficiency, it has wide application in
food industry and processing, especially in the preparation of beer, wine, cheese and bread.
Enzymes are extracted from edible plants/plant parts and animal tissues or from
microorganisms like bacteria, yeasts, and fungi. Rennet is a natural enzyme from the stomach
of calves and other domestic animals which used in the preparation of cheese. Other
industrial revolutionary growth of the enzyme industry may include the baking, beverages,
brewing and dairy products. Additionally, enzymes also find applications in detergents,
leather, textile pulp, and paper industries. Some of the enzymes as used in food industry are
mentioned following:
1. Alpha-amylaseis used to solubilize the carbohydrates found in barley and other cereals
used in brewing.
2. Beta-glucanasecause breakdown of glucans in malt and other materials.
3. Lipaseused to shorten the time for cheese ripening. It is employed in the production of
enzyme-modified cheese/butter from cheese curd or butterfat.
4. Papain is widely used as a meat tenderizer.
5. Chymosin helps in the curdling of milk by breaking down kappa-caseins in cheese

making.
6. Microbialproteasesused in the production of fish meals, meat extracts, texturized

proteins, etc.
7. Pectinaseused in treatment of fruit pulp to facilitate juice extraction. It also helps in the
clarification and filtration of fruit juice.
8. Lactaseused as additive for dairy products for individuals lacking lactase.
9. Glucose oxidasecauseconversion of glucose to gluconic acid to prevent Maillard reaction

(reaction that gives browned food a particular flavor) in products caused by high heat
used in dehydration.
10. Cellulase cause conversion of cellulose waste to fermentable feedstock for ethanol or
single-cell protein production.

8.5.2 Enzymes in drink and brewing industry
Enzymes are novel alternatives to chemical or mechanical methods for improving yield and
quality in the beverage industry. Some enzyme i.e. pectinases, amylases, cellulases, and
xylanases are used in the extraction and clarification of fruit juices. In beer production, sugar
is converted into alcohol. During mashing, enzymes from malt, which is germinated barley,
act on the starch of different grains to form sugars. The malting requires grains and heat for
drying, by passing this process will save energy as well as agricultural land. B. subtilis
proteases and used to solubilize proteins from barley adjuncts for production of wort. Haze
formation due to proteinaceous substances in beer is also hydrolysed by microbial proteases.
Following are some of the enzymes as used in drink and brewing industry.
1. Alpha amylases are used for the production of ethanol.
2. Carbohydrase enzymes are used as a wine enzyme that helps to reduce grape-juice
viscosity and fouling of ultrafilter membranes.
3. Cellulases used in the beer industry to hydrolyzed cellulose and barley β-glycans during
wheat are added as an adjunct.
4. Esterases play a prominent role in various food industries and alcoholic beverage
industries. It is widely used to produce fragrances and flavours and in modification of oil
and fat of fruit juices.
5. Papain is used in post-fermentation stages for beer production.
6. Tannase is extensively used as a clarification of beer and fruit juices. It is used in the
manufacturing of coffee-flavored soft drinks and manufacturing for instant tea.
7. Xylanases enzyme employed for clarifying the wine and to reduce the viscosity in
beverage industries.
8. Pectinase enzyme is used in the beverage industry for making wine. This enzyme
influences the viscosity of wine, the colour, and the flavour of the finished products.
8.5.3 Enzymes in cheese making
Cheese is way of preserving milk for long periods of time. Cheese-making is a long and
involved process that makes use of bacteria, enzymes and naturally formed acids to solidify
milk proteins and fat and preserve them. Once turned into cheese, milk can be stored for

many months or years. Acids and salts play role as main preservatives that give cheese its
longevity. There are four types of enzymes used to produce cheese including -
1. Rennet
During cheese making, milk is first inoculated with lactic acid bacteria and rennet. Rennet
contains a enzyme named as rennin helps in modifying proteins in milk. Specifically, rennin
converts a common protein in milk called caseinogen into casein, which does not dissolve in
water. The casein precipitates out as a gel-like substance that we see it as curd. The casein gel
also captures most of the fat and calcium from the milk. So the lactic acid and the rennet
cause the milk to curdle, separating into curds and whey.
2. Proteases
In cow milk, whey protein is found which is breakdown by certain enzyme known as
proteases. Thus, proposes help in breakdown of these proteins and peptide bonds. During the
production of the soft cheese, the whey protein is separated from the milk after curdling.
3. Catalase
This enzyme helps in breakdown hydrogen peroxide into hydrogen and water. This is used in
making cheese in order to preserve the natural milk enzymes that are beneficial to the flavour
development of the cheese product.
4. Lipases
Lipases help in breakdown of milk fatty acids during cheese making process. For stronger
flavoured cheeses they are prepared using lipases. These flavours come from the fatty acids
that were produced when the milk fats are hydrolysed.
8.6 SYRUPS FROM CORN STARCH

Corn syrup is one of the food corn made from starch of corn. This is a generic name for a
whole spectrum of nutritive sweeteners prepared from corn starch. Corn syrup is a purified
and concentrated mixture of the saccharides obtained from the hydrolysis of corn starch. The
corn starch contains very rich amount of sugar glucose, maltose and some higher
oligosaccharides. Practically, corn syrup is the liquid hydrolysate of mono-, di, and higher
saccharides can be prepared from starch, wheat, tapioca, and potatoes etc. It is also known as
the glucose syrup and generally used to soften texture volume, prevent crystallisation of sugar

and enhance flavour. High fructose corn syrup (HFCS) is a liquid alternative sweetener to
sucrose. According to fructose content, the fructose – glucose syrup is divided into three
specifications HFCS42, HFCS55 and HFCS90.
8.6.1 Production of corn syrups
For corn syrup production, corn starch is chiefly used as row material. In some industries,
corn powder is directly converted into corn syrup. But in case of high quality corn syrup, corn
starch is best for producing corn syrup. In brief, the process of corn syrup production is
mentioned below:
8.6.1.1 Raw material preparation
The corn variety popularly known as “yellow dent corn” is majorly used as source of corn
syrup preparation. It is one of the common variety cultivated in mid western portion of the
United States and other part of world. Other materials used during the process of converting
corn to corn syrup including sulphur dioxide, hydrochloric acid or various enzymes and
water. For raw material preparation, the corn starch should have high amount of corn starch.
Ideally, the specification should be as starch content (85.4%), moisture (14%), fat (0.1%),
protein (0.4%), and ash 0.1%.
8.6.1.2 Mixing process
The mixing process helps corn starch or starch milk to adjust condition for liquefaction. This
process should be done under controlled temperature and pH (5.3 - 5.6). A suitable amount of
water should be added to get desirable concentration of medium.
8.6.1.3 Liquefaction
The starch milk is fully contacted with steam through the injection port, rapidly heats up, the
starch is fully liquefied which inactivated by a high temperature and high pressure jet.
8.6.1.4 Saccharification
After liquefaction, the pH value is reduced to between 4.2 and 4.5, after the solution is cooled
up to 60 ℃. The liquefied material keeps reaction for a certain time under the action of
enzyme. The required dextrose equivalent (DE) value of the glucose should be 15-20%. The
reaction time for saccharification is usually between 24-48 hour depending on enzyme
reaction speed. In this step, Glucoamylase enzyme help in releasing single glucose units from

the ends of dextrin molecule. Syrups of 95% glucose or higher are manufactured by this
process.
8.6.1.5 Filtration and decolourization
Glucose gets through the filter to remove protein and other impurities, then at the appropriate
temperature passes by active carbon to decolouring, finally send to filters to remove activated
carbon and send to the next section.
Fig. 8.7 Process of making corn starch syrup
8.6.1.6 Evaporation
The glucose is totally cleaned through safety filter machine then sent to evaporator for
concentrating to reach the required DS as final product. The corn syrup powder, also called

corn syrup solids, is prepared by a drum or spray dryer to remove 97% of the water. This
produces a crystalline corn syrup powder.
8.6.2 Applications of corn starch
Corn syrups have wide commercial applications in preparation of food stuffs as mentioned
below:
1. It is widely used in food, canned foods, jam, dairy products, beverages, tobacco, cold
drinks, fruit juices, preserve foods and wines etc.
2. It act as thicker, sweetener and humectant of commercial foods such as breads, cereals,
yogurts, soups and condiments etc.
3. It retains moisture and maintain freshness of food products.
4. Corn syrup is used in the production of HFCS which include baked foods, sodas, yogurts
and condiments. HFCS enhances sweetness, texture and flavour, stabilise colour and
cause cost reduction of products.
5. HFCS is effective in enhancing texture and sparsity of ice cream and other dairy products
i.e. chocolate milk.
6. It can replace cane sugar with similar concentration. While, its flavour is similar to
natural juice.
7. The application in other sectors such as pharmaceutical, agriculture, animal feed, poultry
feed is yet not reported.
8.7 ENZYMES AS TARGET FOR DRUG DESIGN

Enzymes are known to catalyse thousands of biochemical reaction types. They act as a target
for drugs for the desired therapeutic effect, which called as biological targets. Enzymes offer
unique opportunities for drug designing that are not available to cell surface receptors,
nuclear hormone receptors, ion channels, transporters. Drugs acting on enzymes by two ways,
first inhibition of enzymes (inhibitors) and second inactivation of enzyme (inactivators). In
this respect, first one is more common. The enzyme-inhibitor or inactivator drugs have do so
either via covalent reaction with nucleophilic enzymatic residues or by noncovalent binding.
Such drugs prevent the formation of the product by inhibiting the enzyme - substrate
interaction via competitive, non-competitive or uncompetitive interactions. Few examples of

such targeted enzymes are microsomal enzymes in neonatal jaundice and cushing’s disease,
transmembrane receptors such as receptor tyrosine kinases, JAK-STAT receptors, receptor
Serine-Threonine Kinases, Toll-like receptors and TNF-α receptors etc.
8.7.1 Mechanism of inhibition of enzymes by drugs
The enzyme-substrate reaction is well known. In this, substrate molecule reacts with the
enzyme to form an enzyme-substrate (ES) complex which at the end results in the formation
of the final products. Drugs inhibiting enzymes prevent the formation of the product by
inhibiting the enzyme-substrate interaction competitively, non-competitively or
uncompetitively. The type of inhibition by the drug is hence classified accordingly.
8.7.1.1 Competitive Enzyme Inhibition by Drugs
Competitive inhibition is caused by compounds which resemble the chemical structure and
molecular geometry of substrate molecule. In this type of inhibition, the inhibitor binds only
to the enzyme and not to the enzyme-substrate complex. The inhibitor gets strongly stuck on
the enzyme which prevents any substrate molecules from further reacting with the
enzyme.However, competitive inhibition is usually known to be surmountable if sufficient
substrate molecules are available to ultimately.
8.7.1.2 Non-competitive inhibition by drugs
Non-competitive inhibition is caused by a substance that reacts with the enzyme at the
allosteric site. It binds to the enzyme as well as the ES complex with equal affinity. Drugs
need not have a similar structure as that of the substrate of the enzyme. This type of inhibition
is unsurmountable. Due to change in shape of active site, the substrate can no longer react
with the enzyme to give the response. Non-competitive inhibition is usually irreversible but
in few cases reversible inhibition have been observed e.g. carbonic anhydrase inhibition by
acetazolamide.
Disulfiram is a non-competitive inhibitor of the enzyme aldehyde dehydrogenase. It forms an

active metabolite diethylthiomethyl carbamate (DETMC). Conversion of acetaldehyde to
acetic acid is stopped. Acetaldehyde accumulation causes nausea, vomiting, flushing, etc.
which is used as a favouring factor in process of alcohol de-addiction. Disulfiram like
reaction is also seen with metronidazole, schlorpropamide, glibenclamide, tolbutamide,

griseofluvin, cefotetan, cefoperazone, etacrynic acid and urinary antiseptics such as
nitrofurantoin because of inhibition of Aldehyde Dehydrogenase by them.
8.7.1.3 Uncompetitive Inhibition by drugs
Uncompetitive inhibition occurs when a drug binds reversibly to the enzyme when the
substrate is already bound to the active site. The inhibitor binds to the enzyme-substrate
complex. Increasing the concentration of substrate will not overcome the inhibition in this
case. The level of inhibition depends on the sufficient substrate being present at the active site
to make E-S complex. Initially, reaction rate is speeded up to the formation of the E-S
complex till a point after which the rate slows down and never reaches Vmax. Hence, the
enzyme will not reach Vmax. The Lineweaver-Burk double reciprocal plot for this set of data
doesn’t show inhibitor line converging on the same point on the X (1/S) axis or the Y (1/V)
axis i.e. Km and Vmax is also reduced. Following are few examples of targeted enzymes at
various sites i.e. suicide inhibitor drugs, microsomal enzymes, transmembrane receptors
linked to intracellular enzymes etc.
8.7.1.4 Suicide Inhibitor drugs
Suicide inhibition, also termed as suicide inactivation or mechanism-based inhibition.It

causes an irreversible form of enzyme inhibition wherein an enzyme binds an inhibitor and
forms an irreversible complex with the enzyme. For example β-lactamase inhibitors are given
with β-Lactams to inhibit β-Lactamase enzyme and hence prevent hydrolysis of β-Lactam
drugs like penicillins. Clavulanic acid covalently bonds to a serine reside present in the active
site of the β- lactamase, which results in restructuring of the clavulanic acid molecule.
8.7.1.5 Microsomal enzymes as drug targets
Microsomal enzymes are enzymes which are typically found in the endoplasmic reticulum of
hepatocytes. They play a very important role in the metabolism of drugs and some
endogenous substrates. These enzymes can be induced or inhibited by certain drugs in order
to affect the metabolism of certain endogenous substrates or drugs.
8.7.1.6 Transmembrane receptors linked to intracellular enzymes
Transmembrane receptors act in cell signaling by binding to extracellular molecules. Signal

transduction is a process through membrane receptors which involves some external
reactions, in which the ligands binds to a membrane receptor, and the corresponding internal

reactions, in which intracellular response is triggered. Majority of the enzyme-linked
receptors are protein kinases or are associated with it.There are various types of
Transmembrane receptors linked to intracellular enzymes i.e. receptor tyrosine kinases, JAK-
STAT receptor, receptor serine-threonine kinases, toll-like receptors (TLR), TNF-α receptors
(Tumor necrosis factor alpha receptors) etc.
8.7.2 Applications
Pharmaceutical industries use enzymes as drug targets to manufacture drugs e.g.

dihydrofolate reductase inhibitors such as methotrexate, phosphodiesterase inhibitors such as
theophylline, etc. Role of enzymes is also extended to therapeutics for example
serratiopeptidases as antii-inflammatory, β-lactamases for penicillin allergy, tissue
plasminogen activators as fibrinolytics. Various anti-cancer drugs have been designed to
resolve cancerous diseases.
8.8 CLINICAL USES OF ENZYMES

Enzymes act as markers in various disease such as myocardial infarction, jaundice,
pancreatitis, cancer, neurodegenerative disorders, etc. They can provide insight into the
disease process by diagnosis, prognosis and assessment of response therapy. The major
applications and uses of such enzymes in clinical sectors are mentioned below.
8.8.1 In medical device cleaning
Enzyme based detergents is used for cleaning of various reusable medical devices. In which
two enzymes are chiefly used named as protease and lipase. Proteases breakdown the protein
rich soils like blood etc. While, lipases used for fatty soils like adipose tissues. Some other
examples i.e. amylases and cellulases help in breakdown of starch and cellulose polymers.
8.8.2 In medicine
Enzymes are typically used as medicines to interchange enzyme deficiencies in patients such
like blood coagulation factors to treat bleeder’s disease, cleaning of wounds, healing,
improving metabolism, in diagnosis of diseases etc. Some popular enzymes i.e. proteases,
carbohydrases, and lipases are widely used in pharmaceutical and healthcare sectors.

8.8.3 In treatment of disorders
In terms of disorders, enzymes are used in three cases including 1) to break the internal blood
clots, 2) to dissolve the hardening of walls of blood vessels, and 3) to dissolve the wound
swelling to promote healing. In some disorders like low blood pressure, or head or spinal
injuries, there are chances of formation of blood clots. Thus, only way is to dissolve the clots.
These clots are usually removed by dissolution by enzymes that can break them. Similarly,
when there is atherosclerosis, hardening and thickening of blood vessel walls. The best way
out at this junction is to decrease the fat intake and also dissolve the formed thickenings.
Enzymes like serratiopeptidase work well at this place. For wound healing, the swelling
formed might be painful and tend to form pus. Enzymes such as trypsin, chymotrypsin,
serratiopeptidase are used to dissolve the swelling.
8.8.4 Used to assist metabolism
In geriatric patients, the digestive capacity is low due to insufficient secretion of digestive
enzymes. In such cases, the patients can be experienced malnutrition, constipation, bloating
etc. To improve such digestive condition, enzymes like papain are administered orally after
food for better digestion.
8.8.5 To assist drug delivery
Some drugs have higher penetration capability inside the tissues. For this, some enzymes i.e.
hyaluronidase are used with such drugs in intra-muscular injection forms. Hyaluronidase is
one of kind of natural enzyme to assist sperms in penetration of female internal reproductive
organ and fertilise with ova.
8.8.6 To diagnose disorders
Enzymes of the liver, kidney, skeletal muscle, heart, etc. leak into blood during related
disorders. Measuring the amount of the corresponding protein in high or low levels in blood
indicates the particular disorder. For example, creatine kinase is for muscle weakness and
injury. Similarly, polymerase chain reaction (PCR) help to diagnose genetic diseases in the
prenatal stage for disorders like sickle cell anaemia, Huntington’s disease, beta-thalassemia
etc.

8.8.7 In preparation of toothpaste
Enzymes of papaya and pineapple are used in the dentifrice. They are found to remove the
stain on teeth to give white and sparkling teeth.
8.9 ENZYMES THERAPY

Enzyme therapy is a therapeutic tool for improvement of body’s ability to maintain certain
disorders or diseases related to any enzymatic biological processes. The biological processes
may relate to metabolism deficiencies, immune system, cancer, cardiovascular disease,
microbial system etc. The enzymes act as a target for drug designing for the desired
therapeutic effects, known as biological targets. Currently, the enzyme therapy is being
studied in treatment of the virus SARC-CoV2 and its associative other viruses, the causative
agents of CoVID-19 disease.
8.9.1 How the enzyme therapy works?
The enzyme-based therapy may be systemic or non-systemic. They have multiple

administration routes including oral, topic, respiratory or intravenous. Diseases caused by
absence or dysfunction of enzymes are the main target of enzyme replacement therapy
(ERT). The use of enzymes for therapy is highly disease specific and varies with each and
every diseased condition especially while considering ERT. These medical conditions are
tried to restore the lost or altered enzyme activities via administration of enzymes
intravenously. Such diseases or disorders may include lysosomal storage disorders, cancer,
Alzheimer’s disease, irritable bowel syndrome, exocrine pancreatic insufficiency, and
hyperuricemia.
8.9.2 Benefits behind enzyme therapy
Enzymes have been associated with multiple futuristic therapies including cancer, microbial
infection, wound healing and gene therapies, lysosomal storage disorders, Alzheimer’s
disease, irritable bowel syndrome, exocrine pancreatic insufficiency, hyperuricemia,
8.9.3 Limitations of enzyme therapy
The limitations of enzyme therapy are mentioned below:
1. The modification of targeting lysosomes

2. Low availability of enzymes in some tissues
3. Short half-life of the enzyme
4. Lack of ability to permeate the blood-brain barrier
5. Side effects that arise from immunogenicity of the enzyme, and
6. An inefficient mode of delivery and a high cost of enzymes etc.
8.10 ENZYMES AND RECOMBINANT DNA TECHNOLOGY

The recombinant DNA technology (RDT) provides a molecular tools to produce recombinant
DNA (rDNA) by using a set of enzymes known as recombinant enzymes e.g. DNA ligase,
restriction enzymes, DNA polymerases, exonuclease etc. The functionality of these enzymes
are quite diverse and may used as powerful tools in various areas i.e. genetic engineering,
molecular biology, proteomics, bioinformatics etc. The multiple functions of these enzymes
may include host-controlled restriction, chain elongation, ligation, protection of their own
DNA from cleavage, methylation etc. A detailed description of enzymes used in RDT with
their functionality has been mentioned in given table 8.3.
Table 8.3 Enzymes used in recombinant DNA technology
Enzyme(s) Function
Type I Restriction endonucleases It has both methylation and endonuclease activity. It cut DNA
about 1000 bp away from the restriction site e.g. EcoKI
Type II Restriction It cuts DNA at restriction site itself e.g. EcoRI, Hind III
endonucleases
Type III Restriction It cuts DNA about 25 bp away from the restriction site e.g.
endonucleases EcoPI
DNA polymerases It helps in elongation of DNA strand via addition of nucleotide

to free 3’OH end. DNA polymerase I isolated from E. coli as
used in gene cloning and Taq polymerase isolated from
Thermusaquatics used in PCR.

Reverse transcriptase It is used to synthesise complementary strand (cDNA) from

mRNA. It is isolated from retrovirus.
Exonuclease III It removes nucleotide residues from the 3’ end of a DNA

strand
DNA ligase Join two DNA molecules or fragments
Polynucleotide kinase It add a phosphate group to the 5’ OH end of DNA strand or

permit ligation
Terminal transferase Add homopolymer tails to the 3’OH ends of linear duplex
Alkaline phosphatase It removes terminal phosphates from either the 5’ or 3’ end or

both.
Ribonuclease H It removes mRNA from DNA-RNA heteroduplex which used

by mRNA to synthesise cDNA
8.11 SUMMARY
Enzymes are specific, versatile, and efficient biocatalysts which participate in reaction and
cause lowering of activation energy. Industrial fermentation is one of the important step in
enzyme production in which the substrates are converted into products. The factors influence
this process may depend on temperature, substrate, pH, aeration and agitation, inhibitors etc.
The essential steps for industrial enzyme production can be categories into two parts
including (1) upstream and (2) downstream processes. Upstream process includes inoculum
development, media preparation, cell culture, cell Separation and harvest. While, the
downstream process includes the recovery of enzymes from the fermented broth.
Enzyme immobilization is a widespread technology to achieve more stable, active and

reusable enzymes. It can be defined as the confinement of enzyme molecules onto/within a
support or matrix via physically or chemically or both for retaining full activity. There are six
major types of principal techniques for immobilization of enzymes include adsorption,
covalent binding, entrapment, cross-linking or copolymerization, metal linked
Immobilization, and encapsulation. The advantages of enzyme immobilization includes the
protection from degradation and deactivation of enzymes, retention of enzymes, enzyme-free

products, recycling or repetitive use of enzymes, enhanced stability and efficiency of
enzymes, use as controlled release agents, ability to stop the reaction rapidly by removing the
enzyme from the reaction, development of multi-enzyme system and minimum reaction rate
and high enzyme substrate ratio.
In industrial application, enzymes are widely used in various products including baking,
beverages, brewing and dairy products. Additionally, enzymes also find applications in
detergents, leather, textile pulp, and paper industries. The enzymes used in such processes
include alpha-amylase, beta-glucanase, lipase, papain, chymosin, pectinase, lactase, glucose
oxidase, cellulase etc. The enzymes used in cheese making are rennet, proteases, catalase,
lipases etc. respectively. Enzymes are also useful in drug designing. They act as a target for
drugs for the desired therapeutic effect, which called as biological targets. Enzymes offer
unique opportunities for drug designing that are not available to cell surface receptors,
nuclear hormone receptors, ion channels, transporters. The drug inhibitions of enzymes are
classified into three major types including Competitive inhibition, non-competitive inhibition,
and Uncompetitive inhibition. Enzymes act as markers in various disease such as myocardial
infarction, jaundice, pancreatitis, cancer, neurodegenerative disorders, etc. They can provide
insight into the disease process by diagnosis, prognosis and assessment of response therapy.
Currently, recombinant DNA technology (RDT) provides a molecular tools to produce
recombinant DNA (rDNA) by using a set of enzymes known as recombinant enzymes e.g.
DNA ligase, restriction enzymes, DNA polymerases, exonuclease etc. The functionality of
these enzymes are quite diverse and may used as powerful tools in various areas i.e. genetic
engineering, molecular biology, proteomics, bioinformatics etc. The multiple functions of
these enzymes may include host-controlled restriction, chain elongation, ligation, protection
of their own DNA from cleavage, methylation etc.

1. Which enzyme cause conversion of cellulose waste to fermentable feedstock for ethanol or
single-cell protein production -
A) Pectinase B) Cellulase C) Lactase D) Glucose oxidase
2. Which enzyme is used as a clarification of beer and fruit juices?

A) Papain B) Cellulase C) Tannase D) Carbohydrase
3. The enzyme used to synthesise complementary strand (cDNA) from mRNA named as -
A) Type I restriction enzyme B) DNA ligase C) Reverse transcriptase D) DNA

gyrase
4. DNA molecules or fragments can be ligated by -
A) DNA ligase B) Alkaline phosphatase C) Reverse transcriptase D) None of these.
5. The first immobilized enzyme was amino acylase isolated from -
A). Aspergillusoryzae B) Clostridium spp. C) Asergillus niger D) Penicillium spp.
6. Enzymes are specific, versatile, and efficient biocatalysts which participate in reaction.
A) Amylases B) papain C) cellulases D) Pectinases and pectinesterases
7. Phytase, the enzyme used in -
A) Paper and pulp industry B) meat industry C) dairy industry D) None of

these
8. It removes nucleotide residues from the 3’ end of a DNA strand -
A) DNA ligase B) Type III Restriction endonucleases
C) Exonuclease III D) None of these
9. Pepsin, trypsin and pancratin isolated from -
A) Animal B) Plant C) Fungi D) Bacteria
10. For commercial production of enzymes the selected microorganisms should be -
A) genetically stable B) genetically unstable C) Morphologically stable

D) Morphologically unstable
B. Fill in the blanks
1. ………………………….. is widely used as a meat tenderizer.
2. Taq polymerase isolated from a bacterium named as …………………….. used in PCR.
3. Adsorption is one of the simplest, ……………. and ………………… method.
4. Calcium alginate is an insoluble material chiefly used in ………………… of enzymes.

5. Rennet contains a enzyme named as ………………… helps in modifying proteins in
milk.
6. HFCS stands for……………………….
7. The enzyme known as ……………… converts a common protein in milk called

caseinogen into casein.
8. The first immobilized enzyme was amino acylase isolated from ……………… for the
production of L-amino acids.
9. Invertase, the enzyme used in food industry isolated from …………………
10. In …………………. method, enzymes are physically entrapped inside a porous matrix.
C. True/False
1. Enzymes are non-specific and inefficient biocatalysts which cannot participate in

reaction. (True/False)
2. Disulfiram is a non-competitive inhibitor of the enzyme aldehyde dehydrogenase.

(True/False)
3. High fructose corn syrup (HFCS) is a liquid alternative sweetener to sucrose.

(True/False)
4. Corn syrup is one of the food corn made from starch of wheat. (True/False)
5. Rennet contains a enzyme named as rennin helps in modifying proteins in milk. (True/
False)
6. Catalase helps in breakdown of hydrogen peroxide into hydrogen and water. (True/False)
7. The preparation of cheese is not a way of preserving milk for long periods of time.
(True/False)
8. Lipases help in breakdown of milk fatty acids during cheese making process.
(True/False)
9. Carbohydraseenzymes are used as a wine enzyme that helps to reduce grape-juice

viscosity. (True/False)
10. Papainis used in post-fermentation stages for beer production. (True/False)

1. PCR - (A) Severe Acute Respiratory Syndrome

Coronavirus 2
2. cDNA - (B) Gluconobacter oxydans
3. HFCS - (C) Join two DNA molecules or fragments
4. Encapsulation - (D) Jack Bean
5. Papain - (E) High fructose corn syrup
6. Urease - (F) enzyme replacement therapy
7. Dextrinase - (G) Reverse transcriptase
8. ERT - (H) A method of immobilization
9. DNA ligase - (I) Papaya
10. SARC-CoV2 - (J) Polymerase chain reaction
Answer Key
1. B 2. C 3. C 4. A 5. A 6. D 7. A 8. C 9. A 10. A
B. Fill in the blanks
1. Cellulose 2. Thermusaquatics 3. oldest, reversible 4. immobilization
5. rennin 6. High fructose corn syrup 7. Renin 8. Aspergillusoryzae
9. Saccharomyces spp.
10. entrapment
C. True and False -
1. False 2. True 3. True 4. False 5. True 6. True
7. False 8. True 9. True 10. True

D. Match the following -
1. J 2. G 3. E 4. H 5. I 6. D 7. B 8. F 9. C 10. A
8.13 GLOSSARY
ERT - Enzyme Replacement Therapy
HFCS - High fructose corn syrup
TLR - Toll-like receptor
PCR - Polymerase chain reaction
SARC-CoV2 - Severe Acute Respiratory Syndrome Coronavirus 2
RDT - Recombinant DNA technology
cDNA - Complementary DNA
rDNA - Recombinant DNA
DETMC - Diethylthiomethyl carbamate
8.14 REFERENCES
1. Fasim A, More VS, More SS (2021) Large-scale production of enzymes for
biotechnology uses. Current Opinion in Biotechnology 69: 68-75.
https://doi.org/10.1016/j.copbio.2020.12.002
2. Crueger W, Crueger A (2017) Cruegers Biotechnology: A Textbook of Industrial

Microbiology (3rd Ed.). Medtech publication. ISBN: 978-9385998638
3. Singhania RR, Patel AK, Pandey A (2010). The Industrial Production of Enzyme. In:
Industrial Biotechnology Sustainable Growth and Economic Success. Soetaert W and
Vandamme EJ (Ed.). Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. pp: 207-225.
https://doi.org/10.1002/9783527630233.ch5
4. Mohamad NR, Marzuki NH, Buang NA, Huyop F, Wahab RA (2015) An overview of
technologies for immobilization of enzymes and surface analysis techniques for
immobilized enzymes. Biotechnol Biotechnol Equip. 29(2):205-220.
https://doi.org/10.1080/13102818.2015.1008192

5. Cacicedo ML, Manzo RM, Municoy S, Bonazza HL, Islan GA, Desimone M et al. (2019)
Chapter7 Immobilized Enzymes and Their Applications. Singh RS, Singhania RR,
Pandey A, Larroche C (Ed.) In Biomass, Biofuels, Biochemicals, Advances in Enzyme
Technology, Elsevier pp. 169-200. ISBN 9780444641144. https://doi.org/10.1016/B978-
0-444-64114-4.00007-8
6. Raveendran S, Parameswaran B, Ummalyma SB, Abraham A, Mathew AK, Madhavan

A, Rebello S, Pandey A (2018) Applications of Microbial Enzymes in Food Industry.
Food Technol Biotechnol. 56(1):16-30. https://doi.org/10.17113/ftb.56.01.18.5491
7. Featherstone S (2015) Ingredients used in the preparation of canned foods. In: A

complete course in canning and related processes (4th Ed.), Vol. 2: Microbiology,
packaging, HACCP and ingredients. pp. 147-211. https://doi.org/10.1016/B978-0-85709-
678-4.00008-7
8. https://en.wikipedia.org/wiki/Corn_syrup
9. Geronikaki A (2021) Recent trends in enzyme inhibition and activation in drug design.
Molecules 26 (1): 17. https://doi.org/10.3390/molecules26010017
10. Robertson JG (2007) Enzymes as a special class of therapeutic target clinical drugs and
modes of action. Curt Open Struct Biol 17(6): 674-679.
https://doi.org/10.1016/j.sbi.2007.08.008
11. Hemalatha T, UmaMaheswari T, Krithiga G, Sankaranarayanan P, Puvanakrishnan R

(2013) Enzymes in clinical medicine: an overview. Indian J Exp Biol. 51(10):777-788
12. Meghwanshi GK, Kaur N, Verma S, Dabi NK, Vashishtha A, Charan PD et al. (2020)
Enzymes for pharmaceutical and therapeutic applications. Biotechnology and Applied
Biochemistry 67(4):586-601. https://doi.org/10.1002/bab.1919
13. https://en.wikipedia.org/wiki/Enzyme_replacement_therapy
14. de la Fuente M, Lombardero L, Gomez-Gonzalez A, Solari C, Angulo-Barturen I, Acera

A, et al. (2021) Enzyme Therapy: Current Challenges and Future Perspectives. Int J Mol
Sci. 22(17):9181. https://doi.org/10.3390/ijms22179181


1. Crueger W, Crueger A (2017) Cruegers Biotechnology: A Textbook of Industrial
Microbiology (3rd Ed.). Medtech publication. ISBN: 978-9385998638
2. Verma PS, Agarwal VK (2009) Molecular Biology. S. Chand & Company Pvt. Ltd.,
India. ISBN: 978-8121931915
3. Lewin B, Stone MH (2007) Gene IX. Jones and Bartlett Publishers, Inc. ISBN:978-
0763752224

1. For commercial production what types of criteria should be followed for screening of
microorganisms?
2. Explain the essential steps of purification of enzymes.
3. Describe about clinical uses of enzymes.
4. How the enzyme therapy works? Explain in detail with suitable examples of diseases or
disorders cured by enzyme therapy.
5. Give a detail note on use of enzymes in recombinant DNA technology.
6. What do you understand of enzymes? Describe the various steps of large scale production
of enzymes.
7. What is immobilization of enzyme? Explain various methods of immobilization with help

of suitable examples.
8. Give detailed notes on “use of enzymes in food industry”.
9. Explain the role of enzymes in process of cheese making with suitable examples.
10. How the enzymes act as target for drug designing? Explain the mechanism of inhibition
of enzymes by drugs.

BLOCK III: BIOPHYSICAL CHEMISTRY
UNIT 9: BIOENERGETICS
CONTENTS:
9.1Introduction
9.2 Objective
9.3 Biological Energy Transformation obey the law of thermodynamic
9.3.1 Gibbs free energy, G
9.3.2 Enthalpy, H
9.3.3 Entropy, S
9.4 Chemical equilibria and the standard stat
9.5 Relation between equilibrium constant and ∆G
9.6 Standard state conversions in biochemistry
9.7 Cells require sources of free energy
9.8 AThermodynamically unfavouarable reaction can be driven by a favourable one
9.9 Role of ATP in Biological system ( Hydrolysis of ATP)
9.10 ATP is Continously Frormed and consumed
9.11 Structure Basis of the high Phosphoryl transfer potential of ATP
9.12 Synthesis of ATP
9.13 Summary
9.14 SAQs Type questions
9.15 Glossary
9.16 References

9.1 INTRODUCTION
The branch of biochemistry dealing with the energy needed to create and break chemical
connections in living molecules is called bioenergetics. Energy exchanges, as well as energy
transformations and transductions, are all being explored in biological organisms. Through a
variety of metabolic processes, all living things in the universe may acquire energy. Because
the function of energy is fundamental to such biological activities, growth, development,
anabolism, and catabolism are among the most important processes in the study of living
entities.
Energy is exchanged between living tissues and cells and the environment to keep living
organisms alive. Autotrophic organisms, for example, may be able to get energy from
sunshine without eating or breaking down nutrients (photosynthesis). Heterotrophs, for
example, rely on food for nutrients in order to maintain their energy levels via metabolic
processes like glycolysis and the citric acid cycle, which break down chemical bonds in food.
As a result of the first law of thermodynamics, autotrophic and heterotrophic organisms
interact in a global metabolic network. Heterotrophs derive their energy by eating autotrophs
(plants). As energy is transmitted and transformed, chemical connections in a living creature
are destroyed and new ones are established.
Energy becomes available for labour (such as mechanical activity) or other activities when
weak connections are broken and stronger ones are established (such as chemical synthesis
and anabolic processes in development). When our connections are stronger, we may be able
to discharge usable energy. The goal of metabolic and catabolic processes is to make ATP
from readily available starting materials (in the environment) and then break them down (into
ADP and inorganic phosphate) for use in biological activities. The ratio of ATP and ADP
concentrations determines the "energy charge" of a cell. The cell can use ATP to conduct
work if there is more ATP than ADP available, but if there is more ADP than ATP, the cell
must synthesise ATP by oxidative phosphorylation.In living organisms, oxidative
phosphorylation from energy sources such as sunlight or oxygen is the primary source of
ATP. When ATP is hydrolyzed (broken down) into adenosine diphosphate and inorganic
phosphate, the terminal phosphate connections become weak. The energy released here is not
part of the phosphoanhydride link between the terminal phosphate group and the remainder
of the ATP molecule, but rather comes from hydrolysis's thermodynamically favourable free

energy. Similar to a battery, an organism's ATP stockpile is used to store energy in cells. All
living species need the chemical energy generated by such molecular bond rearrangement to
fuel their biological activities.
9.2 OBJECTIVE
The objective of this unit, you will be able to-
 After completing this unit, you will be able to understand how standard free energy
changes in biological reactions.
 A process in which ATP is hydrolyzed
 How do you make ATP from ADP?
9.3 BIOLOGICAL ENERGY TRANSFORMATIONS OBEY THE

LAWS OF THERMODYNAMIC
Many quantitative findings by physicists and chemists on the interconversion of various
forms of energy led to the creation of two key thermodynamic ideas in the nineteenth century.
Any physical or chemical change requires energy conversion, according to thermodynamics'
first law. The overall quantity of energy in the cosmos remains constant; it may be transferred
from one location to another, but it cannot be generated or destroyed. The second rule of
thermodynamics states that the universe's entropy is rising in all natural processes, implying
that the cosmos is always heading toward greater chaos.
Living things, despite the second rule of thermodynamics, are made up of a collection of
molecules that are substantially more ordered than the material from which they are built, and
they preserve and develop order. The second rule, on the other hand, is scrupulously followed
by all living beings. To see whether the biological systems' second rule applies, we must first
identify the systems and the environment in which they function. Three thermodynamic
variables indicate the energy changes that take place during a chemical process.
9.3.1 Gibbs free energy, G: Gibbs free energy, also known as Gibbs function, Gibbs
energy, or free enthalpy, is a phrase used to measure the amount of work done in a
thermodynamic system at constant temperature and pressure. Gibbs free energy is represented
by the symbol"G." Its energy is often expressed in joules or kilojoules. Gibbs free energy is

the most work that can be extracted from a closed system.While conducting studies to
anticipate the behaviour of systems when they are connected or if a process may occur
concurrently and spontaneously in 1876, an American scientist called Josiah Willard Gibbs
discovered this feature. "Available energy" was a term used by Gibbs to describe his free
energy. It's the quantity of energy that can be put to use in a thermodynamic system. Gibbs
free energy is calculated by subtracting the system's enthalpy from the temperature and
entropy product. Here's how to figure it out:
G = H – TS
Where, G = Gibbs free energy, H = enthalpy, T = temperature, S = entropy
Gibbs free energy is a state function hence it doesn’t depend on the path. So change in Gibbs
free energy is equal to the change in enthalpy minus the product of temperature and entropy
change of the system.
ΔG = ΔH – Δ(TS)
If the reaction is carried out under constant temperature {ΔT=O}
ΔG = ΔH – TΔS
This equation is called the Gibbs Helmholtz equation.
ΔG > 0; the reaction is non-spontaneous and endergonic
ΔG < 0; the reaction is spontaneous and exergonic
ΔG = 0; reaction is at equilibrium
9.3.2 Enthalpy, H: The enthalpy, or H, is the heat content of a reacting system. This
number reflects the quantity and kinds of chemical bonds in the reactants and products. The
term "exothermic" refers to a chemical reaction that generates heat. Because the heat content
of the products is lower than that of the reactants, ΔH is usually negative. Endotners are heat-
absorbing devices with positive ΔH values.
9.3.3 Entropy, S: Entropy, S, is a numerical representation of the randomness or disorder

in a system. The reaction is considered to occur with a gain in entropy when the products are
less complicated and disordered than the reactants.

ΔG and ΔH are measured in joule per mole or calorie per mole (recall that 1 cal = 4.184 J)
and entropy is measured in joule/mole Kelvin (j/mole. K).
Table 9.1: Some physical constant and used in thermodynamics
Boltzmann constant, k = 1.381 x 10-23J/K
Avogadro’s number, N = 6.022 x 1023mol
Faraday constant, J = 96,680 J/V. mol
Gas constant, R = 8.315 Jmol. K (= 1.987 cal/mol. K)
Units of ΔG and ΔH are J/mol (or cal/mol)
Units of ΔS are J/mol. K ( or cal/mol.K)
1 cal = 4.185 J
Units of absolute temperature, T, are Kelvin, K
25'C = 298K
At 25°C, RT = 2.479 kJ/mol (= 0.592 kcal/mol)
Changes in free energy, enthalpy, and entropy are quantitatively related in biological systems
(including constant temperature and pressure) by the equation
ΔG=ΔH-TΔS
where ΔG is the change in Gibbs free energy of the reacting system, ΔH is the change in
enthalpy of the system, T is the absolute temperature, and as is the change in entropy of the
system. When entropy grows, S has a positive sign, while ΔH, as previously stated, has a
negative value when heat is emitted by the system to its surroundings. ΔG tends to be
negative in any of these situations, which are indicative of a favourable process. In reality,
ΔG is always negative in a spontaneously operating system (Table 9.2).
The second law of thermodynamics implies that all chemical and physical activities increase
the entropy of the universe, but it does not specify that this entropy growth must occur inside
the responding system. As cells expand and divide, the order they generate in their
surroundings far surpasses the order they create inside themselves. Living beings maintain

their internal order by absorbing free energy from their surroundings in the form of nutrients
or sunshine and returning it as heat and entropy.
Table 9.2: Variation of response spontaneity (sign of ∆G) in comparison to the signs of
∆H and ∆S
∆H ∆S ∆G= ∆H-T∆S
- + The reaction is both enthalphically favoured (exothermic) and

entropically favoured. It is spontaneous (exergonic) at all
termperatures.
The reaction is enthalpically favoured but entropically

- -
opposed. It is spontaneous only at temperatures below T=
∆H/∆S.
The reaction is enthalpically opposed (endothermic) but
+ + entropically favoured is spontaneous only at temperatures

above T= ∆H/∆S.
The reaction is both enthalpically and entropically opposed. It

is unspontaneous (endergonic) at all temperatures.
+ -
9.4 CHEMICAL EQUILIBRIA AND THE STANDARD STAT

If entropy changes with concentration, then free energy must change as well. As a
consequence, the concentrations of both reactants and products have an impact on the free
energy change of a chemical process. Because many biological processes function in either
direction depending on the relative concentrations of their reactants and products, this
phenomenon is very important.
9.5 RELATION BETWEEN EQUILIBRIUM CNSTANT AND

GIBBS FREE ENERGY (∆G)
The relationship between the concentration and the free energy of a substance A is
approximately

GA bar denotes A's partial molar free energy or chemical potential (the bar denotes (2) the
quantity per mole), GA0 denotes A0 partial molar free energy in its standard state, R the gas
constant, and [A] the molar concentration. As a consequence, the general reaction has been
positive.
The free energy change is
and
Due to the associative nature of free energies, the overall energy change of a reaction is equal
to the sum of the free energies of the products minus the free energies of the teactants. We
obtain the following when we put these connections into equation (1):
When all of the reactants and products are in their traditional states, ∆G is the free energy
change of the process. As a result, the expression for the free energy change of a reaction has
two parts: (i) a constant term whose value is solely determined by the reaction taking place,
and (ii) a variable term whose value is determined by the concentration of the reactants and
products, the reaction's stoichiometry, and the temperature. Because the forward reaction's
free energy completely balances the reverse reaction's, there is no net change in a reaction at
equilibrium. As a result, ∆G = 0, and equation
∆G= - RT In Keq ……..5
Keq is the reaction's well-known equilibrium constant.
The subscript "eq" denotes the equilibrium concentration of reactant and product (Because
the equilibrium condition is generally obvious from the context of the event, equilibrium

concentrations are often presented without this subscript.). As a consequence, conventional
free energy data may be used to determine the reaction's equilibrium constant and vice versa.
By replacing equation for equation, it is possible to observe how the equilibrium constant
fluctuates with temperature (1). i.e. in equation (5), ∆G = ∆H- T∆S
In the standard state, H0 and S0 are the enthalpy and entropy, respectively. y = mx + b, the
equation for a straight line, is the form of equation (7). From observations of Keq at two (or
more) different temperatures, a van't Hoff plot of In Keq versus 1/T may be used to derive the
values of ∆H0 and ∆S0 (and consequently ∆G). This method is usually more practical than
directly measuring H and S using calorimetry.
9.6 STANDARD STATE CONVERSIONS IN BIOCHEMISTRY

It is required to establish ∆G values relative to some reference state that is arbitrarily assigned
the height of zero, which we refer to as the heights of geographic areas, in order to compare
free energy changes for distinct processes. When the temperature is 25°C, the pressure is 1
atm, and the solute's activity is 1, the solute is said to be in its standard state, according to
physical chemistry norms (A substance's activity is its concentration adjusted for non-ideal
behaviour at greater concentrations than infinite dilution).
In most biological processes, the concentrations of reactants and products are so low
(millimolar or less) that their activities are roughly approximated by their molar
concentrations. Biochemists have developed a slightly modified standard state convention
because biological processes occur around neutral pH.
(i) Even though pure water has a concentration of 55.5 M, its activity is given a value of 1.
Because the [H2O] component may be disregarded, the free energy equations for reactions in
dilute solutions containing water as a reactant are simplified.
(ii) At the physiologically appropriate pH of the biochemical standard state, pH 70 (neutral

pH, where [H+] = 10-7 M), rather than pH O ([H] = 1 M), the physical chemical standard
state, where many biological compounds are unstable, the hydrogen ion activity is given a
value of 1.

9.7 CELLS REQUIRE SOURCES OF FREE ENERGY

Isothermal systems, which include cells, operate at a generally constant temperature (they
also function at constant pressure). Because heat can only accomplish work when it moves to
a zone or object with a lower temperature, heat flow is not a source of energy for cells. The
Gibbs free energy function is a mathematical function that determines the amount of energy
that cells may and must utilise, allowing for prediction of chemical process direction,
accurate equilibrium position, and theoretical work output under constant temperature and
pressure. Heterotrophic cells derive their free energy from food molecules, whereas
photosynthetic cells get it from solar rays absorbed. At constant temperatures, both types of
cells convert this free energy into ATP and other energy-rich molecules capable of powering
biological functions.
9.8 THERMODYNAMICALLY UNFAVOUARABLE REACTION

CAN BE DRIVEN BY A FAVOURABLE ONE
As previously stated, the most relevant thermodynamic notion in biology is free energy. Only
if ∆G is presentIt is a negative shift in free energy. Remember that ∆G is the formula for
forming products C and D from substrates A and B.
As a result, the kind of reactant (as described by the ∆G0 term, the standard free energy
change) and their concentrations have an impact on the process' ∆G (expressed by the
logarithmic term). At pH 7, ∆G0 represents the change in free energy. For a chemically
connected chain of events, the total of the free energy changes at various stages equals the
overall free energy change, which is a crucial thermodynamic component. Take a look at the
process in the presence of standard ions.

Because ∆G is positive, A cannot change into both B and C.However, under normal
condition, it is conceivable to transform B to D thermodynamically.Because free energy
changes add up, the conversion of A to C and D has a ∆G0 of -3 kcal/mol, indicating that it
might occur spontaneously under normal conditions. As a consequence, a thermodynamically
favourable reaction linked to it might cause a thermodynamically unfavourable reaction. A
common chemical intermediary known as B connects the processes in this example. To link
an uphill and downhill reaction, there are two major ways. By storing free energy in an active
protein structure, a thermodynamically unfavourable process may be pushed.
In a number of methods, proteins have been exploited as energy conversion devices. The
phosphoryl potential of ATP is converted into mechanical energy by molecular motors such
as myosin, kinesin, and dynein. ATP phosphorylates and subsequently dephosphorylates the
sodium-potassium pump, which causes active Na+ and K+ transport across membranes. The
pump's affinity for the transported ions changes as a result of this reaction cycle, as does the
orientation of its ion-binding sites with reference to the cell's inside and outside walls.
Bacteriorhodopsin's capacity to capture photons and employ them to pump protons across the
cell membrane highlights the diversity and strength of proteins in energy transduction.
Ionic gradients may also be employed to link uphill and downstream activities. For example,
the electrochemical potential of Na+ might be employed to transport nutrients like
carbohydrates and amino acids into cells or to pump Ca2+ out of cells. Proton gradients
generated by the oxidation of fuel molecules or photosynthesis power most of the ATP
production in cells.Membrane-bound proteinos control all of these energy conversion
processes by cycling through conformational modifications.
9.9 ROLE OF ATP IN BIOLOGICAL SYSTEMS (HYDROLYSIS

OF ATP)
It takes a lot of energy to keep the human body working well because it is such a complex
thing. The energy source for usage and storage at the cellular level is adenosine triphosphate
(ATP). Adenosine triphosphate (ATP) is a nucleoside triphosphate that consists of three
serially connected phosphate groups, a nitrogenous base (adenine), and ribose sugar. The
connection between the second and third phosphate groups in ATP is regarded as the "energy
currency" of the cell because it produces swiftly releasable energy. In addition to generating

energy, ATP hydrolysis performs a variety of activities in the cell, including signalling and
DNA/RNA synthesis. Catabolic pathways such as cellular respiration, beta-oxidation, and
ketosis provide energy for ATP production.
The mitochondrial matrix produces the bulk of the ATP during cellular respiration, with each
molecule of glucose oxidised yielding around 32 ATP molecules. Ion transport, muscular
contraction, nerve impulse transmission, substrate phosphorylation, and chemical synthesis
are just a few of the processes that ATP is responsible for. A large amount of ATP is required
for these and other actions. As a result, 100 to 150 moles of ATP must be hydrolyzed each
day in the human body for cells to operate properly. The relevance of ATP as a critical
molecule in cell activity will be discussed in the following sections.
Because of the phosphate groups that interact through phosphodiester connections, ATP is an
effective energy storage molecule that may be used as "money." Electronegative charges
resist the phosphate groups, resulting in high-energy bonds. The phosphate-phosphate bonds
still have a lot of energy stored in them. As a result, metabolic activities hydrolyze ADP,
AMP, and free inorganic phosphate groups. ATP hydrolysis into ADP has a Gibbs-free
energy of -7.3 cal/mol. ATP must be replenished on a regular basis to keep the cell operating.
The intracellular concentration of ATP ranges from 1 to 10 uM on a regular basis. The cell's
ATP level is kept constant by a number of feedback systems. A frequent regulation approach
is to stimulate or inhibit the ATP synthase enzyme. When cellular ATP levels are high
enough, it inhibits two essential enzymes in glycolysis: phosphofructokinase-1 (PFK1) and
pyruvate kinase, functioning as a negative feedback loop to restrict glucose breakdown.
Autonomic process control, brain glia interactions, pain management, and vascular tone
modulation are only some of the purinergic responses that ATP may cause.
Fritz Lipmann and Herman Kalckar first recognised the importance of ATP in energy
exchange in biological systems in 1941. ATP is a nucleotide consisting of a purine base
adenine, a pentose-sugar ribose, and a triphosphate unit (Fig. 9.1). It contains two oxygen to
phospnorous bonds between two phosphate units which are represented by wavy lines. These
phosphate bonds are called nigh energy phosphate bonds, The active form of ATP is usually a
complex of ATP with Mg2+ Or Mn2+. In considering the role of ATP as an energy carrier, we
can focus on its triphosphate moiety. ATP is an energy rich molecule becouse of the presence

of four negatively charged oxygen atoms very close to each other. These four negatively
charged O-atoms experience very high repulsive energy.
Fig.9.1 AMP, ADP and ATP molecules
When ATP is hydrolyzed to adenosine diphosphale (ADP) and orthophosphate (Pi) or

adenosine monophosphate (AMP) and pyrophosphate, a large quantity of free energy is
released owing to the decrease in repulsive forces (PPi).
ATP+H20ADP + Pi+H+ ∆Gº'= -7.3kcal/mol
ATP+H20 AMP+PPi+H+∆Gº'= -7.3 kca/mol
The ∆Gº’ for these reactions is determined by the medium's ionic strength as well as the
quantities of Mg2+ and Ca2+. We'll choose -7.3 kcal/mol as a starting point. Under typical
cellular conditions, the actual ∆G for these hydrolyses is about -12 kcal/mol.. The enzyme
enzvme adenvlate kinase converts ATP, AMP, and ADP to ADP (also known as myokinase).
ATP + AMP ADP + ADP

Free energy released during ATP hydrolysis is used to fuel activities that need it, such as
muscle contraction. When chemotrophs oxidise fuel molecules or phototrophs catch light,
ADP and Pi are converted to ATP. The ATP-ADP cycle is the most basic energy exchange
mechanism in biological systems. Many cell processes, including muscle movement, the
export of molecules from all cell activity, nutrient intake, nerve impulse transmission, and so
on, may be carried out using the energy supplied by ATP hydrolysis. As a consequence, ATP
is the focal point for all cell processes.
Anaiogous nucleoside triphosphates including guanosine triphosphate (GTP), uridine

triphosphate (UTP), and cytidine triphosphate (CTP) are responsible for a variety of
metabolic functions (CTP). GDP, UDP, and CDP are diphosphated nucleotides, whereas
GMP, UMP, and CMP are monophosphated. The transfer of the terminal phosphoryl group
from one nucleotide to the next can be catalysed by enzymes. Nucleoside diphosphate kinase
is a multifunctional enzyme that catalyses the phosphorylation of nucleoside diphosphates.
It's worth emphasising that ATP is the main cellular energy transporter, despite the fact that
all nucleotide triphoaphaets have the same energetic value. NAD + and FAD, two essential
electron carriers, are also ATP derivatives. The significance of ATP in energy metabolism
cannot be overstated.
9.10 ATP IS CONTINUOUSLY FORMED AND CONSUMED

ATP is the primary immediate donor of free energy in biological systems, rather than
functioning as a long-term storage form of free energy. One ATP molecule is used up in a
normal cell within a minute after its creation. The ATP has an extremely high turnover rate.
In a 24-hour period, a resting adult consumes roughly 40 kg of ATP. During high-intensity
exercise, ATP consumption might reach 0.5 kg per minute. Constant ATP production from
ADP allows for motion, active transport, signal amplification, and biosynthesis (Fig.9.2).
Chemotrophs manufacture ATP by oxidising fuel molecules, whereas phototrophs gather the
free energy in light to make it. Pumping protons over a membrane to generate a proton-

motive force is the energy-saving event in both systems. ATP generation is aided by the
proton gradient.
Fig. 9.2 In biological systems, the ATP-ADP cycle is the most basic method of energy
exchange.
9.11 STRUCTUAL BASIS OF THE HIGH PHOSPHORYL

TRANSFER POTENTIAL OF ATP
Let's compare the energy of ATP hydrolysis to that of a phosphate ester like glycerol 3-
phosphate:
The quantity of glycerol 3-phosphate hydrolysis ∆Gº' is substantially less than that of ATP
hydrolysis. This demonstrates that ATP has a higher tendency than glycerol 3-phosphate to
transfer its terminal phosphoryl group to water. Glycerol 3-phosphate's phosphoryl potential
(phosphoryl group-transfer potential) is lower than that of ATP.

What is the structural basis behind ATP's greater phosphoryl potential? Because ∆Gº' is
determined by the difference in free energy between the products and reactants, it is crucial to
examine the structures of both ATP and its hydrolysis products, ADP and Pi. Electrostatic
repulsion and resonance stablization are the two most important qualities. The ATP
triphosphate unit contains four negative charges at pH 7. Due to their proximity, these
charges are aggressively hostile to one another. When ATP is hydrolyzed, the repulsion
between them is lessened. Furthermore, ADP and Pi have a higher level of resonance stability
than ATP.
The γ-phosphoryl group of ATP has just a few resonance forms, while orthophosphate has a
lot of resonance forms of comparable energy (Fig. 9.3). Because the two phosphorus atoms
fight for electron pairs on oxygen, forms like those seen in fig. 9.4 are uncommon. Also, an
oxygen atom next to a positively charged phosphorus atom has a positive charge, which is
electrostatically unfavourable.
9.12 SYNTHESIS OF ATP

The proton-motive force may now be used to produce ATP. The use of submitochondrial
particles formed by ionic rupture of the inner mitochondrial membrane has benefited
oxidative phosphorylation studies.ATP synthase is shown as spherical projections from the
outer surface of these inside-oul vesicles in electron micrographs. In undamaged
mitochondria, these 85-diameter projections may be detected on the matrix side of the inner
mitochondrial membrane. In 1960, Efrain Racker found that mechanical agitation might be
used to remove these knobs. The stripped submitochondrial particles can still transport
electrons along their electron transport chain, but they can no longer synthesise ATP. On the
other hand, the separated 85 spheres catalyse ATP hydrolysis. According to Racker, the
insertion of these ATPase spheres into the stripped submitochondrial particles restored their
capacity to produce ATP. These spheres are dubbed F1. The fundamental function of the F1
unit is to catalyse the synthesis of ATP. The ATPase activity of solubilized F1 is the reversal

of its typical action when there is no proton gradient. F1 is made up of five polypeptide chains
with a stoichiometry of α3β3γδε. This assembly has a mass of 378 kD, as shown in table 9.4.
Table 9.4: Fundamental function
Subunit Mass (kd) Role Location

F1 378 Contains catalytic site for Spherical headpiece
ATP synthesis On matrix side
α 56
β 52
γ 34
δ 14
ε 6
F0 25 Contains proton channel Transmembrane
21
12
8
F1 Inhibitor 10 Regulates proton flow Stalk between F0
And ATP synthesis and F1
Oligomycin-sensitivity-
Conferring proton (OSCP) 23
Fc2 (F6) 8
The other key component of ATP synthase is F0, a hydrophobic segment that penetrates the
inner mitochondrial membrane. The proton channel of the complex is designated F0. It's
made up of four polypeptide chains of varying lengths. The 8-kd chain, of which there are six
per F1, is believed to create the transmembrane pore for protons. The stalk between Fo and F1
contains a number of other proteins (Table 9.4). One of them exposes the complex to
oligomycin, an antibiotic that limits ATP generation by interfering with the proton gradient's
function. The enzyme ATP synthase is known as FoF1. ATPases ADP and orthophosphate
are converted to ATP by ATP synthase, a catalytic enzyme.
ADP3- + P2-1 + H+ ATP4+ + H20
The real substrates are Mg2+ ADP and ATP complexes, as in earlier phosphoryl transfer
reactions with these nucleotides. ADP's terminal oxygen atom collides with Pi phosphorus

atom to form a pentacovalent intermediate that degrades to ATP and H 2O [Figure (4)]. The
apices of a trigonal bipyramid are occupied by the attackin oxygen atom of ADP and the
departing oxygen atom of Pi.
Fig. 9.4 A pentacovalent phosphoryl intermediate is formed during ATP synthesis. The
opposing apices of the trigonal bipyramid contain ADP's attacking oxygen atom and
Pi's leaving oxygen atom.
The attacking and leaving groups in the catalytic mechanism of ribonuclease A are also in-
line. What effect does the intake of protons have on the production of ATP? One theory is
that supercharged protons travelling via Fo are channelled to F1 catalytic site, where they
remove oxygen from Pi, tipping the balance toward ATP generation. In the absence of a
proton motive force, however, isotopic exchange studies demonstrated that enzyme-bound
ATP forms quickly. In H2O18, ADP and Pi were delivered to ATP synthase, which created
ATP and subsequently hydrolyzed it, allowing H2 O18 to be integrated into Pi (Fig. 9.5).
Fig. 9.5. Enzyme-bound ATP is produced from ADP and Pi in the absence of a proton-
motive force.

18
The rate of incorporation of O into Pi implies that roughly equal quantities of bound ATP
and ADP are in equilibrium at the catalytic site, even in the absence of a proton gradient. In
contrast, adenosine triphosphate does not exit the catalytic site until protons have gone
through. The proton gradient, according to Paul Boyer, is responsible for releasing rather than
producing ATP from the synthase. In addition, he discovered that this enzyme's nucleotide-
binding sites interact. ADP and Pi are transported from one site to another, causing ATP to be
released from the other. ATP synthase, to put it another way, participates in the catalytic
process alongside other enzymes.
9.13 SUMMARY
The summary of this chapter is:
 Energy exchange between living species' tissues and cells, as well as their
environment, keeps them alive.
 Every living entity in the cosmos has the ability to extract energy through a number of
metabolic processes. Every physical or chemical change requires energy conversion,
according to the basic rule of thermodynamics.
 According to the second rule of thermodynamics, the cosmos is moving toward

greater disorder. Living creatures, on the other hand, faithfully follow the second rule.
Gibbs free energy is the amount of work that can be done in a thermodynamic system
while the temperature and pressure remain constant.
 The Gibbs free energy is the difference between the change in enthalpy and the
product of the temperature and entropy changes in the system. Living things maintain
internal order by receiving free energy from their surroundings in the form of
nutrients or sunshine and returning an equivalent amount of energy to them.
 The Gibbs free energy function is a thermodynamic concept that describes how much
energy cells can and must use. It allows for the prediction of chemical processes'
directions, as well as their exact equilibrium positions and the amount of work they
can accomplish under constant temperature and pressure.
 The molecule ATP is essential to the cell's everyday functions. When ATP is
hydrolyzed, it produces ADP or AMP as well as free inorganic phosphate groups. The
intracellular concentration of ATP ranges between 1 and 10 uM on a regular basis.

The nucleotide adenosine triphosphate (ATP) is made up of adenine and the sugar
pentose ribose. Two oxygen-to-phospnorous linkages exist between two phosphate
units.
 The active form of ATP is usually a combination of ATP with Mg2+ or Mn2+. There
are two main techniques for linking uphill and downhill reactions. A
thermodynamically unfavourable process can be pushed forward by storing free
energy in an active protein structure. The ability of bacteriorhodopsin to collect
photons and use them to pump protons across the cell membrane exemplifies the
variety and power of proteins in energy transduction.

A. Fill in the blings
i. ATP is produced primarily through…………………..from energy sources such as

sunlight or oxygen in living organisms.
ii. Gibbs free energy is the most work that can be extracted from a ……………………..
iii. The second law of thermodynamics implies that all chemical and physical activities
increase the…………………………… but it does not specify that this entropy growth must
occur inside the responding system.
B. Match thev following
a. ATP i. Oligomycin-sensitivity-Conferring proton
b. OSCP ii. Adenosine Diphosphate
c. ADP iii. Adenosine Monophosphate
d. AMP iv. Adenosine Triphosphate
C. True and false
i. The proton-motive force may now be used to produce ATP. True/ False
ii. The ATP triphosphate unit contains Two negative charges at pH 7. True/ False
iii. When ATP is hydrolyzed to adenosine diphosphale (ADP) and orthophosphate (Pi) or
adenosine monophosphate (AMP). True/ False

iv. Fritz Lipmann and Herman Kalckar first recognised the importance of ATP in energy
exchange in biological systems in 1941. True/ False
D. Multiple choise quations
i. Catabolic pathways such as cellular respiration, beta-oxidation, and ketosis provide energy
for
A. ATP production
B. ATM production
C. DNA production
D. RNA production
ii. The reaction is both enthalphically favoured (exothermic) and entropically favoured. It is
spontaneous (exergonic) at all termperatures if,
A. H positive and S negative
B. H negative and S positive
C. Both Hand S negative
D. Both Hand S positive
iii. Value of the Boltzmann constant, k
A. 1.381 x 10-23 J/K
B. 2.381 x 10-23 J/K
C. 1.381 x 1023 J/K
D. 3.181 x 10-20 J/K
iv. The disorder or randomness movement of molecule in a system is called
A. Enthalpy
B. Gibbs free energy
C. Entropy
D. Internal energy

v. Heat contant of a system is known as
A. Enthalpy
B. Gibbs free energy
C. Entropy
D. Internal energy
vi. The ATP triphosphate unit contains four negative charges at pH
A. 7
B. 8
C. 6
D. 5
vii. ΔG > 0; the reaction is non-spontaneous and,
A. Equlibrium
B. Exothermic
C. Endothermic
D. All of these
viii. NAD stand for,
A. Nicotinamide Adenine Dinucleotide
B. Nicotinamide Adonosine Diphosphate
C. Nicotinamide Adenine Disulphate
D. Nicotinamide acid Dineturation
Answer
A. i. oxidative phosphorylation, ii closed system, iii entropy of the universe
B. a. iv b. i c. ii d. iii
C. i True ii False iii True iv True
D. i A ii B iii A iv C vA vi A vii C viii A

9.15 GLOSSARY
ATP = Adenosine Triphosphate
ADP = Adenosine Diphosphate
AMP = Adenosine Monophosphate
OSCP = Oligomycin-Sensitivity-Conferring Proton
GTP = Guanosine Triphosphate
UTP = Uridine Triphosphate
CTP = Cytidine Triphosphate
NAD =Nicotinamide Adenine Dinucleotide
FAD = Flavin Adenine Dinucleotide
NMP = N Methyl Pyrrolidone
NDP = nucleoside diphosphate
9.16 REFERENCES
1. Nelson, D. L., Cox, M. M. (2013), Lehninger: Principles of Biochemistry. New York:
W.H. Freeman and Company, . Sixth ed., 24.
2. Green, D. E.; Zande, H. D. (1981), "Universal energy principle of biological systems and
the unity of bioenergetics". Proceedings of the National Academy of Sciences of the United
States of America. 78, 5344–5347.
3. Nelson, D. L., Cox, M. M., (2013) Lehninger: Principles of Biochemistry. New York:
W.H. Freeman and Company, . Sixth ed., 27.
4. Ferrick D. A., Neilson A., Beeson, C., (2008). Advances in measuring cellular
bioenergetics using extracellular flux. Drug Discovery Today, 13, 268–274.
5. Nelson, D. L., Cox, M. M.(2013) Lehninger: Principles of Biochemistry. New York: W.H.
Freeman and Company, . Sixth ed., p. 506.

6. SchmidtR., K. (2020). "Oxygen Is the High-Energy Molecule Powering Complex
Multicellular Life: Fundamental Corrections to Traditional Bioenergetics”. ACS Omega 5:
2221–2233.
7. Meurer F, Do H. T., Sadowski G., Held C., (2017) Standard Gibbs energy of metabolic
reactions: Glucose-6-phosphatase reaction and ATP hydrolysis. Biophys Chem., 223, 30-
38.
8. Beis I., Newsholme E. A., (1975), The contents of adenine nucleotides, phosphagens and
some glycolytic intermediates in resting muscles from vertebrates and
invertebrates. Biochem J., 152, 23-32.
9. Bonora M., Patergnani S., Rimessi A., De Marchi E., Suski J. M., Bononi A., Giorgi C.,
Marchi S., Missiroli S., Poletti F., Wieckowski M. R., Pinton P., (2012), ATP synthesis
and storage. Purinergic Signal, 8, 343-57.
10.Cárdenas C., Miller R. A., Smith I., Bui T., Molgó J., Müller M., Vais H., Cheung K. H.,
Yang J., Parker I., Thompson C. B., Birnbaum M. J., Hallows K. R., Foskett J. K.,(2010),
Essential regulation of cell bioenergetics by constitutive InsP3 receptor Ca2+ transfer to
mitochondria. Cell., 23, 270-83.
11.Pablo H. T. J., Verónica D. M., (2004), Sympathetic co-transmission: the coordinated

action of ATP and noradrenaline and their modulation by neuropeptide Y in human
vascular neuroeffector junctions, Eur J Pharmacol, 500, 27-35.
12.Coco S., Calegari F., Pravettoni E., Pozzi D., Taverna E., Rosa P., Matteoli M., Verderio
C., (2003), Storage and release of ATP from astrocytes in culture, J Biol Chem., 10,
1354-1362.
13. Gurtu-Gurtu,(2016), Biophysical Chemistry, A Pragati Edition, 224.

1. J.P. Allen, (2008), Biophysical chemistry, welley-blackwell, ISBN: 978-1-4051-2436-2.
2. A. upadhyay, K. upadhyay, N. Nath, (2009), Biophysical chemistry principles and

techniques, Himalaya Publishing House, ISBN : 978-81-83188-65-4.
3. A. Cooper,(2004), Biophysical chemistry, The Royal society of chemistry, Thomas House,

Science Park, Milton Road, Cambridge CB4 0WF, UK, ISBN: 0-85404-480-9.


1. Explain how biochemical processes change their standard free energy.
2. In biochemistry, mention the standard state conventions.
3. Discuss the process of ATP hydrolysis.
4. How is ADP converted into ATP?
5. Determine the relationship between the equilibrium constant and the change in free energy
in the system.
6. Demonstrate that biological energy change is governed by thermodynamic rule.
7. How do cells demand free energy sources?
8. Explain how ATP functions in biological systems.
9. Explain how biological processes use the ATP-ADP cycle.

UNIT 10: BIOPOLYMER INTERACTIONS,

THERMODYNAMICS OF BIOPOLYMER SOLUTIONS
CONTENTS:
10.1. Introduction
10.2. Objective
10.3 Forces involved in biopolymer interactions
10.4 Electrostatic charge and molecular expansion
10.5 Hydrophobic Interactions
10.6 Osmotic pressure
10.7 Membrane equilibrium
10.8 Size of Biopolymers
10.9 Methods for Determining Particle Shape
10.10 Molecular Weight Determination of Biopolymers
10.11 Summary
10.1 INTRODUCTION
Properties of biopolymer depend upon intermolecular forces like van der Walls forces and
hydrogen bonds existing in the macromolecules. Although these intermolecular forces are
present in simple molecules also, their effect is less significant in them as compared to that in
macromolecules. This is due to the accumulative effect of these forces all along the long
chains of the polymers. Apparently, longer the chain, more intense is the effect of
intermolecular forces.
Several different-types of bonds are involved in maintaining the four levels of protein
structure. The covalent bond in proteins is of two main types. The first is the peptide bond
uniting amino acid monomers in the primary sequence. The second is the disulfide bond (S-S
bridge) which, as we have just seen, is established between the -SH groups of two cysteine
residues and is responsible for some aspects of secondary and tertiary structure.

The solution of a polymer is a function of molecular structure, composition, and molecular
weight. Polar polymers are usually more soluble in polar solvents, e.g., polyvinyl alcohol, in
water, where as non-polar polymers are more soluble in non-polar solvents, e.g., polystyrene
in toluene.
In crystalline polymer the inter-molecular crystalline forces must be overcome by the solvent.
Cross -linked polymers swell in a compatible solvent rather than dissolve. The rate of
solution decreases with increasing molecular weight and increasing length of side chain
launching.
∆Gsol. = ∆Hsoln. − T∆Ssoln.
The dissolution of a polymer is favored if H < TDS.
Thus for solubility to occur ΔH must be negative or if positive must be very close to zero.
∆H − ∆E = ∅1 ∅2 (δ1 δ2 )2
where ∆E is the change in internal energy on forming a solution, ∅1 and ∅2 are volume
fractions of polymer and solvent and δ1 and δ2 are solubility parameters of the polymer and
the solvent. Thus for solubility for solubility.
1/2
δ = (δd + δp + δn )
where δd ,is due to dispersion or Vander Waals forces, δp , is permanent dipole interaction
and δn is due to hydrogen bonds.
This is more helpful where two non-solvents can form a solution mixture. This approach is
important in the coating fields. Thus the viscosity of a polymer solution or the stability of a
suspension of polymer particles are affected by the solvent. Crystallization and
morphological changes are induced by solvent/Various solution methods are used for
determination of molecular weights of macromolecules.
10.2 OBJECTIVE
After going through this unit you will be able to:
 Understand the Forces involved in biopolymer interactions
 Understand the electrostatic charge and molecular expansion

 Understand the concept of Osmotic pressure.
 Understand the meaning of Membrane equilibrium.
 Molecular mass, size and shape of biopolymers.
10.3 FORCES INVOLVED IN BIOPOLYMER INTERACTION

Various kinds of weak interactions are important in the establishment of secondary and
tertiary structure. These weak bonds are all non-covalent; the main types are as follows:
Ionic or electrostatic bonds result from the attractive force between ionized groups having
opposite charges. Hydrogen bonds result when a H+ (proton) is shared between two
neighboring electronegative atoms. The H+ can be shared between nitrogen or oxygen atoms
that are close to each other. Hydrogen bonds have many important biochemical functions.
They are essential for the specific pairing between nucleic acid bases, thus providing the
main force that holds the two DNA strands together as well as allowing the specific copying
of DNA into RNA. Hydrogen bonds in DNA and protein play important part. Thus the forces
that stabilise biopolymer structures are as follows:
(a) Hydrogen bonding: Weak force of attraction between partially positive hydrogen and a
partially negative atom such as oxygen, fluorine or nitrogen on the same or another molecule.
(b) Ionic bonding: Side chain cross-linking can occur as a result of bonding between anionic
and cationic side chains.
(c) Covalent bonding: The most common form of inter-chain bonding is the disulfide bond
formed between the sulfur atoms of two cysteine residues. The insulin consists of two
polypeptide chains linked together by this type of bridges.
(d) Hydrophobic bonding: Several amino acid residues have hydrophobic (water-hating)
side chains. Proteins in aqueous solutions fold so that most of the hydrophobic chains become
clustered inside the folds. The polar side chains which are hydrophilic (water-loving) lie on
the outside or the surface of the protein.
10.4 ELECTROSTATIC CHARGES AND MOLECULAR

EXPANSION

Hydrophobic interactions involve the clustering of molecular groups, which associate with
each other in such a way that they are not in contact with water. In globular proteins the side
chains of the most hydrophobic amino acid tend to aggregate inside the molecule, and the
hydrophilic groups protrude from the surface of the structure. The hydrophobic residues tend
to repel the water molecules that surround the protein, thereby causing the globular structure
to be more compact.Van der Waals interactions occur only when two atoms come very close
together. The closeness of two molecules can induce charge fluctuations, which may produce
mutual attraction at very short range.The essential difference between a covalent and a non-
covalent bond in the amount of energy needed to break the bond. For example, breaking a
hydrogen bond requires only 4.5 kcal mole -1, as compared with 110 kcal mole -1, for the
covalent O-H bond present in water. Although each individual bond is weak, larger numbers
of them can produce stable structures, as in the case of double-stranded DNA. Covalent
bonds are generally broken by the intervention of enzymes whereas non-covalent bonds are
easily dissociated by physicochemical forces.
10.5 HYDROPHOBIC INTERACTIONS

When a nonpolar substance is added to an aqueous solution, it does not dissolve but instead is
excluded by water. The tendency of water to minimize its contact with hydrophobic molecule
is called the hydrophobic effect. Many large molecules such as proteins and cellular
membranes, assume their shapes in response to the hydrophobic effect.
Consider the thermodynamics of transferring a nonpolar molecule from an aqueous solution

to a nonpolar solvent. In all the cases, the free energy change is negative indicates such
transfers are spontaneous processes (Table 10.1).
Table 10.1: Thermodynamic changes for transferring hydrocarbons from water to

nonpolar solvents at 25o C
Process ΔH(kJ.mol-1) -TΔS (kJ.mol-1) ΔG (kJ.mol-1)

CH4 in H2O CH4 in C2H6 11.7 -22.6 -10.9
CH4 in H2O CH4 in CCl4 10.5 -22.6 -12.1
C2H6 in H2O C2H6 in Benzene 0.2 -25.1 -15.9

Benzene in H2O Liquid Benzene 0.0 -17.2 -8.0
Toluene in H2O Liquid Toluene 0.0 -20.2 -20.2
Entropy or randomness is a measure of the order of a system. If entropy increases when a

nonpolar molecule leaves an aqueous solution, entropy must decrease when the molecule
enters water. This decrease in entropy when a nonpolar molecule is solvated by water is an
experimental observation, not a theoretical conclusion. Yet, the entropy changes are too large
to reflect only the changes in the conformations of the hydrocarbons. Thus, the entropy
changes must arise mainly from some sort of ordering of the water itself. What is the nature
of ordering?
The extensive hydrogen bonding network of liquid water molecule is disrupted when a
nonpolar group intrudes. A nonpolar group can neither accept nor donate hydrogen bonds, so
the water molecules at the surface of the cavity occupied by the nonpolar group cannot
hydrogen bond tp other molecules in their usual fashion. In order to recover their lost
hydrogen-bonding energy, these surface water molecules orient themselves to form a
hydrogen bonded network enclosing the cavity (Fig.10.1).
Fig. 10.1 Orientation of water molecules around a nonpolar solute. In order to maximize
their number of hydrogen bonds, water molecules form a ‘cage’ around the solute.
The unfavourable free energy of hydration of a nonpolar substance caused by its ordering of
the surrounding water molecules has the net result that the nonpolar substance tends to be
excluded from the aqueous phase. This is because the surface area of a cavity containing an
aggregate of nonpolar molecule is less than the sum of the surface areas of the cavities that
each of these molecules would individually occupy. The aggregation of the nonpolar groups

thereby minimizes the surface area of the cavity and, therefore, the entropy loss of the entire
system. In a sense, the nonpolar groups are squeezed out of the aqueous phase.
10.6 OSMOTIC PRESSURE

The properties of solution that depend on the number of particles without any consideration
of their kind are called colligative properties (Colligates-collected together). Thus most of the
thermodynamic properties like lowering of vapour pressure, elevation of bonding point,
depression of freezing point, osmotic pressure etc., and come under colligative properties.
Various colligative properties are used for determining molecular weight of substances in
solution particularly osmotic pressure.
Osmotic is defined as the process of net diffusion of water molecules from a dilute solution or
pure water (solvent) itself to a more concentrated solution, when both are separated by a
semipermeable membrane. This membrane allows the water to diffuse but not the solute.
Thus, separated from water by semipermeable the membrane. Water molecules diffuse in
both directions across the semipermeable membrane, but a net diffusion or osmosis of water
from the dilute to the concentrated solution results from a larger number of water molecules
diffusing in that direction than in the reverse direction. Water continues to flow into the more
concentrated solution across the membrane in this way until the hydrostatic pressure rises so
high on the concentrated side of the membrane to cause a transmembrane diffusion of water
in the opposite direction at the rate as the osmotic inflow. This hydrostatic pressure which
exactly balances the osmotic influx of water from pure water to concentrated solution is
called the osmotic pressure of that solution.
Thus, osmotic pressure (g) can also be defined as the pressure which has to be exerted on the
concentrated solution which has to be exerted on the concentrated solution, separated from
pure water by a semipemeable membrane. In order to counteract and stop the osmotic inflow
into the solution. It equals the difference between the hydrostatic pressures on the two sides
of membrane. Osmotic pressure is a colligative property of a solution. A rise in the number of
solute particles in the solution increases the number of solvent particles bound by solute
particles in complexes of solvent particles bound by solute particles in complexes called
solvates. Osmotic pressure may be considered to be caused by the higher partial pressure of
solvent molecules on that side of the semipermeable membrane as has higher concentration
of the solvent.

 Osmotic pressure of a dilute solution is directly proportional to other colligative

properties like this fall in freezing point, lowering of vapour pressure, etc.
 Osmotic pressure is inversely proportional to the MW of the solute.
Colloid osmotic pressure of plasma proteins partly counteracts the filtering effect of blood
pressure and retains water in the plasma. In Kwashorkor, hepatic cinnboses and nephrosia a
fall in plasma concentration of albumin, refuses the colloid osmotic pressure of blood and
lowers the relation of water in circulation leading to oedema.
Methods of Measuring Osmotic Pressure: There are three methods for measuring the
osmotic pressure viz. static method, dynamic method and half sum method.
(I) Static Method
In the static method, the solvent is allowed to diffuse through the membrane until there is no
further interchange in the internal head h. The osmometer measures the equilibrium
difference of level. Corrections for the effect of surface tension and the resultant equilibrium
concentration of the solution due to passage of solvent through the membrane has to be
applied. Sometimes during the establishment of equilibrium, which usually is a long period,
the concentration of the solution near the membrane may increase due to adsorption of
solvent. This can be avoided by the proper production and storage of the membrane in the
solvent. This is a simple method but takes unusually long period for the attainment of the
equilibrium.
(II) Dynamic Method
In the dynamic method, the solvent flows through the membrane. The rate of penetration is
measured by applying an external gas pressure to the solution. The interpolated pressure for
zero rate will be equal to osmotic pressure. By establishing an equilibrium pressure which
remains constant for quite a good period give more reliable results. This is a quick method
but requires a leak tight complex cell.
(III) Half Sum Method
The internal head h is adjusted initially to be close to the expected equilibrium, say slightly
above the equilibrium value. Frequent readings of the depression in volume with time are
taken. After sufficient time the cure between h and time is plotted as represented by x. The
experiment is repeated by adjusting the head slightly below the equilibrium value and the

increase in volume with time is noted till the g curve represented by Y becomes asymptotic.
By calculating the one half the sum of the ordinates of x & y at several values, a new curve A
is obtained which converges to a constant value. Since in both the cases, the change in
volume involved is small, the equilibrium concentration is assumed equal to the critical
concentration.
10.7 MEMBRANE EQUILIBRIUM

It was Pfeiffer who first deposited cupric ferrocyanide on the pores of earthenware pot and
used it as a semipermeable membrane. Since then a large number of substances ranging from
animal bladder to cellulose have been used as semipermeable membrane. The semipermeable
membrane is critically important in osmometry. The reliability of osmotic measurements
depends to a considerable extent on proper choice of' membranes. Selection of a membrane
involves reconciliation of high permeability towards the solvent with virtual impermeability
to the smallest solute molecules present in the solution. The membrane should not swell to
any great extent in the solvent and it should have sufficient fine pores to allow the solvent
molecules to pass through freely.
The most convenient material used is cellulose in the form of a non-water proofed cellophane
sheet or specially treated films of denitrated cellulose nitrate. Generally cellophane
membrane is used because treatment of cellophane with ammonia solution or other reagents
increases the pore size of the membrane very little. It is possible to prepare membranes of
varying porosity depending on the swelling and solvent transfer treatment. Hookway has
reported some fast membranes permeable to solutes of molecular weight 50000, while
nitrocellulose membrane can be used up to molecular weights of 2000. To condition the
membrane in water for use with organic solvents, it is essential at first to wash it with 25, 50,
75 and 100 per cent alcohol or acetone solutions and then displace the alcohol or acetone by
similar washings with the desired organic solvent. Precaution should be taken that the
membranes is not allowed to dry out. It should always be stored in the solvent.
Theories of Semipermeable Membranes: There are three theories of semipermeable

membranes, viz. sieve theory, solution theory and adsorption theory.
(I) Sieve theory
Traube considered the semipermeable membranes as atomic or molecular sieves or bundles

of capillaries through which larger molecules find it difficult to diffuse. According to him, the

only difference between various membranes (copper ferrocyanide, parchment, etc.) is in the
size of their pores. This theory fails to explain as to why a rubber membrane is impermeable
to water but permeable to a large molecule like benzene and pyridine.
(II) Solution theory
Liebig and Hermite postulated that a membrane will be permeable to substances that dissolve
in it and impermeable to those that do not dissolve which explains why rubber is permeable
to benzene, toluene, pyridine, etc., which are soluble in rubber and diffuse through it, but
water which is insoluble does not pass through it. It can be concluded from the above that
substances that diffuse through the membrane, first dissolve in the membrane. Although this
proved to be a necessary criterion, but it was not all. Bigelow and Bartell observed osmotic
effects with inert substance where neither solution nor chemical reaction was possible: Porus
cups, with very fine pores or pores clogged with substances, acted as semipermeable
membranes. Silica, carbon, metallic copper, silver, gold can be compressed into disks with
very fine pores also acted as semi-permeable membrane. Bartell concluded that copper limit
of the pores should be 9.0 x 10-3 cm.
(III) The adsorption theory
Wieser and other workers have shown that inert membranes sometimes take up relatively
more of the solvent than the solute. This is known as negative adsorption and the solution
gets more concentrated. Mathieu observed similar phenomenon with a number of solutions
using porus plates as membranes. He concluded that with sufficiently fine capillaries only
water would be adsorbed. Similarly sugar was negatively adsorbed by copper ferrocyanide
membrane. Thin palladium foil is permeable to hydrogen but impermeable to nitrogen. Thus
a semipemeable membrane acts like a solvent than like a sieve. Irrespective of the mechanism
by which the semipermeable membrane operates, the chemical potential of the diffusing
component is the same on both sides, of the membrane.
10.8 SIZE OF BIOPOLYMERS

We know that under an ultramicroscope, the ture image of the particle does not appear, but
only a ‘halo’ of light surrounding the particle, only indicating its position appears. Lobry de
Bruyn considered that biopolymer particles would not have a diameter less than 5-10

mµ(1mµ= 10 -7cm), since smaller particles do not polarize light. There are several methods
by which the size of particles may be determined indirectly.
(I) From Electromagnetic Theory
According to electromagnetic theory of light, the wavelength (λ) which suffers maximum
absorption on passing through a solution of biopolymer, e.g.,protein, is connected with the
radius of the particle by the expression:
√3. λ
r=
4πn
Where n = refractive index of the dispersion medium.
(II) From Tyndall Effect
The volume of a particle in a sol of a biopolymer may be approximately determined by means

of Tyndall light, the intensity of which may be measured by ‘Tyndall meter’ or
photographically. Rayleigh (1871) showed that the intensity of scattered light (I s) by a
suspension from a beam of known intensity (I) is given by the realtion
V2
Is = I. 2 4
d λ
Where V is the volume of particles, d is the distance between the particle and the observer
and λ is the wavelength of the scattered light.
As the expression involves the volume term, the radius of particle (r) can be calculated by the
relation, V = 4/3 πr3.
(III) From Brownian Motion
Protein or gelatin particles suspened in a liquid medium exhibit Brownian motion. They tend
to settle down due to gravity. Due to the influence of both these effects, the colloidal particles
distribute themselves in a vertical column according to the equation
2.303 RT n1 4
log10 = πr 3 g(h2 − h1 )(ρ − ρ′ )
N n2 3
Where N is the Avogadro’s number, n1 and n2 are the number of particles at heights h1 and
h2 of the vertical column, ρ and ρ’ are the density of dispersed phase and dispersed medium
and r is the radius of the particles. Thus, the value of r,i.e.,the size of particle can be
calculated.

(IV) From Scattering of Light
Zsigmondy used ultamicroscope for determining the size of particles. Each spot of light seen
in an ultramicroscope corresponds to a particle. So the number of particles in a given volume
of a solution can be counted. The obseravation is made a number of times and an average is
taken. The length and breadth of the field of vision are measured by an eye piece micrometer.
The depth is measured by rotating the slit through 90 o. from this data the exact volume of the
solution containing the observed number of particles can be measured. Thus, the number of
particles per unit volume of the solution can be calculated. Let the number be n.
Next, a known volume of polymer solution is evaporated to dryness. From the weight of the
residue, the mass of the particles per unit volume is calculated. Let it be m gm. Now,
assuming the particles to the spherical and density (d) of the particles to be the same as that in
the bulk state, the volume of solid phase is m/d.
m 4 3 m 1/n
= πr . n or r = (3 )
d 3 4πdn
10.9 METHODS FOR DETERMINING PARTICLE SHAPE

Biopolymer particles may have several shapes. The most common types of shapes are shown
in figure. Although it becomes very difficult to give a classification of biopolymers based on
particles shape, yet the following type of classification has been found to be of great value.

There are several methods for the determination of biopolymer shapes and sizes. The most
important methods are given as follows:
(I) X-ray method
As most lyophilic biopolymers have crystalline properties, it is possible to use X-ray methods
to find the size and shape of the particles. The X-ray method allows particle size
determinations down to approximately 10 -7 cm of edge length, assuming the particle to be
cubic in shape.
The size of the biopolymers can also be determined by small angle scattering but it is found
that the latter is advantageous over the broadnening of diffraction lines in the respect that
small angle of scattering depends upon the density fluctuations in the sample. The small
angle diffraction patterns are due to the action of monochromatic X-rays scattered by the
separate particles of the sample. The scattering depends solely upon the electron distribution
within the sample, which in turn is measured by the boundries of grains, their size and shape.
In other words, scattering pattern is formed by separate electron packets which have the size
of the grains of the sample. This type of scattering has been observed by Hosemann (1939),
Guinier (1930) and Kratky (1938).
According to Yodwitch (1951), it is possible to determine the size of the particles from peak
analysis and from stop analysis of the experimental curves.
(II) Electron diffraction method
The size of the smallest particles which can be determined by X-ray method is about 10 A0
the method cannot be improved even by using short wavelengths. Electron diffraction method
has been found to be most suitable for particle size determination in the range of 100-10 A0,
and even below 10 A0 i.e., 1 mµ. so, for the determination of the size of slightly smaller
particles, electrons can be successfully used, and it has been found to be more accurate than
X-ray method.
According to de Broglie (1924), the material particles also possess the properties of waves.
The wavelength of diffracted electron is given as follows:
150
γ = √( ) × 10−8 cm
V
Where V is the applied voltage

From this equation, it is clear that the wavelength of the beam of electrons is shorter if
voltage is higher. This property of shorter wavelength is of great importance in the
determination of size of very small particles.
The diffraction methods are used for calculation of grain sizes and determination of shapes.
An electron microscope as the name implies, is a device to magnify minute objects which can
not be seen by naked eye, where a beam of electrons is employed instead of light rays as in an
ordinary microscope. The electron microscope therefore depends upon the following two
principles:
(i) A beam of electrons under a constant voltage is having the properties of a beam of light
and at the voltage used; the wavelength corresponds to 0.05 A0
(ii) Electrons can be focused by suitable electric and magnetic fields, very much like light
rays get focused by glass lenses.
Electron microscope ia an apparatus in which the glass or quartz lenses of light microscope
are replaced by electrostatic or electromagnetic lenses and in which an electron beam instead
of a beam light is used. The voltage and current are closely controlled. The biopolymer to be
studied is kept on a very thin collodion film and a focused beam of electrons is passed
through. The image can be seen on a fluorescent screen or recorded on a photographic plate.
(III) From radius of gyration:
The particles in a solution which scatter light by diffraction are small as compared to the
wavelength of the incident light. In a solution of macromolecules, the dimensions of the
solute molecules are of an appreciable magnitude and so they cannot behave as point sources.
Individual centres with in a particle scatter light in their own way resulting in interference of
the radiation scattered. The scattering envelope in such a case is asymmetric. If the
macromolecule has a linear dimension greater than about 1/10 th the wavelength of the
incident light, the scattering becomes progressively more pronounced in the forward
direction. The ration of the scattered light in the forward direction to that in the backward
direction is a function of the size and shape of the particles.
The radius of gyration, Rg, gives an approximate measure of the effective radius of the
macromolecule. The value of Rg is obtained from the scattering of incident light at low angles
by X-ray scattering.

10.10 Molecular Weight Determination of Biopolymers

It was Avogadro who developed the concept of a mole. According to him a mole consists of
amount of substance containing the same number of atoms as are present in 12 gm of C-12.
This has a value 6.0229×1023. The weight of a mole in grams is numerically the same as the
weight of a single molecule in atomic mass units (amu). The weight of one gram atom of an
element is called gram-atomic weight and of one mole of molecules is referred to as gram
molecular weight. They are simplified as atomic and molecular weight. One amu is equal to
1.66×10-24 gm. The molecular weight is the sum of all the atoms present in the molecule.
10.10.1 Molecular Mass of Polymers
Natural polymers such as proteins contain chains of identical length. Therefore, their
molecular masses are singular in nature. On the other hand, during the process of synthesis of
polymers, the growth of a polymer chain depends upon the availability of the monomer units
in its neighbourhood which differs from one place to another in the reaction mixture. Hence
all the polymers are heterogeneous with respect to molecular weight. The values of molecular
weight of the same polymer will differ with the solvent and method of determination. A
series of polymeric compounds having the same chemical structure but differing only in
molecular weight is known as polymeric homologous series. The molecular weight so
determined will give the value of average molecular weight. Depending on the method of
determination, different average molecular weights are obtained. They are given as follows:
(a)Number average molecular weight ̅̅̅̅

Mn
(b)Weight average molecular weight ̅̅̅̅̅

Mw
(c)Viscosity average molecular weight ̅̅̅̅

Mv
(d)Z − average molecular weight ̅̅̅̅

Mz
In the case of mono-dispersed systems:
̅̅̅̅
Mn = ̅̅̅̅̅
Mw = ̅̅̅̅
Mv = ̅̅̅̅
Mz
while they differ very much in polydisperesed system.

̅̅̅̅̅
(a)Number Average Molecular Mass (M n)
It is obtained by dividing the sum of masses of all the molecules of different monomer units
of different masses by the total number of molecules. We can understand it by considering a
polymer made up of three monomeric units of masses M1, M2 and M3.If N1 molecules of
monomer of mass M1, N2 molecules of mass M2 and N3 molecules of mass M3 constitute the
polymer then,
the total mass of N1, molecules = N1M1 (i)
the total mass of N2 molecules = N2M2 (ii)
the total mass of N3 molecules = N3M3 (iii)
Adding (i), (ii), and (iii), we will get the total mass.
Total molecular mass = N1M1 + N2M2 + N3M3 (iv)
and total molecules = N1 + N2 + N3 (v)
Number Average Molecular mass=N1M1+N2 M2+N3M3 ÷ (N1+N2+N3)
∑ Ni Mi
̅̅̅̅̅
(M n) =
√Ni
where ∑ Ni = N1 + N2 + N3 … ….
where ∑ Ni Mi = N1 M1 + N2 M2 + N3 M3 … …
̅̅̅̅̅
(M n ) is generally determined by osmotic pressure measurement, depression in freezing
point and elevation in boiling point.
(𝐛)𝐖𝐞𝐢𝐠𝐡𝐭 𝐚𝐯𝐞𝐫𝐚𝐠𝐞 𝐦𝐨𝐥𝐞𝐜𝐮𝐥𝐚𝐫 𝐰𝐞𝐢𝐠𝐡𝐭 ( ̅̅̅̅̅

𝐌𝐰 )
It is obtained by multiplying the sum of total molecular masses of different monomeric units
by their respective molecular masses, adding all the molecular masses and then dividing by
the total mass of all the molecules.

If a polymer consists of N1 molecules of monomeric unit of molecular mass M1,N2 molecules
of another monomeric unit of molecular mass M2 and N3, molecules of the third monomeric
unit of molecular mass of M3 then
Weight average molecular weight ( ̅̅̅̅̅

Mw )
N1 M1 × M1 + N2 M2 × M2 + N3 M3 × M3
= … … (vi)
N1 M1 + N2 M2 + N3 M3
∑ Ni Mi2
or ( ̅̅̅̅̅
Mw ) = … … (vii)
∑ Ni Mi
where, ∑ Ni Mi2 = N1 M12 + N2 M22 + N3 M32 … …
and , ∑ Ni Mi = N1 M1 + N2 M2 + N3 M3 … …
( ̅̅̅̅̅
Mw ) is generally determined by ultracentrifugation or sedimentatione.g. the number
average molecular mass and the weight average molecular mass of a polymer sample
containing 30 molecules of molecular mass 10,000, 30% of molecular mass 20,000 and the
remaining 40% of molecular mass 30,000, will be be
30 × 10000 + 30 × 20000 + 40 × 30000

̅̅̅̅
Mn = = 21000
30 + 30 + 40
30 × 10000 × 10000 + 30 × 20000 × 20000 + 40 × 30000 × 30000

̅̅̅̅̅
Mw = = 24286
30 × 10000 + 30 × 20000 + 40 × 30000
Ratio of ̅̅̅̅̅
Mw and ̅̅̅̅̅
Mw is called polydispersion index (PDI)
̅̅̅̅̅
Mw
PDI = ̅̅̅̅
Mn
For natural polymers, PDI = 1 whereas for synthetic fibres, PDI >1.
10.10.2 Molecular Weight Determination
The molecular weights can be determination by ebiometry, cryscopy, osmometry, end group
determination, light scattering, ultra centrifuge and dilute viscometry (Table 10.2.)

Molecular Symbol Method of Molecule

weight Determining
size of
Number average n1 M1 n2 M2 n3 M3 Colligative Small

̅̅̅̅
Mn = + + +⋯
∑ ni ∑ ni ∑ ni method
Weightaverage n1 M12 n2 M22 n3 M32 Light Large

̅̅̅̅̅
Mw = + + +⋯
∑ ni M i ∑ ni M i ∑ ni M i scattering
Average n1 M13 n2 M23 n3 M33 Sedimentatio n Large

̅̅̅̅
Mz = + + +⋯
∑ ni Mi2 ∑ ni Mi2 ∑ ni Mi2 method
a
Viscosity ̅̅̅̅
Mv = [η]K −1 ̅̅̅̅
Mv Viscometer
method
Table 10.2 Methods of molecular weight determination
Where n = number of molecules in sample.
n1, n2, n3 = number of molecules of molecular weight M1, M2, M3
The molecular weights can be determined directly or in solution.
(I) Direct measurement
The method for direct determination of molecular weights can only be applied to gases or
volatile liquids and solids. They are known as:
1. Vapour density method
2. Low pressure effusion of gases
3. Mass spectrometry
These methods are useful for gaseous or volatile substances. Hence, molecular weights of
biopolymers are generally determined by solution methods.
(II) Solution Methods
There are two types of important methods of determining the molecular weight of substances
in solution, viz. chemical method and physical method.

(III) Chemical Method
Chemical methods are important for the molecular weight determination of macromolecular
compounds. Application to this method requires that the structure of polymer should contain
a known number of the chemically determinable functional groups, radicals or elements per
molecule. In polymers these functional groups occur as end groups (δ) except branched
polymers. In linear polymers the quantitative determination of all end groups, e.g., a polyester
where-OH groups is at one end and- COOH group at the other end, can be estimated by
acidimetric titrations. Therefore determination of one of the functional groups suffices for the
evaluation of number average molecular weight. Katchalski determined the molecular weight
of polyamino acids by the chemical, as well as by other, methods and found them in
reasonable agreements.
In case the polymer is formed by chain transfer the number of transfer agents molecules in
the polymer can be determined by chemical analysis. From these the molecular weight of the
polymer can be computed.
1 1 (S)
=( + C1 )
DPn ( )
DPn 0 (M)
where DPn is the number average of polymerization, (1/(DPn)0) is the reciprocal of DPn
without chain transfer agent or solvent, and (S) & (M) are the concentrations of chain transfer
agent and monomer respectively and C1 is the chain transfer constant.
Km Ks Ki
Cm = , Cb = and C1 =
K b, Kb K b,
Where Km, Ks and Ki are the velocity constants of chain transfer with monomer, solvent and
initiator respectively.
In the case of branched chain polymer, the end group determination establishes the branching
of chains. Kern19 polymerized styrene in presence of bromobenzoyl-peroxide and determined
the amount of bromine in the polymer. He found that each molecule contains three to four
atoms of bromine. In another sample polystyrene containing hydroxyl as end groups and the
presence of end groups was ascertained by infrared spectroscopy. The molecular weight was
found to be 17,300.

Temperature (0C)
In the case of branched chain polymer, the end group determination establishes the branching
of chains. Kern19 polymerized styrene in presence of bromobenzoyl-peroxide and determined
the amount of bromine in the polymer. He found that each molecule contains three to four
atoms of bromine. In another sample polystyrene containing hydroxyl as end groups and the
presence of end groups was ascertained by infrared spectroscopy. The molecular weight was
found to be 17,300. In another method, the presence of unique structural unit in small amount
was the sufficient data for finding the molecular weight, e.g., a protein molecule may contain
just one atom of ion. If Aa is the atomic weight of this trace element and x is the analytically
determined percentage, then the molecular weight M
M = 100Aa/x
Hemoglobin from mammalian blood contain 0.335 per cent ion
100 × 55.85
M= = 16700
0.335
Similarly small amounts of amino acids, sulphur, and other specific constituent can be used to
determine the molecular weight of the polymer.
Tablet 10.3 gives the molecular weight determined by chemical method.
S. No Biopolymer Molecular weight

01 Egg Albumin 44,000
02 Hemoglobin 16,700
03 Insulin 12,000, 6000
04 Edestin 46, 000

In the case of copolymerization of two monomers of M1 and M2 the copolymer will be
formed on the basis of their respective reactivities r 1 and r2.
r1 = K11/K12 and r2 = K22/K21
where K11, K12 are the rate constants for the reactions M1, free radical and monomers M1, M2
and K22, K21 are the rate constant for the reactions M2 free radical with monomer M2, & M1.
Generally r1 × r2 < 1 but sometimes r1 = r2 = 1, then random copolymers will result. If r 1 & r2
are low and r1 r2 is approaching zero, alternating copolymers will result.
If M0 = M1 charged/ M2 charged and P0 = mole of initial monomer of M1/ mole of initial

monomer of M1
then it can be shown that,
(P0 − 1) + [(1 − P0 )2 + 4P0 r1 r2 ]1/2

M0 =
2r1
Therefore if the composition M o is allowed to copolymerize, a copolymer of desired

composition can be obtained and the molecular weight can be computed.
The chemical methods only find use in condensation polymers which have average molecular
weight of the order of 25,000. This method is not sensitive to large molecular weights
sometimes some of end groups not considered for computing the molecular weight becomes
consequential, especially in the case of cross-linked and highly branch chained structures.
(IV) Physical Methods
Various physical methods depend on either the evaluation of thermodynamic properties or the
kinetic behavior or the combination of the two, in dilute solutions. Polymer solutions exhibit
large deviation from their limiting infinite dilution behavior. Hence, not only, all the
experiments are carried out at low concentrations but they are invariably extrapolated to
infinite dilution. In the case of chain molecules, a polymer molecule is assumed to have a
symmetrical statistical distribution of chain elements about a centre of gravity and the volume
occupied by this distribution is many times the actual molecular volume. Thus the volume
over which an individual polymer molecule exerts its influence depends on the chain length
and on the interaction between the polymer and the solvent. Hence the solution taken for

physical measurements should be so dilute that each of the molecule couples separate portion
of the volume. This will not only require very sensitive equipment to measure small physical
changes, but the polymer should also be very properly fractionated, otherwise the molecular
weight obtained by different methods will vary in wide range.
Various physical methods depend on either the evaluation of thermodynamic properties or the
kinetic behavior or the combination of the two, in dilute solutions. Polymer solutions exhibit
large deviation from their limiting infinite dilution behavior. Hence, not only, all the
experiments are carried out at low concentrations but they are invariably extrapolated to
infinite dilution. In the case of chain molecules, a polymer molecule is assumed to have a
symmetrical statistical distribution of chain elements about a centre of gravity and the volume
occupied by this distribution is many times the actual molecular volume. Thus the volume
over which an individual polymer molecule exerts its influence depends on the chain length
and on the interaction between the polymer and the solvent. Hence the solution taken for
physical measurements should be so dilute that each of the molecule couples separate portion
of the volume. This will not only require very sensitive equipment to measure small physical
changes, but the polymer should also be very properly fractionated, otherwise the molecular
weight obtained by different methods will vary in wide range.
(V) Colligative Properties
The dilute solutions show more or less ideal behavior as the heat and volume changes,
accompanying the mixing of solute and solvent are negligible for all practical purposes.
Dilute solution obey Raoult's law.
Dilute solutions containing non volatile solute exhibit some special properties which depend
only on the number of particles in the solution irrespective of their nature. These properties
are termed as:
Colligative properties and these include:
(i) Lowering in the vapour pressure
(ii) Elevation in the boiling point
(iii) Depression in the freezing point
(iv) Osmotic pressure

The important of these properties lies in the fact that they provide methods for the
determination of molecular weights of dissolve solute.
A. Lowering in Vapour pressure
When a nonvolatile solute is dissolved in a solvent, its vapour pressure decreases, Von Babo
showed that although both vapour pressure of pure solvent (p o) and vapour pressure of
solution (ps) increase with increase of temperature yet the ration, po – ps/po remains the same
at all temperature. While p - ps is the lowering in vapour pressure, po – ps/po is termed relative
lowering of vapour pressure. According to Raoult's law, the relative lowering of vapour
pressure of a dilute solution is equal to the mole fraction of the solute present in the dilute
solution.
Now, if 'n' moles of solute be dissolved in N moles of the solvent, the
mole fraction of the solute will be = n / n+N

p0 − ps n
∴ According to Raoult ′s Law , = … … (i)
p0 n+N
If a solution is made by dissolving w.gm. of the solute (molecular weight m) in W gm of the

solvent (molecular weight M) the mole fraction of the solute will be,
w⁄
= M
w⁄ + W⁄
m M
As in dilute solution the amount of solute is very small, w/m can be neglected in the
denominator as compared to W/M, the equation (i) becomes,
p 0 − ps w M
=
p0 mw
So, measuring relative lowering of vapour pressure, the molecular-weight of the solute can be
determined.
Relative lowering of vapour pressure is determined experimentally by Ostwald and Walker

method.
B. Elevation in Boiling Point (Ebullioscopic method) and Depression of Freezing Point

(Cryoscopic method)
We know, the boiling point of a liquid is the temperature at which its vapour pressure is equal
to the atmospheric pressure.

Similarly, the freezing point is defined as the temperature at which the vapour pressure of its
liquid is equal to the vapour pressure of the corresponding solid.
As the vapour pressure of a pure solvent is higher than that of the solution hence the elevation
in boiling point and depression in freezing point will be proportional to the mole fraction of
the solute.
If ΔTb = Elevation in Boiling Point and ΔTf = Depression in Freezing point
Then,
po − ps ∆P
∆Tb ∞ × where ∆P = po − ps
p0 po
∆P
and ∆Tf ∞
po
Now, according to Rooult's Law
∆P w M
= ×
po m W
w M w M
So, ∆Tb × po ( × ) and Tb × po ( × )
m W m W
As for the pure solvent po and M are constant, therefore
w i w w
∆Tb ∞ × or ∆Tb = K b ( )…… (ii)
m w mw m. W
w w
∆Tf ∞ or ∆Tf = K f ( )…… (iii)
mW m. W
where, Kb is pulsation constant, and Kf is depression constant
When, w/m =1 (i.e. one mole of nonvolatile solute is dissolved in 1 gm of the solvent) ΔT b =
Kb (i.e. Kb is equal to the elevation in B.P. when 1 mole of solute is dissolved in 1 gm of the
solvent)
and, ΔTf = Kf (i.e. Kf is equal to the depression in F.P. when 1 mole of solute is dissolved in 1
gm of the solvent)
For practical purpose, in place of kb or kf, kb and kf are used, where.
kb = Modal elevation constant = The elevation in B.P. when 1 mole of solute is dissolves in
1000 gm of the solvent.

Thus, 1000 kb = kb
Similarly.
kf = Model depression constant = The depression in F.P. when 1 mole of solute is dissolved =
100 gm of the solvent.
Thus, 100 kf = kf
So (ii) and (iii) relation will be-
1000k b w
∆Tb = molality × k b or =
m. W
1000k f w
∆Tf = molality × k f or =
m. W
Elevation in B.P. is experimentally determined by Landsberger method and depression in F.P.

is experimentally determined by Beekmann's method. In both of these methods Beckmann
thermometer is used to record small change in temperature. Knowing the value of ΔT b and
ΔTf and the strength of the solution molecular weight of the solute (m) can be determined.
A. Osmotic Pressure
For dilute solutions, according to Van't Hoff theory, the equation PV = nST holds good.
If w gm of solute (molecular weight m) be present in V liters of solution, then
n = w/m and V = V1
Thus, the equation PV = nST becomes,
w w×S×T
PV1 = ST or m =
m PV1
where, P = Osmotic pressure, T = absolute temperature
S = Molar Solution Constant = 0.082 lit atm. K-1 mol-1
Knowing the value of P experimentally, the value of m (molecular weight of the solute) can
be determined.
Osmotic pressure is determined using Berkeley and Hartley method.

Extent of Hydration: Solubility
As has been pointed out earlier the solubility of a polymer is a function of molecular
structure, composition, and molecular weight.
Polar polymers are usually more soluble in polar solvents, e.g. poly vinyl alcohol, water,
whereas non-polar polymers are more soluble in non-polar solvents, e.g. polystyrene in
toluene.
In crystalline polymers the intermolecular crystalline forces must be overcome by the solvent.
Cross-linked polymers swell in a compatible solvent rather than dissolve. The rate of solution
decreases with increasing molecular weight and increasing length of side chain launching.
As a matter of fact polymer solutions exhibit large deviations from their limiting infinite
dilution behavior. Hence, not only, all the experiments are carried out at low concentrations
but they are invariable extrapolated to infinite dilutions. In the case of chain molecules, a
polymer molecule is assumed to have a symmetrical statistical distribution of chain elements
about a centre of gravity and the volume occupied by this distribution is many times the
actual molecular volume. Thus the volume over which an individual polymer molecular
exerts its influence depends of the chain length and on the interaction between the polymer
and the solvent. Hence, the solution taken for physical measurements should be so dilute that
each of the molecule occupies separate portion on the volume.
Sedimentation Equilibria
On ultracentrifuging the polymer solution, several boundaries are observed revealing the
presence of different components in the polymer.
This method is useful only where there is sufficient difference in the molecular weights. This
fact is used in Sedimehtaism equilibrium method for molecular weight determination.
In this method at the equilibrium stage rate at which the solute is driven outwards by the
centrifugal force is equal to the rate at which it diffuses inwards due to concentration
gradient.
I
The sedimentation rate = Cw 2 × M(1 − vp ) ( ) … … (i)
f
KRT dc
The diffusion rate = − …… (ii)
f dx

dc (1 − vp )w 2 dx
=− …… (iii)
C RT
Integrating (ii)
CRTln(c2 c1 )
̅̅̅̅̅
Mw = …… (iv)
2
(1 − vp )w 2 (x2− x12 )
This method requires the time for equilibrium which was found to be too long with
substances with molecular weight greater than 500. Shortly after the centrifuge is brought to
speed a determination of concentrations at the top meniscus and bottom of the cell, gives the
equilibrium values.
(VI) Hydrodynamic Methods
A. Diffusion
The transport of molecule in the absence of bulk flow is called diffusion. It is the directed
thermal motion of molecules or fine particles from places of high concentrations. This
random movement was observed by Brown and is known as Brownian movement. This is
brought about from the bombardment of the dispersed particles by the molecules of
dispersion medium.
Ficks has shown that if an amount dw i.e., the number of grams of macromolecules is
transferred across the boundary of area A in the direction X in time dt, it is proportional to the
concentration gradient dc/dx
dw dc
= −DA
dt dx
where D is known as diffusion coefficient.
The force that derives the molecules to more dilute region is given by
RT 1 dc
f= .
N C dx
This is balanced by the frictional force exerted by the viscous solution. Stokes found that for
a spherical molecule of radius r the force for a viscous flurid of viscosity η is given by
dx RT 1 dc
f = 6πrη =− .
dt N C dx

dx RT 1 dc
or C =− .
dt N 6πrη dx
dw RT 1 dc
=− .
dt N 6πrη dx
RT 1
∴D= .
N 6πrηN
The volume of the spherical molecules is 4/3 πr 3Nd. Therefore the molecular weight is given
by 4/3 πr3Nd where d is the density,
M = 4/3 πr3Nd
If the is molecule is non-spherical, then
Dspherical fDnon−spherical
= = asymmetric factor
Dnon−spherical fspherical
Fick's second law state that
dc d2 c
= D 2 …… (v)
dx dx
The rate at which the boundary between the solution of the polymer and the solvent get
blurred is measured and then D can be calculated. Integrating Eq. (v) we obtain Wiener's
equation
dc C0 −x 2
= exp …… (vi)
dx √4πDT 4πDt
where Co is the concentration of the solution in glcm3, t is the time for the beginning of
diffusion and x is the distance of the gradient from the boundary (Figure 10.4)
There are two methods of determining D, namely
(1) Free boundary spreading, and (2) Diffusion through porous plate.
Rectangle cells (Figure 10.3) are used to study free boundary diffusion. A sharp boundary can
be easily formed by using a sliding joint to superimpose the solvent on the solution. The
boundary spreading is observed by refraction changes in a polarization diffusion meter at
certain levels x at infinite time interval (Figure 10.4).

dn dn A
− =
dxmax dxmax √4πDt
where n is the refractive index and A is the area under curve.
Figure 10.3(a)
From equation (vi) can be seen that D depends on concentration, Extrapolating to zero
concentration, Do can be obtained.
For a wide range of polymers
D0 = K θ M −b
where K θ is a constant for the given polymer solvent system and b is a parameter.
Lamm has designed micro apparatus to measure D which requires only I cc of solution.
A. Sedimentation Velocity Method
The macromolecules having a large size and heavy mass, settle out of dispersion under the
gravitational force. The force F causing sedimentation of spherical particles is given by
4 3 dx
F= πr (p − pθ )g = 6πηr
3 dt
where r is the radius of the particle, p and p o are the densities of the particle and suspension
medium respectively, η is the viscosity of the solution and dx/dt is the velocity of
sedimentation.
dx (p − pθ )g
= 2r 3
dt 9μ

Since the retarding force is equal to the sedimentation velocity
√9 𝑑𝑥 𝜂
𝑑𝑡
𝑟=
2(p − pθ )g
The radius of the particle can be determined by the path it traverses in a definite time. It was
observed that a particle of radius 10-7 mm with a density of 2.5 gm per cm3 will take about
100 years to settle down. Wiegner, Kelly and Stamm have designed equipment to measure
the sedimentation velocity of colloidal particles.
Svedberg and others developed analytical ultracentrifuges to determine the velocity of

sedimentation. A particle of mass m at a distance x from the centre of rotation will experience
a centrifugal at force, fc. given by
fc = m × w 2
Figure 10.3 (b) Figure 10.4
where w is the angular velocity in radian per second, i.e. 2π times the number of revolutions
per second
dx
f = v(p − pθ )w 2 x
dt
according to Stokes law
dx
v(p − pθ )w 2 x = 6πηr
dt
where r is the radius of a given macromolecule in a given solution is its sedimentation

coefficients
dx/dt m(1 − vp )g
S =
w 2x 6πηr

on integration –
lnx2 lnx1
S=
w 2 (t 2 − t1
The value of w2 x comes out to be 2.36×108 cm sec-2 in a centrifuge with 60,000 rpm and at a
distance of 6 cm which is 240,000 times greater than the acceleration in the earth's field.
If one mole of the substance is sedimenting, then
M = vpN
p 2 dx
or M(1 − )w x = 6πηr
p0 dt
dx/dt η 1 − vp0
∴ M= .
w 2 x η0 1 − p
Where V is the partial specific volume of the dispersed phase η and p is the viscosity and
density of the polymer solution andη0,po is the density of the medium.
RTS
∴ M=
D(1 − vp)
Where D is the diffusion coefficient.
For precise measurements S, D and V are extrapolated to infinite dilution. This method takes
very long time. The solution to be studied is placed in a cell with thick quartz windows. A
beam of light is passed through the solution placed in the cell of ultracentrifuge.This beam of
light falls on a photographic plate placed beyond the cell when the cell is rotated at velocity
of 50,000 rpm, the interface between the solution and the solvent gradually shifts with the
sedimentation of the particles and the light is absorbed to different extent at different heights
of the cell. By measuring the optical densities at different time intervals, the sedimentation
velocity can be measured. The curve of distribution of concentration gradient along the height
of the cell at different time integrals is plotted. Then the curve between lnx and t is plotted
which comes out to be a straight.
The slope of the curve gives the sedimentation constant 4.5 extra polating lasting it to infinite
dilution So is obtained
S0 = K 3 M1−b

Where Ks is a constant for a given polymer solvent and b has the same interpretation as in
diffusion. The values of b+K3 are given in the literature. Thus M can be calculated.
C. Viscosity
A shear stress when applied to a body displaces a plane in the body parallel to itself relative
to other parallel planes in the body. The fluids begin to flow as soon as the stress is applied.
Even solids somewhat flow when the stress is maintained for a long time. This slow flow of
solids is called creep. Under high stress creep passes over into plastic deformation of solids.
Silicone polymer gives bouncing putty which is a hybrid of solid and liquid in regard to its
flow properties. Viscosity is a measure of resistance that a body offers to flow. Maxwell
showed that the relaxation time t is given by
T =η/K
Where η is the shear viscosity and K is shear elasticity, liquid of complex structures display
considerable elasticity due to coiling and uncoiling of molecular chains.
Streamline Flow: If the speed of the flow is not very fast, and the liquid moves under a pure
shearing motion it is called the laminar or streamline flow. When the liquid moves with high
velocities, the flow becomes turbulent. The Reynolds number R N attains a value 103 to 104
Pva
RN = . where a gives the dimensions of the flowing object.
η
ῦa
RN = r . where r is tube radius and ῦ the average velocity of the fluid..
η
Restricting to isotropic bodies, let us consider the plane X having planes X 1 & X11 above and
below at a distance l, known as mean free path. Let n be the number of molecules of mass m
per unit volume. Let dv be the difference in velocities of two layers as a distance dx.
Therefore the net rate of upward flow of momentum through any given plane is given by
1 dv dv
= − nmῦ1 = η
3 dt dx
Where ῦ is the root men square velocity, The rate of down flow and transfer of momentum
per unit area of the plane x from above is
1 dv dv
+ nmῦ1 =η
3 dt dx

This change of momentum is balanced by a force f acting on area A of the moving plane.
f 1 dv dv
= nmῦ1 = η
A 3 dt dx
Where η is called the coefficient of viscosity
1
η = nmῦl
3
In the case of gases
3RT 1/2
ῦ=( ) and mn = pM/RT
M
1 pM 3RT
∴η= ( ) lgmsec −1 cm−1 = 2.96 × 10−25 √MRT ld2
3 RT M
Where d is the molecular diameter and p is the pressure and N is Avogadro's number. Poise is
the unit of viscosity. If a force of 1 dyne cm-2 causes a plane to slide past a parallel surface 1
cm-2 apart with a velocity of 1 cm sec -1, then the viscosity will be 1 poise.
Electrophoresis and Rotational Motions
Each protein has a characteristic isoelecric point, and this property can be used in the
separation of proteins. In the technique called isoelectric focusing, proteins are subjected to
electrophoresis on a pH gradient. Each protein moves until it reaches a pH equal to its
individual isoelectric point. At that moment, migration in the electrical field stops because the
net charge of the protein is zero.
The techniques of isoelectric focusing and SDS polyacrylamide gel electrophoresis have been
combined to produce two-dimensional separation of proteins. Several hundred cellular
proteins can be resolved from one another. This technique is increasingly used in cell
biology, and its great resolving power is due to the use of two independent properties of
proteins. The proteins are first separated by isoelectric focussing (This is the dimension)
which separates proteins according to their charge (isoelectric point). The proteins are
subsequently separated by electrophoresis (this is the second dimension) in polyacrylamide
gels containing SDS, which separates proteins according to their size (molecular weight).
This technique results in a series of spots distributed throughout the polyacrylamide gel (if
the same property of proteins had been used in both dimensions, the spots would be
distributed along a diagonal).

When the detergent sodium dodecyl sulfate (SDS) is used in electrophoresis, the proteins are
separated mainly according to their molecular weight. This is because SDS binds to the
proteins, giving them large numbers of negative charges due to the sulfate. Thus, most of the
proteins charges will come from the SDS, minimizing the role of charge differences between
individual proteins (differences which would otherwise affect electrophoretic mobility), and
all the proteins migrate according to their size. The larger proteins move more slowly than the
smaller ones because they encounter more resistance when traversing the molecular proess
within the polyacrylamide gel used for electrophoresis. SDS electrophoresis is widely used as
a method for determining molecular weights of proteins.
In summary, the vapous pressure methods are applicable to vapours that follow perfect gas
equation. In this case also Victor Meyer's method is of higher precision. These can be applied
to oligomers only whose critical temperature and critical pressure are known.
Among the ebulliometric and cryoscopic methods, the cryoscopic methods are more precise.
Let us suppose a solution of concentration o 1 gm / 100 ml. of solvent which at M = 100
correspond to a number of moles n = 0.01. If cryoscopic constant is 5, then the depression in
freezing point will be 0.050C. If molar mass is M = 106, the value of n=10 -6. Hence Tf = 5 x
10-6. No existing method can measure such insignificant changes in temperature. The
conventional cryoscopic method can determine the molecular mass from 15,000 to 30,000.
The osmometry can be used for determining molecular masses from 10 4 to 106.
Viscometric methods are not recommended for determining absolute molecular masses but
only changes in molecular masses during various processes (polymerization, degradation,
etc.)
The method of sedimentation in the ultra-centrifuge is an absolute method for measuring the
molecular mass of a polymer.
The light scattering method is more precise and makes possible to calculate the value of the
mw molecular mass of polymers without assumption of the shape of macromolecules in a
solution.
10.11 SUMMARY
By going through this unit you must have achieved the objectives laid down at the start of the
unit. Let us sum up what we have discussed so far:

 Out of the various forces involve in biopolymers hydrophobic interactions involve

the clustering of non-polar groups, which associate with each other in such a way
that they are not in contact with water.
 It is possible for the polymers to have the average molecular weight but different
molecular weight distribution (MWD).
 Statistically, we can express the average in terms of number or weight.

Consequently, the molecular mass of a polymer is expressed as number average
̅̅̅̅n ) or weight average molecular mass (M
mass (M ̅̅̅̅̅
w ).
 ̅̅̅̅n ⁄̅̅̅̅̅
The ratio of the weight and number average molecular masses (M Mw ) is called
'Poly-dispersity index' (PDI). In natural polymers which are generally mono
dispersed, the PDI is unity.
 ̅̅̅̅
Mn is determined by employing methods which depend upon the number of
molecules present in the polymer sample, viz colligative properties like osmotic
pressure, depression in freezing point and elevation in boiling points. On the other
hand methods such as light scattering and ultra-centrifuging depend on the mass
of the individual molecules and yield weight average molecular mass.
 Osmosis is defined as the forces of net diffusion of water molecule from a dilute
solution or pure water (solvent) itself to a more concentrated solution, when both
are separated by a semi permeable membrane.The membrane allows the water to
diffuse but not the solute.
 The hydrostatic pressure which exactly balances the osmotic influx of water from
pure water to concentrated solution is called the osmotic pressure of that solution.
 Molecular weights are determined either directly or in solution.
 The method for direct determination of molecular weights can only be applied to
gases or volatile liquids and solids.
 However, molecular weights of biopolymers are generally determined by solution

methods. The important methods involve use of colligative properties viz (i)
lowering in the vapour pressure (ii) elevation in the boiling point (iii) depression

in the freezing point and (iv) osmotic pressure for the determination of molecular
weight.
 The hydrodynamic methods for determination of biopolymer molecular weight,

include (i) diffusion method,(ii) sedimentation method and (iii) viscosity method.
 However SDS electrophoresis is widely used as a method for determination of

molecular weights of proteins.
10.12 SAQs
Multiple Choice Questions (MCQ)
1. Molecular mass of biopolymers are expressed as a/an
(a) Average
(b) Median
(c) Mode
(d) Percentage
2. The polydispercity index (PDI) of natural polymers is
(a) 0
(b) 2
(c) 1
(d) 1.5
3. What are colligative properties useful for
(a) Determining boiling and melting temperature
(b) Determining molar mass
(c) Determining equivalent weight
(d) Determining van’t Hoff factor
4. What is the necessary condition for osmosis to takes place
(a) Semi permeable membrane

(b) Same concentration of solvent
(c) High temperature
(d) Pressure greater than osmotic pressure
5. Which of the following is not a biopolymer?
(a) Proteins
(b) Cellulose
(c) Rubber
(d) RNA

1. Explain thermodynamics of biopolymer solutions.
2. Explain the forces involved in biopolymer interactions.
3. Define osmosis and membrane equilibrium
4. What role do electrostatic charges play in molecular expansion.
5. How will you determine the size of biopolymers? Mention any two methods.
6. What are the different methods to determine the shape of particles of biopolymers?
7. What are the different methods to determine the molecular mass of biopolymers?
10.14 REFERENCES
1. Gurtu-Gurtu, Biophysical Chemistry, A Pragati Edition
2. J.P Allen, Biophysical Chemistry
3. N.Mahanta and P.S.Kalsi, Biophysical Chemistry
Answers of MCQ
1. a 2. c 3. b 4. a 5. c

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MSCCH-603

M.Sc. III Semester

BIO-INORGANIC, BIO-ORGANIC AND

Phone No. 05946-261122, 261123

Prof. A. B. Melkani Prof. Diwan S Rawat

Dr. Hemant Kandpal Dr. Charu C. Pant

Prof S.P. S. Mehta Prof. Viveka Nand

Dr. Shalini Singh Dr. Charu C. Pant

2. Dr. Deep Prakash

Published by : Uttarakhand Open University, Haldwani, Nainital- 263139

BLOCK-1 BIOINORGANIC CHEMISTRY

Unit 1 Metal Storage and Transport 1-48

BLOCK-2 Bioorganic Chemistry

Unit 4 Introduction 119-165

BLOCK-3 BIOPHYSICAL CHEMISTRY

Unit 9 Bioenergetics 299-322

BLOCK I: BIOINORGANIC CHEMISTRY

UNIT 1: METAL STORAGE AND TRANSPORT

UTTARAKHAND OPEN UNIVERSITY Page 1

UTTARAKHAND OPEN UNIVERSITY Page 2

UTTARAKHAND OPEN UNIVERSITY Page 3

(i) What is the hemoglobin, its function and its structure

(ii) What is the myoglobin, its function and its structure

(iii) You will be able to function of ferrin and transferritin

1.3 ELEMENTS IN LIVING SYSTEMS

1.4 PORPHYRIN RING

Fig. 1.1 Pyrrole ring

UTTARAKHAND OPEN UNIVERSITY Page 4

Fig 1.2 Structure of porphyrin ring

UTTARAKHAND OPEN UNIVERSITY Page 5

Fig 1.4 Formation of heme group

Other metal-containing protoporphyrins are not as common as the iron-containing

UTTARAKHAND OPEN UNIVERSITY Page 6

 Porphyrin is a macrocyclic compound. Structurally, porphyrin ring consists of four

 Porphyrin ring act as both diacidic as well as dibasic in nature.

 Porphyrin ring contain eleven (11) conjugative double bond.

 With a total of 26 pi-electrons, of which 18 pi-electron form a planar, continuous

 The electronic transition in porphyrin ring is 𝜋 − 𝜋 ∗.

 The complexes which containing porphyrin ring colour appears due to 𝜋 − 𝜋 ∗

UTTARAKHAND OPEN UNIVERSITY Page 7

1.5.1 Some point noted about metalloporphyrin are:

iii. Matelloporphyrin ring the number of conjugated double bond is 11.

1.6 ROLE OF IRON IN LIVING SYSTEM

(b) Iron sulpher protein e.g. rubredoxin, ferridoxin, nitrogenase.

(c) Di-iron oxo bridged compound e.g. haemerythrene, ribonucleotide etc.

1.7 METAL STORAGE TRASNPORT AND

UTTARAKHAND OPEN UNIVERSITY Page 8

1.7.2 Storage of iron-ferritin

(i) Role of ferritin

(ii) Structure of Ferritin

UTTARAKHAND OPEN UNIVERSITY Page 9

Fig 1.5 Structure of ferritin

1.7.3 Transport of iron-transferrins

UTTARAKHAND OPEN UNIVERSITY Page 10

 Transferrin binds Iron is in +3 oxidation stste.

 Fe2+ is oxidation to Fe+3 by ceruloplasmin ( a blue copper protein).

 Transferrin delivers ions when it is reduced to Fe+2 in bone marrow, Fe+2 is

 Ferritin contain two compound,

I. Apoferritin- 24 protein chain each with 175 amino acid.

II. Iron core- it contain 4500 Fe+3 ions.