BCHET-149

MOLECULES OF LIFE
Indira Gandhi National Open University
School of Sciences

Block

3
ENZYMES
UNIT 7
Enzymes: Nature and Classification 151

UNIT 8
Enzymes: Activation and Inhibition 164

UNIT 9
Enzymes: Drug and Action 188
Course Design Committee

Prof. K.K. Arora, Dept. of Chemistry, Zakir School of Sciences,

Husain College, University of Delhi, Delhi IGNOU, New Delhi 110068

Dr. Sunita Joshi (Retd.), Dept. of

Prof. M.S. Nathawat
Biochemistry, Daulat Ram College,
University of Delhi, Delhi Prof. Sunita Malhotra
Prof. B.I. Fozdar
Dr. Bhupinder Mehta, Dept. of Chemistry, Prof. Javed A. Farooqi
Swami Shraddhanand College, University Prof. Sanjiv Kumar
of Delhi, Delhi Prof. Lalita S. Kumar
Prof. Kamalika Banerjee

Block Preparation Team

Prof. Lalita S. Kumar Prof. Seemi Bashir Ahmed (Editor)
School of Sciences, IGNOU Department of Chemistry,
Jamia Millia Islamia, New Delhi
Dr. Swati Omanwar, Ex. Consultant
School of Sciences, IGNOU

Course Coordinator: Prof. Lalita S Kumar

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Acknowledgements: Sh. Sarabjeet Singh for CRC preparation

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Material partially adapted from the Biochemistry Course (CHE-09).

November, 2021
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 BLOCK 3: ENZYMES
In continuation to the study of biomolecules in this course, another class of biomolecules are
discussed. Thus, in Block 3 you will study about another very important biomolecules viz.,
enzymes. These molecules are responsible for almost all the physiological processes in the
cell, hence the living beings.

Block 3 comprises three units. In Unit-7, ‘Enzymes: Nature and Classification’ an

introduction to the nature of enzymes is discussed. The unit deals with the nomenclature,
classification and characteristics of enzymes. Unit 8 is on ‘Enzyme: Action and Regulation’
which discusses the mechanism of enzyme action, enzyme kinetics, factors affecting enzyme
reaction rate and the regulation of enzyme activity. Unit 9 on ‘Enzymes and Drug Action’
deals with the significant role of enzymes in medicine. The unit differentiates medicine from
drug, pharmacokinetics from pharmacodynamics and explains the important drug-receptor
theory of drugs in a living system. It also explains the process of enzymes as drug targets
and the concept of structure activity relationship.

Expected Learning Outcomes

After studying this block, you should be able to:

 describe the nomenclature, classification and characteristics of enzymes,

 explain the mechanism of enzyme action, enzyme kinetics, factors affecting enzyme
reaction rate and the regulation of enzyme activity, and

 explain the drug-receptor theory and the mechanism involved in enzymes as drug
targets.
Unit 7 Enzymes: Nature and Classification

UNIT 7

ENZYMES: NATURE AND

CLASSIFICATION

Structure
7.1 Introduction Catalytic Efficiency

Expected Learning Outcomes Specificity of Action

7.2 Nomenclature of Enzymes Regulation of Enzyme Activity

7.3 Classification of Enzymes 7.5 Summary

7.4 General Characteristics of 7.6 Terminal Questions
Enzymes 7.7 Answers
Chemical Nature

7.1 INTRODUCTION
In Unit 6, you learnt about an important class of biomolecules, called proteins.
The variety of functions which the proteins perform in the cell are crucial to life
and undeniably, the most important of these functions is carried out by a group
of proteins called enzymes. An enzyme is a biological catalyst. It speeds up
or catalyses a biochemical reaction. You studied in Unit 1 about the living cell.
The cell depends upon a large numbers of biochemical transformations for its
own survival and hence that of the organism to which it belongs. Since each
biochemical reaction requires a separate enzyme to catalyse it, a cell must
contain a large number of different enzymes. The enzymes regulate the speed
and direction of all biochemical changes. Thus, understanding enzymes and
their properties is important in understanding metabolism, the latter would be
discussed later in this course.

In the beginning of this unit of you will study about the nomenclature of
enzymes. As the number of enzymes is very large, it is very essential to
understand their various types. It is dealt under the classification of enzymes.
In order to understand the functioning in the living celI it is very important to
understand the characteristics of enzymes that have been explained next in
the unit. You will study more about the mechanism of functioning of enzymes
in the next unit. 151
Block 3 Enzymes

Expected Learning Outcomes

After studying this unit, you should be able to:
 define enzymes and explain the basic nature of these molecules;
 explain the different ways of naming enzymes;
 describe the chemical nature and catalytic efficiency of enzymes;
 explain the specificity of action and regulation of enzyme activity; and
 classify enzymes into six major groups on the basis of their biochemical
actions.

7.2 NOMENCLATURE OF ENZYMES

The term enzyme was introduced by W. Kühne in 1878 for certain substances
in yeast (in zymin) that were responsible for fermentation. These are a
specialised class of proteins, and, as already mentioned, these act as
biocatalysts in metabolic reactions. These are responsible for most of the
Metabolic reactions
consists of catabolic
activities that take place within the system and till now more than two
and anabolic thousand enzymes have been identified. In nature, enzymes help millions of
reactions. In catabolic chemical reactions to occur at extraordinary speeds and under moderate
reactions larger
conditions. In the absence of these biocatalysts the reactions would proceed
molecules are broken
down to produce at a very low rate and under rigorous conditions. As enzymes are important in
energy, whereas in maintaining life, they have many medicinal and commercial uses also. For
anabolic reactions, example, determining the level of particular enzymes in blood gives a clue to
the cell uses energy
to produce molecules the extent of heart muscle damage after a heart attack. On the commercial
which it needs for side, enzymes have been used for centuries in industrial production, as in
growth and repair. fermentation to make alcoholic beverages.

Although systematic names are used to designate the enzymes, more often
common names are employed to identify them. The common name of an
enzyme is generally derived by naming the substrate and the type of reaction
it catalyses and adding the suffix – ase to it. For example, the oxidation of
lactate to pyruvate involves removal of hydrogen atoms from adjacent atoms,
i.e., dehydrogenation. Nicotinamide adenine dinucleotide (NAD) is the
oxidising agent used in this reaction. The enzyme which catalyses this
reaction is named as lactate dehydrogenase or LDH in short.
OH
– + O
CH3 - CH - COO + NAD Enzyme LDH
– +
CH3 - C - COO + NADH + H
Lactate Dehydrogenation
(substrate) (reaction type) Pyruvate

O
urease
H2N - C - NH 2 + H2O CO2 + 2NH 3
Urea Carbon Ammonia
dioxide

+ dehydrogenase
CH3CH2OH + NAD CH3CHO + NADH
Ethanol Ethanal
152
Unit 7 Enzymes: Nature and Classification

This nomenclature worked fine initially when the number of enzymes was not
that large. However, with large number of enzymes this system of
nomenclature did not work well due to overlap and other problems. Therefore
the International Commission on enzymes was set up by the International
Union of Biochemistry (IUB) in 1957 to impart the nomenclature of enzymes.
The Commission on Enzymes (EC) considered the question of a systematic
and logical nomenclature for enzymes and recommended two types of
nomenclature; one systematic and the other working or trivial. The
nomenclature has been revised and updated from time to time.

In the systematic way all the known enzymes are given code numbers
depending on their classes and sub-classes etc. These are collected in a list
called as enzyme list, in which the common names follow immediately after
the code number and are described as recommended names. Thus,
enzymes have been assigned code numbers with four parts:

 the first number indicates the type of reaction catalysed, as per the
classification scheme described above.
 The second number shows the subclass which refers to the substrates
which participate in the reaction or the bond attacked.
 The third number is indicative of sub-sub-class, denoting the exact
specification of the reaction removed.
 The fourth number refers to the serial number of the enzyme, in its sub-
sub-class.

As an illustrative example, the number of lactate dehydrogenase is 1.1.1.27

and for hexokinase it is 2.7.1.10.

You would note that although systematic names describe the enzyme system
accurately, they are otherwise not very convenient for day to day use. Hence,
old trivial names/common names continue to be used in the biochemical
literature. The classification of enzymes is based on the coding system which
you would study in the following subsection.

7.3 CLASSIFICATION OF ENZYMES

The enzyme commission has classified enzymes by assigning an EC
(enzyme) commission number to these which consist of four parts as
mentioned in the previous subsection. The following three principles are to be
followed in classifying enzymes.

1. An enzyme is to be named by adding the suffix ‘–ase’ to the name of the

substrate on which it acts.

2. The chemical reaction catalysed is specific for an enzyme so it forms the

basis of classifying or naming the enzyme.

3. The enzymes are classified on the basis of the type of reaction catalysed.

Thus the classification is based on the reactions enzymes catalyse and the
substrates they act on. We shall illustrate each class of enzyme with an
example. 153
Block 3 Enzymes

Oxidoreductases

This class of enzymes participates in physiological oxidation-reduction

processes i.e., they catalyse electron transfer. An example of this class is the
enzyme, alcohol: NAD oxidoreductase, which indicates that alcohol acts as
the electron donor and NAD+ as the electron acceptor:

Enzyme +
+ Acetaldehyde + NADH + H
Ethyl alcohol + NAD

This enzyme is also known by its trivial or common name, alcohol

dehydrogenase. You would, however, observe that the common name does
not completely describe the substrates undergoing the reaction.

Transferases

This class of enzymes catalyses the transfer of a chemical group from one
substrate to another. The transferred groups could be amino, methyl, alkyl,
acyl, sulphate or phosphate, etc. A typical example is an enzyme commonly
designated by its trivial name, hexokinase. The systematic name of this
enzyme is ATP: D-hexose 6-phosphotransferase, indicating that ATP is the
phosphate donor, D-hexose is the phosphate acceptor and the transfer occurs
to the hydroxyl on 6-carbon position of hexose.

Hexokinase
ATP + D - Hexose ADP + D - Hexose 6-phosphate

Hydrolases

A very large number of enzymes catalyse hydrolytic reactions. Many of the

digestive enzymes, such as amylase, sucrose, lipase and all the proteases,
which cause the breakdown of food materials, belong to this group. An
enzyme of this group, commonly known by its trivial name, pancreatic lipase,
degrades lipids and is known by its systematic name triacylglycerol
acylhydrolase.

Pancreatic lipase
Triacylglycerol + H2O 2 - Monoacyglycerol + 2 Fatty acids

Lyases

These are enzymes which catalyse the elimination of chemical groups without
hydrolysis, resulting in the formation of a double bond. An enzyme known by
its common or trivial name, fructose bisphosphate aldoase, is an example of
this type. The full systematic name of this enzyme is D-fructose-1, 6-
bisphosphate-D-glyceraldehyde 3-phosphate lyase, signifying that the
substrate, D-fructose-1, 6-bisphophate is degraded in such a way as to give D-
glyceraldehyde-3-phosphate as a product.

Fructose - bisphosphate aldoase D-Glyceraldehyde 3-phosphate

D -Fructose -1, 6-bisphosphate
+ Dihyroxyacetone phosphate

154
Unit 7 Enzymes: Nature and Classification

Isomerases

Enzymes of this class catalyse isomerisations, and include racemases,

epimerases and mutases. An enzyme known by its trivial name,
triosephosphate isomerase, has the systematic name of
D-glyceraldehyde 3-phosphate ketol-isomerase, signifying that the aldose,
D-glyceraldehyde 3-phosphate is converted to its isomer ketose i.e.,
dihydroxyacetone phosphate.

Triosephosphate isomerase
D - Glyceraldehyde 3-phosphate Dihydroxyacetone phosphate

Ligases (synthetases)

The function of this class of enzymes is to join two molecules at the cost of
energy generated by the hydrolysis of a high energy bond. An enzyme known
by its trivial name, isoleucyl-t RNA synthetase, carries the systematic name, L-
isoleucine: t RNAile ligase (AMP-forming). The systematic name indicates
that L- isoleucine is joined to isoleucine-specific t RNA acceptor, the process
being accompanied by splitting of ATP to give AMP and pyrophosphate.

ATP + L-Isoleucine + tRNAile AMP + Pyrophosphate + L- Isoleucyl-tRNAile

SAQ 1
Write T for true and F for false in the following statements:

a) Enzyme urease which catalyses the hydrolysis of urea is nonprotein in

nature. [ ]

b) In alcohol: NAD oxidoreductase NAD acts as an oxidant. [ ]

c) The biochemical reaction catalysed is specific for an enzyme. [ ]

d) The second number in the recommended name of the enzyme is related

to the type of the reaction catalysed. [ ]

SAQ 2
Identify hydrolases from the following enzymes. Tick [ ] mark the appropriate
box.

a) Urease [ ]

b) LDH [ ]

c) Alcohol dehydrogenase [ ]

d) Amylase [ ]
155
Block 3 Enzymes

7.4 GENERAL CHARACTERISTICS OF ENZYMES

Enzymes, for a long time were associated with the whole living organism with
its cellular structures intact. However, Buchner in 1897 demonstrated that
enzymes were organic substances produced by living cells and did not require
intact cellular organisation for their catalytic activity. The exact chemical nature
of enzymes was established thirty years later by Sumner. He showed that
urease, an enzyme which catalyses the hydrolysis of urea, could be
crystallised and was shown to possess properties characteristic of proteins.

O
Enzyme urease
H2N - C - NH2 + H 2O 2NH 3 + CO 2
Urea

This result was confirmed by Northrop and Kuntiz, who crystallised several
enzymes and established that these were proteins in nature. In 1960, the
laboratories of Stein and Moore established the chemistry of the enzyme
Ribonuclease ribonuclease by deducing its amino acid sequence and a few years later,
catalyses the Merrifield (1969) achieved its total synthesis on the basis of its known amino
breakdown of acid sequence. This provided the ultimate proof that enzymes are no different
ribonucleic acid.
from other chemicals of nonbiological origin. Let us study the chemical nature
and some other characteristics that are very specific to enzymes with more
clarity in the following subsections.

7.4.1 Chemical Nature

A complete understanding of the chemistry of an enzyme came with the work

of Philips (1965) who determined the three-dimensional structure of
Lysozyme is an lysozyme. Using this information, Philips and his group proposed a chemical
enzyme which mechanism for the catalytic process of lysozyme. It was for the first time that
degrades bacterial
such a success was accomplished. The primary amino acid sequence and the
cell wall.
three-dimensional structure of many could be achieved. The primary amino
acid sequence and the three-dimensional structure of many enzymes is now
known. This knowledge has also thrown extensive light on the general
features governing enzyme structure, function, regulation and evolution.

Considerable diversity of structure is seen in the enzymes. Many enzymes are

You would recall from
simple protein molecules. This implies that the protein molecule in itself is the
previous unit that
proteins which true catalyst. However, many enzymes require the presence of additional
produce only amino nonprotein molecules for the full expression of their catalytic function, which
acids upon hydrolysis means that these enzymes are conjugated protein molecules. Let us look into
are called simple
proteins, whereas the types of groups associated with the enzymes and the terms used for
conjugated proteins these. Fig. 7.1 gives you a general idea of these terms, as related to the
produce amino acids structure and function of an enzyme.
and other organic or
inorganic substances Apoenzyme: The protein part of an enzyme molecule is known as
upon hydrolysis.
apoenzyme.

Cofactor: The nonprotein part of an enzyme is called a cofactor. These

cofactors are basically the additional chemical groups, which appear in those
enzymes that are conjugated protein molecules. Some cofactors may be metal
156 ions such as Mg2+, Zn2+ or complex organic molecules, such as nicotinamide
Unit 7 Enzymes: Nature and Classification

adenine dinucleotide. Whatever be the nature of a cofactor, both are required

for enzyme activity. You will study more about these in Unit 8.

Fig. 7.1: Schematic representation of an enzyme.

Holoenzyme: The combination of an apoenzyme and the cofactor is known as

the holoenzyme:

Apoenzyme + Cofactor Holoenzyme

Activator: In case the cofactor of an enzyme molecule is a metal ion such as,
Fe2+, Mg2+, Cu2+, K+, Na+, Zn2+, the cofactor is known as an activator.

Coenzyme: When the cofactor of an enzyme is a nonprotein organic

molecule, the cofactor is known as a coenzyme. For example, the B group of
vitamins are coenzymes that are necessary for proper cellular respiration. You
will get some more information about coenzymes in the next unit.

Prosthetic group: A cofactor, which is tightly bound to the apoenzyme, is

generally called a prosthetic group. However, it is possible that what is a
prosthetic group for one enzyme may be simply a cofactor for another.

Substrate: A chemical substance or substances, the transformation of which

is catalysed by an enzyme, is called its substrate. The catalytic transformation
of a substrate by an enzyme involves its prior binding to the enzyme, followed
by a reaction leading to bond making or bond breaking, resulting in a product.

Active site: The part of an enzyme where all the events of the catalytic
process occur is called its active site. Thus, it is the specific area of an enzyme
to which the substrate attaches during the reaction. Besides, the reaction is
also catalysed and the products subsequently released at this site.

The active site consists of a few amino acid side chains, some known as
binding groups and the other as catalytic groups. The binding side chains
can bind to different parts of the substrate by hydrogen bonds and electrostatic
or hydrophobic interactions. Thus, binding amino acids can either be polar or
nonpolar. However, the amino acid side chains of the catalytic groups are of
polar type only, because they bring about changes in the electronic structure
of the substrate, which is a pre-stage to chemical transformations. In Fig. 7.2,
a schematic view of an active site is represented. 157
Block 3 Enzymes

Fig. 7.2: A schematic representation of the active site of an enzyme. The letter R with
subscript shows the position of the amino acids in the protein chain.

We shall now describe the efficiency of an enzyme as a biocatalyst. You will

also learn that enzymes possess enormous catalytic power.

7.4.2 Catalytic Efficiency

Enzymes, in a living cell, perform their functions under very moderate

conditions of temperature and pH. Various chemical reactions, which are
catalysed by enzymes in the living cell under physiological conditions, would
RBCs are rich in the hardly occur outside the cell in the absence of an enzyme. For example, it
enzyme carbonic
anhydrase. They can would take nearly 50 years to digest a single meal without enzymes. Another
absorb CO2 as it is example, which we can cite, is that of the reversible reaction between carbon
produced in the body dioxide and water to produce carbonic acid. This reaction is catalysed by
and transport it back
carbonic anhydrase, an enzyme which is present in most of the tissues,
to the lungs where it
is released as one of especially in erythrocytes (Red Blood Corpuscles, RBCs).
the products of the
body. Carbonic anhydrase
CO2 + H2O H2CO3
Carbonic acid

The rate of this reaction is very low, but with the presence of enzymes it is
increased to about 107 times. Another reaction, which we shall illustrate here is
the decomposition of hydrogen peroxide to oxygen and water.

Peroxidase
2H2O2 2H 2O + O 2
The catalytic Hydrogen
efficiency of an peroxide
enzyme is expressed
in terms of its
turnover number. This reaction is catalysed by the enzyme peroxidise. Hydrogen peroxide is
This number indicates produced as a by-product in many biological oxidations. It is highly toxic and
the number of has to be decomposed quickly to prevent any damage to the cell components.
substrate molecules
which are
This is achieved with the help of peroxidase, which affects an increase in the
transformed in one rate of reaction by about 1010 as compared to the uncatalysed reaction.
unit of time by one
molecule of an It is thus obvious that enzymes increase the reaction rate enormously, which is
enzyme, under of utmost significance in the biological system. Wherever it has been possible
optimal conditions of
temperature and pH.
to make comparisons, a rate enhancement of about 104 to 1014 has been
observed with the presence of enzymes in biochemical reactions. Table 7.1
gives you some examples of rate enhancements brought about by enzymes.
We shall consider various factors that lead to this rate acceleration in the next
158 unit.
Unit 7 Enzymes: Nature and Classification

Table 7.1: Comparison of nonenzymic and enzymic catalysis

Substrate Catalyst Temperature (K) Rate constant (k)

-3 -1 -1
(mol dm ) s
Amide (hydrolysis)
+ -6
benzamide H 325 2.4 × 10
- -6
benzamide OH 326 8.5 × 10
benzoyl-L- -Chymotrypsin 298 14.9
tyrosinamide
+ -7
Urea (hydrolysis) H 335 7.4 × 10
6
Urease 294 5.0 × 10
2+
Hydrogen peroxide Fe 295 56
7
(decomposition) Peroxidase 295 3.5 × 10

The catalytic efficiency of an enzyme is expressed in terms of its turnover

number. This number indicates the number of substrate molecules which are
transformed in one unit of time by one molecule of an enzyme, under optimal
conditions of temperature and pH. You will study about the optimal conditions
of temperature and pH in Unit 8. The turnover numbers of some enzymes are
given in Table 7.2. As it is evident from the table the rate at which an enzyme
catalyses a reaction, will very from enzyme to enzyme.
Table 7.2: Turnover numbers of some enzymes
-1
Enzyme Turnover Number (sec )
Carbonic anhydrase 600, 000
3-Ketosteroid isomerase 280, 000
Acetyl cholinesterase 25,000
Lactate dehydrogenase 1,000
Chymotrypsin 100
DNA polymerase 15

An important aspect of enzyme catalysis is their specificity in acting on a

particular substrate(s), corresponding to a particular reaction or a class of
reactions. In the next subsection, you will study about enzymatic. specificity of
action.

7.4.3 Specificity of Action

An aspect of catalysis that distinguishes an enzymic from its nonenzymic
counterpart, is the specificity displayed by the former in the action on the
substrate. Enzymes are highly specific in their action, both with respect to the
substrate they act upon and the kind of reaction they catalyse. Thus, the same
substrate undergoing two different types of chemical transformations will be
acted upon by two different enzymes. Physiologically the term specificity refers
to the ability of an enzyme to recognise and transform on particular substrate
in a mixture of several substrates. Therefore, it follows that the specificity of
an enzyme for a substrate not only involves the strength of binding of
the substrate to the enzyme but also velocity of the catalysed reaction. 159
Block 3 Enzymes

The degree of specificity varies from one enzyme to another. Some enzymes
have a very low specificity. This enables them to act on a variety of substrates,
provided they contain a particular susceptible bond such as, a peptide bond, a
phosphate ester bond or a carboxylic ester bond. These enzymes are of the
degradative type, such as peptidases, phosphatases or esterases. They
generally participate in digestive process where a narrow specificity would be
expensive for the economy of the organism.

There are other enzymes of intermediate specificity such as those which

display group specificities. Thus, carboxypeptidase A is specific for the
removal of C-terminal amino acids containing a free carboxyl group. Another
enzyme of intermediate specificity is hexokinase which catalyses the
phosphorylation of a variety of D-hexoses. Besides the above mentioned
enzymes of variable specificity, some enzymes show absolute specificity for a
particular substrate only, and the best example of this type is the enzyme
urease, which splits only urea. Many enzymatic reactions also display
stereochemical specificity. For example, D-amino acid oxidase is specific for
D-amino acids only and will not affect L-amino acids. As can be expected
biosynthetic enzymes tend to be highly specific so that anabolic activities are
channelized in a particular direction. The specificity of enzymes can be
explained by two theories described below.

Lock and Key Theory of Specificity

About a century ago, Email Fischer had proposed the “lock and key”
hypothesis to explain the specificity of enzyme-substrate interaction.
According to this, most of the enzymes are specific for only one particular
substrate. This is comparable to a lock which opens up with only one particular
key. Although, this hypothesis still holds good in part, the specificity of an
enzyme with respect to its substrate or a bond to be broken, is determined by
the shape of the active site, its topographical features and the proper
disposition in space of the amino acid residues participating in the process of
substrate binding and catalysis. The ‘lock and key’ theory is depicted
schematically in Fig. 7.3.

Enzyme Possible Only one substrate fits

substrates the active site

Fig. 7.3: In “lock and key” hypothesis, the active site conforms precisely to the
substrate molecule.
160
Unit 7 Enzymes: Nature and Classification

Induced Fit Theory of Specificity

In the light of modern knowledge, one may say that not only have the lock and
key to fit each other but they have to be flexible enough for the best fit. This
constitutes what is known as the “induced fit” theory and can be compared to
a hand slipping into a glove, which then causes or more appropriately
‘induces’ a fit. However, the specificity is still retained as a left-handed glove
will not fit a right and vice versa. This theory is also diagrammatically
illustrated in Fig. 7.4.

Fig. 7.4: In “induced fit” theory, the active site is induced to take a
complementary shape by the substrate molecule.

7.4.4 Regulation of Enzyme Activity

An interesting feature of enzymic catalysis which is not shown by its non-
enzymic counterpart, is its ability to be regulated by small ions or small
molecules which may be substrate, substrate analogues or substances
structurally unrelated to the substrate. Quite often such regulatory molecules
are the end products of a biosynthetic pathway which inhibit the early enzymes
in the pathway, thus regulating their own formation. This is one of the most
fascinating properties of enzymes, which enables fine turning of enzyme
activity in response to changing conditions in the cell. You will learn more
about this aspect of enzyme function in Unit 8.

SAQ 3
Choose the correct option to complete the following statement.
The binding of an enzyme to the substrate involves:
a) only hydrogen bonds.
b) only electrostatic interactions.
c) hydrogen bonds and electrostatic interactions.
d) only van der Waals interactions.

SAQ 4
How is a cofactor different from an activator?

7.5 SUMMARY
 Enzymes are biocatalysts which increase reaction rate enormously. These
are responsible for most of the chemical reactions which take place in the
living organisms. Enzymes are protein in nature, with considerable
diversity of structure. Some enzymes are simple proteins, whereas many
are conjugated proteins. 161
Block 3 Enzymes

 The common name of an enzyme is generally derived by naming the

substrate and the type of reaction it catalyses and adding the suffix – ase
to it. In the systematic way all the known enzymes are given code
numbers depending on their classes and sub-classes etc.

 Enzymes have been grouped into six major classes on the basis of the
nature of the reactions they catalyse. These classes are :
Oxidoreductases, transferases, hydrolases, lyases, isomerases and
ligases. Each enzyme has been assigned a four number identification
code, indicating the major class, type of substrate or the bond cleaved,
the actual reaction catalysed and the serial number of the enzyme in its
subsubclass.

 Many enzymes (conjugated proteins) require nonprotein part without

which these cannot function. These nonprotein parts, called cofactors, are
either metal ions or low molecular weight organic compounds. These
organic cofactors are mostly derived from B group of vitamins. The
organic cofactors are generally known as coenzymes.

 Enzymes are characterised by an active site, where the substrate

(reactant) binds, undergoes transformation and then the products are
subsequently released. The active site consists of amino acid side chains,
some of which act as binding groups while others act as catalytic groups.
The amino acid side chains of catalytic groups are of polar type only.

 Enzymes are characterised by high catalytic efficiency, remarkable

specificity and regulatory properties. Enzymes can increase reaction rates
a billion fold, which enables biochemical reactions to occur under
physiological conditions. They are highly specific, both with regard to the
substrate and the reaction they catalyse. The degree of specificity varies
from one enzyme to another. The “lock and key” and “induced fit”
hypothesis are proposed to explain the specificity of enzyme-substrate
interaction.

 The regulatory properties of enzymes enable fine tuning of enzyme

activity in response to the needs of the cell, under changing physiological
conditions.

7.6 TERMINAL QUESTIONS

1. How does an oxidoreductase differ from a transferase? Illustrate your
answer with an example.

2. Describe the active site of an enzyme.

3. List the characteristics of enzymes and explain how are the enzyme
catalysed reactions different from the chemical catalysed reactions.

4. Name and differentiate between the two models proposed for explaining
the specificity of action of enzymes.

162 5. Differentiate between coenzymes and prosthetic group.

Unit 7 Enzymes: Nature and Classification

7.7 ANSWERS
Self-Assessment Questions
1. a) False b) True c) True d) False

2. a) and d)

3. c)

4. Cofactor: the nonprotein part of an enzyme is called a cofactor. These are

additional chemical groups for example, Mg2+, Zn2+. Activator: when the
cofactor of an enzyme molecule is a metal like Fe2+, Cu2+, it is called an
activator.

Terminal Questions
1. An oxidoreductase takes part in physiological oxidation-reduction process.
On the other hand, a transferase catalyses the transfer of a chemical
group, from one substrate to another. Lactate dehydrogenase is an
example of an oxidoreductase and hexokinase is an example of a
transferase.

2. The portion of an enzyme which binds to the substrate and catalyses its
chemical transformation is called the active site of an enzyme. The active
site has hydrophobic and hydrophilic amino acids which bind to the
substrate by hydrophobic, electrostatic and hydrogen bonded interactions.
Some of the hydrophilic amino acids, which are charged or uncharged
also participate in bond breaking processes.

3. Most of the enzymes are chemically proteinaceous in nature however,

some of these have a nonprotein part too. These are characterised by
high catalytic efficiency, remarkable specificity and regulatory properties.
The enzymes increase the reaction rate enormously as compared to the
chemical catalysed reaction.

4. The “lock and key” and “induced fit” hypothesis are proposed to explain
the specificity of enzyme-substrate interaction. According to “lock and key”
model, most of the enzymes are specific to only one particular substrate.
This is comparable to a lock which opens up with only one particular key.

5. When the cofactor of an enzyme is a nonprotein organic molecule, the

cofactor is known as a coenzyme. A cofactor, which is tightly bound to the
apoenzyme, is generally called a prosthetic group.

163
Block 3 Enzymes

UNIT 8

ENZYMES:
ACTION AND REGULATION

Structure
8.1 Introduction Effect of Temperature
Expected Learning Outcomes Enzyme Inhibition
8.2 Mechanism of Enzyme 8.5 Regulation of Enzyme
Action Activity
Transition State Theory of Regulation by Substrate of
Chemical Reactions Product
How Enzymes Lower the Allosteric Regulation
Activation Energy?
Regulation by Reversible
8.3 Enzyme Kinetics Covalent Modification of the
Enzyme
Measurement of Reaction Rates
of Enzymes 8.6 Isoenzyme
8.4 Factors Affecting Enzyme 8.7 Enzymes in Health Sciences
Reaction Rate
8.8 Summary
Concentration of Substrate
8.9 Terminal Questions
Concentration of Enzyme
8.10 Answers
Effect of pH

8.1 INTRODUCTION
In the previous unit you studied about enzymes as the important biological
catalysts. Now you are familiar with the nature, classification and some
general characteristics of these molecules. As is clear that enzymes are very
specific for a reaction and act on a specific substrate, it becomes important to
learn how do these act on substartesi.e. the mechanism of enzyme action. In
this unit this is what you are precisely going to study.The transition state
theory and the concept of activation energy explain the action of enzymes on
substrates. The unit begins with the mechanism of action and the role of
coenzymes and cofactors in the biological reactions. You have studied in the
previous unit that cofactors are some of the metal ions attached to the main
164
Unit 8 Enzymes: Action and Regulation

enzymic moiety where as coenzymes are the nonprotein parts. Some vitamins
and minerals

act as coenzymes or cofactors for these biocatalysts. Most of the vitamins and
minerals are basically required to maintain proper cell function, growth and
reproduction.

You are very well familiar with the kinetics of chemical reactions. Enzyme
kinetics is similar to that of other chemical reactions in concept and related to
the reaction rates. You will study the various factors that affect the rate of
biological reactions. The enzymes being biological catalysts need to be
regulated so that the reactions are taken to an appropriate stage. Acting in a
co-ordinated fashion, enzymes regulate the speed and the direction of all
biochemical changes. You will study how do enzyme regulate
biotransformations in the last section of this unit.

The next unit deals with the important role of enzymes in pharmaceuticals.

Expected Learning Outcomes

After studying this unit, you should be able to:

 explain the transition state theory of mechanism of enzyme action;

 explain the concept of activation energy for enzyme catalysed reactions;

 describe the biological role of cofactors and coenzymes;

 describe the effect of substrate and enzyme concentration and alsothe

role of pH and temperature on biological reaction rates;

 distinguish between competitive and non-competitive inhibition; and

 explain the various mechanisms of enzyme regulation.

8.2 MECHANISM OF ENZYME ACTION

In this section let us attempt to understand the mechanism of enzyme activity. All the enzyme
In the next section, you will learn about the factors that influence the rates of catalysed reactions in
enzymatic reactions. the living system are
collectively defined as
In general, the enzyme catalysed reactions proceed through various steps. metabolic reactions.

In the first step the substrate (S) gets attached to the surface of an enzyme
(E), forming an enzyme-substrate complex (ES):
E + S ES

After this complex formation, the substrate becomes activated and the bonds
in the substrate become polarised:

E + S E S*
Activated complex
In the next stage of the reaction, the activated complex undergoes a chemical
change to form an enzyme-product complex: 165
Block 3 Enzymes

E S* EP

The last step involves the release of the products and the enzyme is made
available for further catalysis.
EP E + P

These steps are pictorially depicted in Fig. 8.1.

Fig. 8.1: Diagrammatic representation of various steps in enzyme catalysis.

All the above events take place at the surface of the enzyme, i.e., at the active
site, where the binding and the catalytic groups will be involved in the
transformation of the substrate. The most critical step in enzyme catalysis is
the formation of the activated complex and the interactions between the
enzyme and substrate are generally of noncovalent type, i.e., electrostatic,
hydrophobic, hydrogen bonding, etc. Though, in some cases, actual
covalent bonds are also formed, the interaction between an enzyme and a
substrate has to be enough, so that enzyme-product complex can break apart
to release the product and thus regenerate the enzyme.

We shall emphasise here that the astronomical rate enhancement associated

with enzymes are not a magical effect, but a phenomenon, which can be
understood qualitatively in accordance with the principles of physical and
organic chemistry. It is also generally accepted that usual reactions such as
nucleophilic, electrophilic, homolytic reactions and rearrangement etc. are
involved in the transformation of the substrate in enzymatic reactions. Also the
efficiency of enzymatic reactions has been accounted for by suggesting
factors like proximity effect and orientation effect. However, the precise
quantitative contribution of each of the physico chemical factors to final rate
enhancement in any particular enzyme is still a matter of guess only.

Let us now discuss, in simple terms, the principles underlying catalytic power
of enzymes.

8.2.1 Transition State Theory of Chemical Reactions

A chemical reaction can occur if it is thermodynamically feasible. This means

that the conversion of reactants to products must result in a negative free
166 energy change, i.e., ∆G must be negative under the existing conditions, such
Unit 8 Enzymes: Action and Regulation

as the concentrations of reactants and products. This can be shown as given

in Fig. 8.2.

Fig. 8.2: Change in free energy level during the progress of a reaction.

As is evident from the reaction coordinate, the reactants have to overcome a

free energy barrier before the products can be formed. This energy barrier is
known as the activation energy (E) of the reaction. The molecular structure
and conformation corresponding to the peak position in this profile is called the
transition state of the reaction. In the transition state the chemical bonds are
in the process of being formed or broken. It is, therefore, the most activated
and hence highly unstable entity in the reaction pathway.

The concept of the transition state helps to express the rate of reaction, in
terms of the energy of activation. Since the molecules in the transition state
are the ones which lead to the formation of products, the rate of reaction will
depend on the concentration of molecules in the transition state. For this
concentration to increase, with a corresponding increase in reaction rate, the
free energy of activation has to decrease (Fig. 8.3). For this to happen, the
transition state may be reached with a smaller expenditure of heat or enthalpy
of activation (∆H#)or a smaller decrease in the entropy of activation (∆S#). This
is precisely what enzymes achieve and thus bring about a remarkable
increase in the reaction rate. It should be clear from Fig. 8.2 and Fig. 8.3, that
enzymes only decrease the free energy of activation, so that more and more
of reactant molecules pass the energy barrier to form the products. Thus, the
enzymes do not change the equilibrium point of the reaction which is related to
the free energy of the reaction (∆G; Fig. 8.3) but merely speed up the rate at
which the equilibrium point is reached.

(a) (b)
Fig. 8.3: Effect of enzymes on the activation energy: (a) Nonenzymatic reaction;
(b) Enzymatic reaction. 167
Block 3 Enzymes

We shall now discuss in qualitative terms how enzymes cause a decrease in

energy of activation.

8.2.2 How Enzymes Lower the Activation Energy?

A chemical reaction results from random collisions between reacting
molecules. This means the reactants have to come very close to each other.
In a nonezymatic reaction, the probability of such an event is low. Even if the
reactants collide, most of the collisions are not effective, i.e., they do not result
in a chemical reaction. Only those collisions will be effective and yield products
where the reactant molecules have adequate energy and their participating
groups in the reaction are properly oriented with respect to each other.
However, such as effective collision is a rare event and occurs with a low
probability. Further, the proper orientation of groups causes a decrease in the
entropy of the system. This decrease in entropy contributes to high values for
free energy of activation, which explains the low reaction rate of a
nonezymatic reaction.

An enzyme on the other hand possesses an active site which binds the
interacting molecules, and thus brings them close enough to react (in effect
collide). This is known as the proximity effect and shown in Fig. 8.4.As a
result of this effect extremely high concentrations of reacting molecules are
attained, resulting in a large increase in reaction rates (in effect, increase in
collision rates). Thus, enzymes “collect” substrates from the reaction medium
and “make them to collide”. By constructing model organic compounds the
proximity effect of an enzyme has been mimicked in which two interacting
groups were attached to a single molecule. It has been demonstrated that a
rate increase of about 104fold can be realised by this factor alone.

Fig. 8.4: Proximity effect on reaction rates.

The other condition of fruitful collisions, namely the proper orientation of the
reacting groups, is also realised by the enzymes. The active site of an enzyme
with the help of its binding groups, holds the interacting molecules in a correct
rigid orientation with respect to each other. This is known as the orientation
effect. Thus, substrates are precisely oriented at the active site and hence
168 properly positioned for reaction (in effect collision). The orientation effect is
Unit 8 Enzymes: Action and Regulation

shown in Fig. 8.5. This effect has also been imitated and a rate increase of 104
observed. Thus, a total rate enhancement of 108 results from proximity and
orientation effects alone in enzyme catalysis.

Fig. 8.5: Orientation effect on reaction rates.

With these effects an enzyme overcomes the unfavourable entropic barrier,

which is characteristic of nonenzymic reactions. However, the enzyme pays a
price to overcome the loss of entropy. It is the binding energy of enzyme-
substrate interaction at the active side, which is used to pay for this price. It is
thus easy to understand how the enzyme decreases the free energy of
activation of a reaction.

Another contributing factor to enzyme catalysis is the “induced fit”. This

suggests that when an enzyme-substrate complex is formed, the conformation
of both the enzyme as well as the substrate change. This conformation
change in the substrate produces a strain in the form of distortion of bond
angles and bond lengths which brings it closer to the transition state. This, in
turn, reduces the energy of activation required for converting a substrate into
its products. We may mention here that using model systems, it has been
shown that subjecting a substrate to strain can increase its rate of reaction by
a factor of 108.

Lastly, the catalytic functional groups at the active site also contribute to rate
+
enhancement. You will recall that many organic reactions are catalysed by H
-
or OH ions. However, in a biological medium, with the exception of gastric
secretions, the concentration of these ions is very low. At the active site of an
enzyme, various acidic or basic groups acting as catalytic groups can act as
proton donors or acceptors. They thus effectively catalyse a biochemical
reaction, as their simultaneous action on the substrate can be much more
significant than the chance encounter of a reactant with an acidic or a basic
group in the nonenzymic reaction.

We can summarise here that enzymes are highly effective catalyst because
they bring together interacting molecules in a proper orientation. Also the
functional groups at the active site provide donors and acceptors in high local
concentration. Their groups are also properly positioned to bring about a
reaction. 169
Block 3 Enzymes

SAQ 1
Fill in the blanks with appropriate words.

a) Enzymes ............................. the rate at which equilibrium is attained in a

reaction.

b) Enzymes ................................. the activation energy of a reaction.

c) Enzymes overcome the entropic barrier to chemical reaction by ...... and

........effects.

d) In the ................... state, the chemical bonds are in a process of being

formed or being broken.

In the foregoing section we explained how the enzyme molecule is able to

bring about a rate enhancement. You will now learn about the kinetics of
enzymatic reactions. We shall describe the factors that have a direct effect on
the rate of enzymatic reactions.

8.3 ENZYME KINETICS

Enzyme kinetics deals with rates of enzymatic reactions and the various
parameters that govern these rates, such as concentration of substrates,
concentration of enzyme, pH, temperature and the presence of various
substances that may be inhibitors or activators. Study of enzyme kinetics is
important because it throws some light on the mechanism of action of an
enzyme. Such a study also provides information on the behaviour of an
enzyme in the cellular environment and gives us a clue regarding the
regulatory mechanisms available to the organism for fine control of enzyme
activity.

8.3.1 Measurement of Reaction Rates of Enzymes

The activity of an enzyme can be measured by monitoring the time-
dependence of the chemical change that occurs during an enzymatic reaction.
The enzyme is incubated with the substrate under optimum conditions. The
reaction is observed with the disappearance of the substrate and the formation
of a new product. These changes are monitored by withdrawing small aliquots
and analysing them for the formation of the product or disappearance of the
substrate. In some cases such measurements can be made directly on the
incubation mixture without the necessity of withdrawing aliquots. For example,
in several oxidation-reduction reaction, where NAD or NADP is the electron
acceptor, the progress of the reaction can be continuously monitored by
following the reduction of NAD(or NADP) to the reduced product NADH (or
NADPH) which absorbs light at 340 nm. Same principle is applied to monitor
many other enzyme catalysed reactions, where either the substrate or the
product has a unique and easily measurable absorption spectrum.

Let us now learn the factors affecting the rate of biochemical reactions.
170
Unit 8 Enzymes: Action and Regulation

8.4 FACTORS AFFECTING ENZYME REACTION

RATE
Several factors affect the rate at which enzymatic reactions proceed. These
factors are enzyme concentration, substrate concentration, temperature, pH,
and the presence of any inhibitors or activators. These are discussed in the
following subsections.

8.4.1 Concentration of Substrate

In order to outline the effect of substrate concentration on reaction rate, we The conditions of
shall discuss the reaction between a single substrate and an enzyme for the initial reactions rate is
sake of simplicity. If we consider an enzyme a reactant, then this is equivalent important because it
ensures that very little
to a chemical reaction between two chemical substances i.e., enzyme and its product is present to
substrate. However, there is an important difference between enzymic and initiate the reverse
nonenzymic systems with respect to the dependence of rate on the reactant reaction, which may
complicate results.
concentrations. For example, if we carry out a nonenzymic reaction between
two reactants A and B, keeping the concentration of A constant and changing
the concentration of the B, then the initial rate of product formation will be
directly proportional to the concentration of the reactant B. If on the other hand
the conditions of an enzymic reaction are so arranged that the enzyme
concentration is kept constant and the concentration of substrate is changed,
then the initial reaction rate varies hyperbolically with increasing substrate
concentrations. The rate reaches a maximum velocity at high substrate
concentration which remains unaffected by further increase in substrate
concentrations. This is illustrated in Fig. 8.4.

Fig. 8.4: Variation of reaction rate with substrate concentration keeping enzyme
concentration constant.

The fact that in an enzymic system, as opposed to a nonenzymic system, the

reaction velocity reaches a saturating value at high substrate concentrations
can be explained as follows. In a nonenzymatic reaction, the reaction rate is
dependent on the number of effective collisions between the reactants. The
number of such effective collisions would increase in direct proportion to the
concentration of one of the reactants, if the concentration of the other reactant
is kept constant. On the other hand, in an enzymatic reaction, if the 171
Block 3 Enzymes

concentration of one reactant is kept constant, the effective collision between

the enzyme and the substrate leads to the formation of an enzyme-substrate
complex, in which the substrate is firmly bound to the enzyme at its active site.
This complex then breaks down to give the product (P) and release the
original enzyme. Since the number of active sites is limited by the enzyme
concentration (held constant), the reaction rate will increase with substrate
concentration only till all these sites are filled. Under these conditions the
system will give maximum possible rate for reaction. Thereafter, there will be
no further increase in the rate of reaction.

The shape of the curve as shown in Fig. 8.4, depicting the dependence of
reaction rate on the substrate concentration, is a hyperbola, Michaelis and
Menton were the first to recognise this relation for enzyme catalysed reactions
and put the same in a mathematical form in 1913. This equation for enzyme
kinetics is known as the Michaelis-Menton equation:

[
s
]
m[
a]
x
ν

K


S
Rate of reaction

m



Where [S] is the molar concentration of the substrate and Vmax is the maximum
possible rate for a given enzyme concentration, Km, also called Michaelis
constant, is numerically equal to the concentration of the substrate at that
stage when the observed rate of the enzyme catalysed reaction is one half of
V

the maximum rate i.e.,   1 / 2 max . This will be clear to you from Fig. 8.5. From
the above equation, it is clear the rate will tend to acquire a maximum value
when [S) is very high.

Fig. 8.5 : Km is equal to the concentration of the substrate required to give an

initial reaction rate corresponding to half of Vmax

We shall now briefly explain what Vmax and Km convey in enzyme kinetics.

Significance of Vmax

At Vmax, all the enzyme molecules have formed enzyme-substrate complex

(ES) and are continuously catalysing the conversion of substrate into the
product. Thus at Vmax value the enzyme is fully saturated. Vmax values can be
used to compare the activity of various enzymes, if they happen to catalyse
the same reaction.
172
Unit 8 Enzymes: Action and Regulation

Significance of Km

As we have mentioned already, Km is equal to the concentration of the

V
substrate at   1 / 2 m ax . Since at Vmax all the enzyme molecules have formed
enzyme-substrate complex, it, therefore, follows that the concentration of
substrate (Km) required to convert half of enzyme molecules to ES complex, is
a measure of the affinity of the enzyme for substrate. A small value of Km
signifies the high affinity of the enzyme for the substrate, since a low
concentration of substrate would be needed to saturate the enzyme. Similarly,
a large value for Km would indicate a relatively high concentration of substrate
for saturating the enzyme, thus signifying a low affinity of the enzyme for its
substrate.

You have thus learnt that increase in substrate concentration has a

corresponding effect on the rate up to a certain point, beyond which the rate
remains unaffected to any further increase in concentration of substrate. We
shall now discuss the role of enzyme concentration in the kinetics of enzymic
reactions.

8.4.2 Concentration of Enzyme

In enzymic reactions, the enzyme concentration is generally very low, as
compared to the substrate concentration. The rate, therefore, is proportional to
the concentration of the enzyme, provided a purified enzyme is used and
substrate concentration is very high. Under these conditions, for example, if
enzyme concentration is doubled, the rate of conversion of substrate of
product shows a corresponding increase (Fig. 8.6).

Fig. 8.6: Variation of rate of catalysed reaction with concentration of the enzyme.

Let us now discuss how catalytic activity of an enzyme is affected by the pH of

the solution in which the enzymic reaction occurs.

8.4.3 Effect of pH
An enzyme catalysed reaction is strongly influenced by the hydrogen ion
concentration, i.e. pH of the reaction mixture. Usually a small change in pH
causes an appreciable change in the ability of an enzyme to function as a
biological catalyst. The curve showing rate of enzyme catalysed reaction
against change in pH of the reaction mixture is generally bell-shaped
173
Block 3 Enzymes

(Fig. 8.7). You would observe that enzyme activity is maximum only in a
narrow pH range and decrease at both higher and lower pH.

Fig. 8.7: Effect of pH on the activity of an enzyme.

The pH where an enzyme shows maximum reaction rate is known as its

optimal pH. Most enzymes have a maximum catalytic activity around pH 7,
which happens to be the pH of most of the biological fluids. However, many
enzymes do have maximum activity at higher or lower pH than this. An
important example is that of pepsin, which is a digestive enzyme in the
stomach. It has maximum activity around pH 1.5, which is the pH of gastric
juice. We have listed optimal pH values of some enzymes in Table 8.1.

Table 8.1: Optimal pH values of some enzymes

Enzyme Optimal pH values

some enzymes

Pepsin 1.5

α-Glucosidase 5.4

Urease 6.7

α-Amylase 7.0

Carboxypeptidase 7.5

Trypsin 7.8

Alkaline phosphate 9.5

In qualitative terms, this effect of pH on enzyme activity can be attributed to

several factors, some or all of which may operate at the same time. The
change in pH can cause denaturation of the enzymic protein, rendering it
inactive. Further, you may recall from subsection 7.1 that active site of an
enzyme consists of amino acid side chains which form binding and catalytic
groups. The catalytic activity may be possible only when these side chains are
in a correct state of ionisation. The state of ionisation of these side chains
would in turn depend on the pH of the reaction mixture, thus influencing
enzyme activity. We will illustrate this with an example. Let us suppose an
enzyme needs two charged/ionised side chains (~NH+3) and ~COO-) at its
active site for catalytic. This ionisation state would be possible only in a
particular pH range, corresponding to the optimal pH. This is depicted in
174 Fig. 8.8.
Unit 8 Enzymes: Action and Regulation

Fig. 8.8: Effect of pH affecting enzyme activity.

As you would observe from the above representation, at acidic pH (lower pH

value than optimal pH) the –COO- groups gets protonated to –COOH and at
basic pH (higher pH than optimal pH), the –NH+3 group gets deprotonated to –
NH2. Consequently in both these situations the catalytic activity decreases for
lack of proper combination of the ionisable side chains in the active site.
Lastly, the effect of pH on enzyme activity could also result from a change in
ionisation state of a charged substrate with change in pH, affecting its binding
to the enzyme

You will now learn about the fourth factor which influences the rate in an
enzyme catalysed reaction.

8.4.4 Effect of Temperature

You will recall that increase in temperature leads to increased frequency of For most of the
collisions between reactants. Thus rates of chemical reactions increase with enzymes optimal
increase in temperature. However, in the case of enzymatic reactions this temperature are at or
above those of the
increase occurs only within a narrow temperature range beyond which the rate cells in which they
decrease sharply, suggesting that the enzyme may have an optimal occur.
temperature, where the rate is maximum as shown in Fig. 8.9.

The activity of most of

Fig. 8.9: Effect of temperature on the activity of an enzyme. the enzymes is
usually destroyed by
As you would have observed, enzyme activity increases with temperature. heat. However, some
This, evidently, is due to increase in the number of collisions between the exceptions are the
enzymes of bacteria,
enzyme and substrate molecules, and also due to the energy of these living in hot springs at
collisions. With further increase in temperature, enzyme activity decrease temperatures of 60-
o
sharply. The latter is due to denaturation of the enzyme proteins with heat. We 80 C.
can thus say that though reaction rate increases with temperature, the rate of
inactivation of the enzyme also increases simultaneously. The concept of 175
Block 3 Enzymes

optimal temperature for enzyme activity does not seem to be a satisfactory

one, since it is the result of an increase in enzyme activity due to increase in
temperature, and loss of activity due to inactivation of the enzyme. Since the
latter factor is time dependent, the optimal temperature will itself depend upon
the time taken for rate determination at a particular temperature. The change
in rate of reaction for any 10o rise in temperature is known as its Q10, or
temperature coefficient value. This value is close to 2 for most chemical
reactions. For enzymatic reactions on the other hand the Q10 values are lower.

8.4.5 Enzyme Inhibition

Enzyme activity is inhibited by several factors, such as unfavourable pH
conditions, rise (or sometimes fall) in temperature or presence of protein
precipitants, such as alcohol, acetone and trichloroacetic acid. Inhibitory
action, caused by these agents is of a general nature and nonspecific. Of
greater interest in terms of structure and function of enzymes, are inhibitors
known as competitive and noncompetitive inhibitors. These will be described
in detail in Unit 9.

SAQ 2
Tick [ ] mark the correct statement.

Increasing the temperature of an enzymatic reaction is accompanied by

a) activity remaining constant [ ]

b) activity increasing continuously [ ]

c) activity decreasing continuously [ ]

d) activity first increasing and then decreasing [ ]

We have described the various factors that alter the rate of an enzymatic
reactions. You will recall that enzymes bring about enormous changes in the
rates of biochemical reactions. This makes life possible for an organism.
however, their importance for a living system is not only limited to increasing
the rates of biochemical reactions. Their activity can also be controlled or
regulated. This is highly significant in a living system, for rates of individual
biocatalytic reactions can be controlled and combined into different metabolic
pathways. These individual metabolic pathways are in turn integrated into an
overall metabolic system in the living organism. Let us now discuss more
about the regulation of enzyme activity in the following section.

8.5 REGULATION OF ENZYME ACTIVITY

Lehninger defined the living cell a self assembling, self adjusting, self
perpetuating isothermal system of molecules that exchanges matter and
energy with its environment. The cell accomplishes this by a process called
176 metabolism, in which many consecutive chemical reactions are organised into
Unit 8 Enzymes: Action and Regulation

metabolic pathways. You will recall that metabolism consists of anabolism and
catabolism. The term anabolism refers to those pathways of metabolism in
which low molecular weight precursors are transformed into more complex
substances which include carbohydrates, lipids, nucleic acids and proteins,
leading to the synthesis of new cellular components and overall growth.
Catabolism on the other hand is the process by which complex and simple
substances are broken down in other metabolic pathways to produce energy,
either in the form of heat or in the form of higher energy compounds. Both the
anabolic and catabolic pathways are interconnected.

It is self evident that such a complex system of biochemical transformations

has to be so regulated that concentrations of certain key metabolites are
controlled, both in terms of time and space, to direct metabolism in a desired
direction, and not allow it to drift into the ultimate products of aerobic
metabolism i.e., carbon dioxide and water. Since metabolism is driven by
enzymes, regulation of metabolism necessitates regulation of enzyme action.
We shall now look at some of the principal mechanisms by which enzyme
activity is regulated.

8.5.1 Regulation by Substrate or Product

The intracellular concentrations of a substrate can sometimes regulate the
action of the corresponding enzyme. Such regulation is possible when the Km
value of the enzyme is much higher than the intracellular concentration of the
substrate, so that enzyme activity is first order with respect to substrate
concentration.

When the product of an enzymatic reaction has a structure resembling that of

the substrate, it may act as an inhibitor of the reaction when it accumulates in
sufficient concentration. However, the significance of such product inhibition in
enzyme regulation cannot be very high since one would expect the product to
accumulate to a high concentration before significant inhibition is achieved.
Also it is difficult to conceptualise the control of a metabolic pathway, if the
product of one particular enzyme inhibits that enzyme independently of the
needs of the whole pathway.

An important biological mechanism which controls enzyme activity is

allosteric regulation. This interaction may bring about inhibition or stimulation
of activity by altering the activity of key enzyme. This is possible when
enzymes interact with the molecules produced in the cell. Let us explain more
about this type of regulation.

8.5.2 Allosteric Regulation

Regulation of some of the biosynthetic pathways occurs by this mode. In such
cases, the first enzyme of a biosynthetic pathway is strongly inhibited by the
end product of that pathway (also called feedback inhibition). For example,
aspartate carbamoyl transferase, the enzyme which catalyses the first step in
pyrimidine biosynthesis in E.coli, is inhibited by the final product of this
pathway, which is CTP, a pyrimidine nucleotide. This regulation is represented
in Fig. 8.10. 177
Block 3 Enzymes

Fig. 8.10: Inhibition of the first enzyme by the end product of the pathway.

Interestingly ATP which is a purine nucleotide, activates this enzyme. This is

shown graphically in Fig. 8.11. Thus, as a result of the opposing action of
these two types of nucleotides, production of purine and pyrimidine
nucleotides is properly balanced for the synthesis of nucleic acids.

Fig.8.11: Effect of CTP and ATP molecules (regulators) on the kinetics of

aspartate carbamoyl transferase from E.Coli.

On the basis of the work carried out on several other pathways, the following
general conclusions regarding this type of regulation have emerged.

 Only the first enzyme of the biosynthetic pathway is affected, due to the
feedback inhibition by the final product of the pathway.

 This final product of the metabolic pathway bears no structural

resemblance to the substrate or product of the first enzyme, thus, providing
a mode of enzyme regulation independent of the substrate or product (also
refer to subsection 8.5.1).

 The regulatory molecules, also called effectors, being structurally different

from the substrate or product of the first reaction, do not bind to the active
site of the first enzyme, but would perform their regulatory function by
binding at another site on the enzyme, called the allosteric enzyme site.
This binding is reversible. Enzymes so regulated are called allosteric
enzymes.

 Such allosteric enzymes do not exhibit normal Michaelis-Menton or

hyperbolic kinetics. In such cases, the plot of velocity against substrate
concentration shows sigmoidal behaviour (Figs. 8.12). The behaviour
indicates that at certain concentrations of the substrate, the enzyme
activity is much more influenced by changes in concentration of the
178 substrate, than would be the case of an enzyme characterised by normal
Unit 8 Enzymes: Action and Regulation

kinetic behaviour. Sigmoidal kinetics of a regulatory enzyme can be

changed to hyperbolic kinetics by subjecting the enzyme to physical or
chemical treatment. This kinetic behaviour is compared in case of
aspartate carbamoyl transferase as given in Fig. 8.12.

Fig. 8.12: Comparison of kinetics of the treated and untreated regulatory

enzymes, aspartate carbamoyl transferase from E.Coli.
 Regulatory enzymes generally posses an oligomeric structure like
haemoglobin (Unit 6) which is an oxygen transport protein of blood. The
multiple subunits which constitute the oligomeric structure of these
enzymes, are held together by weak noncovalent forces. The binding of
the regulator molecule to such an enzyme at the allosteric site brings about
a reversible conformational change i.e., a change of shape in the enzyme
subunits, causing a change in the structure in the structure of the active
site, leading to alternation in enzyme activity. This is depicted in Fig. 8.13
schematically.

Fig. 8.13: Diagrammatic representation of the reversible structural change in a

regulatory enzyme, brought about by a regulator.

You will recall that in the case of haemoglobin, a plot of percent oxygen
saturation against oxygen pressure is sigmoidal in nature (Fig. 6.16). The
sigmoidal kinetics of regulatory enzymes denotes interaction between
subunits, so that changes in the active site structure of the first subunit would
affect active site structures of other subunits through rearrangement of
noncovalent interactions. Such interactions between subunits, in response to
the binding of a regulator molecule to the allosteric enzyme, are called
cooperative interactions.

Regulation of enzyme activity by conformational change in the enzyme,

induced by the binding of regulator to its allosteric site, is possibly the most 179
Block 3 Enzymes

important means of metabolic regulation or control. Another mechanism by

which enzyme activity is regulated, involves reversible covalent modification of
the enzyme. Let us understand this mechanism in the next subsection.

8.5.3 Regulation by Reversible Covalent

Modification of the Enzyme
Several enzymes, crucial to biosynthetic and degradative pathways, are
regulated by reversible covalent modification of their side chains. These
enzymes are present in two different forms, possessing different catalytic
efficiency. These forms are interconvertible by the action of other enzymes,
some of which catalyse the modification of the side chains, whereas others
catalyse reversal of this modification.

We shall illustrate this with two enzymes, phosphorylase and glycogen

synthase. These enzymes are involved in the degradation and biosynthesis of
glycogen, respectively. The enzyme phosphorylase catalyses the following
reaction:

+ phosphorylase
(Glycogen)n (Glycogen)n-1 + -D-glucose 1-phosphate

The enzyme phosphorylase has two forms. The active form a and the inactive
form b. The only structural difference between the two forms is that side chain
of serine, at position 14 in the sequence of the enzyme, is phosphorylated in
the form a but not in the form b. This phosphorylation of the enzyme is
catalysed by the enzyme phosphorylase kinase, in presence of ATP and Mg3+.
The reversal of the process i.e., the conversion of phosphorylase a to
phosphorylase b is accomplished in the presence of another enzyme,
phosphorylase phosphatase, which catalyses the removal of the phosphoryl
group. These actions are schematically represented in Fig. 8.14.

Fig. 8.14 : Schematic representation of the interconversion of phosphorylase a

and phosphorylase b.

Similarly the other enzyme, glycogen synthase, which catalyses the synthesis
of glycogen (as opposed to phosphorylase, which catalyses its breakdown), is
converted to an inactive form by phosphorylation, a process which also leads
to the activation of phosphorylase. It is thus clear that the simultaneous
activation of phosphorylase and inactivation of glycogen synthase in a
reversible manner, are complementary process that allow a well integrated
control of glycogen metabolism.

Reversible conversion of active to inactive enzyme forms is a potent

mechanism of enzyme regulation. The amounts of the active and inactive
180 forms of the enzyme increase or decrease rapidly, because their
Unit 8 Enzymes: Action and Regulation

interconversion is enzyme catalysed. This also results in a larger amplification

of the initial signal, where a series of modifying enzymes act in succession to
promote a final metabolic event. The continuous activation and inactivation of
the enzyme also enables the enzyme system to be more responsive to
metabolic requirements.

SAQ 3
Tick [ √ ] mark the following statements as true or false.

a) Allosteric effectors function by binding not to the active site, but to an

alternative site on the enzyme. [True / False]

b) Enzyme phosphorylation can result either in a more active or a less

active enzyme. [True / False]

c) In the feedback mechanism the end product of the metabolic pathway

does not inhibit the first enzyme of the pathway. [True / False]
d) Reversible interconversion of an active enzyme to the inactive form
does not regulate enzyme activity. [True / False]

You will now briefly learn that some enzymes can exist in more than one form,
which differ in their kinetics, as well as regulatory properties. Genetic factors
are responsible for these different forms, and each form is tuned to a function
in a specific tissue.

8.6 ISOENZYMES
Sometimes different forms of an enzyme, catalysing the same reaction, occur
in a species. They are called isozymes or isoenzymes. These may arise from
minor modification of the same amino acid sequence or they may have
different amino acid sequences. These changes are genetically determined.
The term isoenzyme is reserved for those forms of an enzyme which
catalyse the same reaction, are isolated from the same species and differ
in amino acid sequences because of genetic reasons. The isoenzymes
generally differ in their kinetic and regulatory properties and their distribution is
tissue specific. This suggests that the kinetic and regulatory properties of a
particular isoenzyme are attuned to the metabolic requirements of a particular
tissue, where it is found in abundance. The nomenclature of the isoenzyme is
based on their electrophoretic mobilities towards the anode. Thus, hexokinase
isoenzymes are numbered I to IV, with isoenzyme I having the lowest mobility.

Isoenzymes of lactate dehydrogenase represent an interesting case of the

origin of isoenzymes. All isoenzymic forms of this enzyme consists of four
subunits. These subunits are of two different types, α and β. These combine in
five different ways viz. Α4, α3β, α2β2, αβ3 and β4, to respectively produce five
different isoenzymes, LDH-1 to LDH-5 as shown in Fig. 8.16. LDH-1 has the
highest electrophoretic mobility towards anode. LDH-1 is predominantly
present in heart and LDH-5 is mostly present in skeletal muscle and liver.
These isoenzymes differ both in their heat lability and sensitivity to inhibition
by excess substrate. 181
Block 3 Enzymes

Fig. 8.15: Electrophoretic mobility of isoenzymes of isoenzymes of lactate

dehydrogenase.

Several enzymes such as alkaline phosphatase, hexokinase, amylase, and

glucose 6-phosphate dehydrogenase, exist in isoenzymic forms, which are,
however, not as exhaustively studied as those of lactate dehydrogenase

SAQ 4
Tick [√ ] mark the most appropriate statement.

Isoenzymes are:

a) electrophoretic variants of the same enzyme. [ ]

b) electrophoretic variants produced by modification of side chains. [ ]

c) electrophoretic variants of the same enzyme which are genetically [ ]

determined.

d) different enzymes acting on the same substrate. [ ]

In this unit we mainly discussed the role of enzymes as biocatalysts, regulating

metabolic pathways in the organism. However, you may recall that enzymes
have many commercial, as well as medical uses also. We will attempt to give
you some idea of the significance of enzymes as important “tools” in medical
sciences. Let us, therefore, give you a brief account of their role in health
sciences.

8.7 ENZYMES IN HEALTH SCIENCE

In the present times enzymes are used to diagnose and to treat diseases.
Enzymology is an essential of the day to day life of modern clinicians. The
diagnostic value of certain enzymes arises from their differential distribution
between the blood plasma and cells of other tissues. For example, the
enzymes involved in blood coagulation are found exclusively in the plasma.
On the other hand, many other enzymes are present in much higher
concentrations in the tissue cells, than in blood. These are released into the
blood and various biological fluids only when there is usual destruction of the
cells. Their normal levels in plasma are insignificant, being more than one
million times lower than their concentration in the cells. In case of cell
destruction or injury by such cases as a damaged heart or uncontrolled growth
of cancer cells, the plasma levels of these cellular enzymes are elevated
182
Unit 8 Enzymes: Action and Regulation

significantly. These changes in plasma concentration of particular enzymes

are estimated by the clinicians and used not only to detect cell damage but
also so suggest the site of cell damage. The degree of elevation of plasma
concentrations of these enzymes also give a clue to the extent of cellular
damage. These enzyme assays have become a critical diagnostic tool in the
detection of heart, liver, pancreas, skeletal muscle, bond and malignant
diseases. In Table 8.4 you will find a list of some enzymes, clinical assay of
which are used for detecting particular diseased states.

Table 8.4: Enzymes assayed in medical diagnostics

Enzyme Used in the determination of

Lactate dehydrogenase (LDH) Heart or skeletal muscle damage

Alkaline phosphatase Liver and bone diseases

Serum Glutamate Oxaloacetate Heart and liver diseases
Transaminase (SGOT)

Creatine phosphokinase (CK) Myocardial infarction and muscle

diseases

Acid phosphatase Cancer of the prostate

α-Amylase Pancreatitis

We shall illustrate the use of enzyme assay in myocardial infarction, which in

simple language means a heart attack. In this condition the blood supply to the
heart muscle is blocked and some of the cells of the heart muscle die, thereby,
releasing an abnormal amount of their enzymes into the blood. Monitoring the
blood concentration of three enzymes –CK, SGOT and LDH, helps a physician
in identifying a heart attack and its severity. You will find a graph of the
concentration of these enzymes after a heart attack in Fig. 8.16.

Fig. 8.16 : After a heart attack, concentration of CK increases rapidly and then
falls. SGOT level also increases rapidly then falls. One to two days
afteran attack, LDH concentration begins to increase, gradually rises
for three to four days, and then decreases gradually over the next
seven to ten days.

Enzymes also find extensive uses as laboratory reagents. A common test,

involving use of enzymes, is to measure glucose concentration in urine, as in
case of diabetes. Two enzymes, namely glucose oxidase and peroxidise, are
183
Block 3 Enzymes

used in this test. They are contained on a test strip which is momentarily
dipped in the urine sample. A colour change, on the strip after sometimes, is
compared to a colour scale to measure the concentration of glucose. In this
test glucose oxidase catalyses conversion of glucose to gluconic acid and
hydrogen peroxide. Peroxidase then catalyses the reaction of hydrogen
peroxide and o-toluidine to give a coloured product, the colour intensity of this
indicates the glucose concentration.

glucose oxidase
D-Glucose + O 2 + H 2O Gluconic acid + H 2O

peroxidase
o-Toluidine + H 2O2 Coloured product + H 2O
(schiff base)
Enzymes are also used in treatment of many medical problems, for example,
trypsin and chymotrypsin are used in severe burns, for they decompose the
proteins of the clotted blood, pus and dead skin on the burned areas. Similarly
many nasal sprays contain enzymes to clear up congestion. And probably you
might have used some digestive enzymes when you had a heavy spicy meal,
just to aid your digestion.

SAQ 5
Tick [√ ] mark the most appropriate statement.

Alkaline phosphatase is used in the determination of:

(a) heart or skeletal muscle damage

(b) liver and bone disease

(c) myocardial infarction and muscle diseases

(d) cancer of the prostate

8.8 SUMMARY
 Enzymes are biocatalysts which increase reaction rate enormously. They
are responsible for most of the chemical reactions which take place in the
living organisms.

 Enzymes are protein in nature, with considerable diversity of structure.

Some enzymes are simple proteins, whereas many are conjugated
proteins.

 Many enzymes (conjugated proteins) require a part without which they

cannot function. These nonprotein parts, called cofactors, are either metal
ions or low molecular weight organic compounds. These organic cofactors
are mostly derived from B group of vitamins. The organic cofactors are
generally known as coenzymes.

 Enzymes are characterised by an active site, where the substrate

(reactant) binds, undergoes transformation and then the products are
184
Unit 8 Enzymes: Action and Regulation

subsequently released. The active site consists of amino acid side chains,
some of which act as binding groups while others act as catalytic groups.

 Enzymes are characterised by high catalytic efficiency, remarkable

specificity and regulatory properties. Enzymes can increase reaction rates
a billion fold, which enables biochemical reactions to occur under
physiological conditions. They are highly specific, both with regard to the
substrate and the reaction they catalyse. The degree of specificity varies
from one enzyme to another. The regulatory properties of enzymes
enables fine tuning of enzyme activity in response to the needs of the cell,
under changing physiological conditions.

 Enzymes have been grouped into six major classes on the basis of the
nature of the reactions they catalyse. These classes are: Oxidoreductases,
transferases, hydrolases, lyases, isomerases and ligases. Each enzyme
has been assigned a four number identification code, indicating the major
class, type of substrate or the bond cleaved, the actual reaction catalysed
and the serial number of the enzyme in its subsubclass.

 Enzymes increase reaction rate enormously by lowering the energy of

activation of the reaction. This is achieved by bringing the reactants in
close proximity and in a correct orientation for the reaction to occur. Also at
the active site, substrate is subjected to stress and strain, which facilitates
bond making or breaking. The energy for this is derived from favourable
enzyme-substrate interactions at the active site.

 Various factors govern the rate of enzymic reactions. The plot of enzyme
velocity versus substrate concentration shows a plateau at high substrate
concentration, indicating that the reaction rate after reaching a maximum
value is independent of further increase in substrate concentration.

 The substrate concentration required for half maximal velocity (Vmax) is

called the Michaelis Constant Km. This is useful parameter for determining
catalytic potential of an enzyme under physiological conditions and also
gives an idea of the affinity of the enzyme for its substrate.

 Enzyme activity is proportional to the concentration of the enzyme and is

dependent on pH and temperature of the medium.

 Enzymes are inhibited by reagents which compete with the substrate for
the active site. These substances are known as competitive inhibitors. The
other type of substances which inhibit the enzyme by inactivating it are
called noncompetitive inhibitors.

 Enzymes are regulated by the end product of a biosynthetic pathway. The

final and product inhibits the first enzyme of that pathway by binding to an
allosteric site which is distinct from the active site. The inhibition results
from a conformational change in the oligomeric structure of the first
enzyme.

 In another type of regulation, the amino acid side chains of enzymes are
reversibly modified, mainly by phosphorylation or dephosphorylation. This
produces an active or an inactive enzyme.

185
Block 3 Enzymes

 Sometimes enzymes catalysing the same reaction exist in different

electrophoretic forms in different tissues of the same species. These
different forms are known as isoenzymes and result due to genetic factors.
Lactate dehydrogenase is an interesting example of isoenzymes.

 Enzymes have a diagnostic value in medical sciences. Various enzyme

assays are employed to confirm, locate and also indicate the severity of a
disease in a human being. Enzymes are used as laboratory reagents and
are also employed in treating various diseased conditions.

8.9 TERMINAL QUESTIONS

1. How does an oxidoreductase differ from a transferase? Illustrate your
answer with an example.

2. Describe active site of an enzyme.

3. Discuss the need for regulation of enzyme action.

4. Briefly describe important features of allosteric regulation.

5. Explain what is ‘transition state’ of a reaction.

6. How does an enzyme contribute to the lowering of energy of activation of

a chemical reaction?

7. Describe the origin of isoenzymes of lactate dehydrogenase.

8. What is Km and what does a low value of Km signify?

8.10 ANSWERS
Self-Assessment Questions
1. a) increase b) lower c) proximity and orientation d) transition

2. d)

3. a) True b)True c) False d) False

4. c)

5. b)

Terminal Questions
1. An oxidoreductase takes part in physiological oxidation-reducing process.
On the other hand, a transferase catalyses the transfer of a chemical
group, from one substrate to another. Lactate dehydrogenase is an
example of an oxidoreductase, and hexokinase is an example of a
transferase.

2. The portion of an enzyme which binds to the substrate and catalyses its
chemical transformation is called the active site of an enzyme. The active
site has hydrophobic and hydrophilic amino acids which bind to the
186 substrate by hydrophobic, electrostatic and hydrogen bonded interactions.
Unit 8 Enzymes: Action and Regulation

Some of the hydrophilic amino acids, which are charged or uncharged also
participate in bond breaking processes.

3. Metabolic activities of the cell lead to the biosynthesis of cellular

components and to the generation of energy needed to sustain life. Such
a complex system of biochemical transformations has to be regulated to
channelize crucial metabolites into the required pathway and not allow
them to drift into the ultimate products of aerobic metabolism i.e., carbon
dioxide and water. Since metabolism is driven by enzymes, it stands to
reason that regulation of metabolism is essentially regulation of enzyme
action.

4. An important feature of allosteric regulation is that the end product of a

pathway inhibits the first enzyme of that pathway, by binding to an
allosteric site or a site distinct from the enzymic active site. The inhibition
results from a conformational change in the oligomeric structure of the first
enzyme. Such allosteric enzymes do not show normal kinetic behaviour,
for in their case, plot of rate versus concentration is not a hyperbole but
sigmoidal. The sigmoidal kinetics shows that interaction of the substrate at
one active site affects other active sites of the oligomeric structure through
inter-subunit contacts.

5. The energy profile a chemical reaction shows that the reactants have to
overcome an energy barrier before they converted into products. The
structure and conformation of reactant molecules corresponding to the
peak position of this energy barrier is called the ‘transition state’ of the
reaction. This state represents molecules in their most energised form
where bonds are in the process of being made or broken.

6. Enzymes lower the energy of activation by bringing the reactants in close

proximity, and correctly orienting them towards each other, thus, in effect
increasing their concentration by several orders of magnitude. The
entropic disadvantage which chemical reactions suffer from is thus
overcome by the enzyme. The energy for this process is derived from
favourable enzyme-substrate interactions at the active site. The energy
from these interactions is also used to subject the substrate to stress and
strain to facilitate and bond making and bond breaking processes.

7. Lactate dehydrogenase isoenzymes consists of four subunits of two

types, α and β, which are coded for two different genes. These two types
of subunits combine in different proportions viz., α4, α3β, α2β2, αβ2 and β4,
to give the five isoenzymes.

8. Concentrations of the substrate expressed in mole per litre that gives half
maximal velocity of the enzyme, is known as Km. Small value of Km
indicates high affinity of the enzyme for the substrate.

187
Block 3 Enzymes

UNIT 9

ENZYMES AND DRUG ACTION

Structure
9.1 Introduction Drug Receptors

Expected Learning Outcomes Drug Receptor Theory

9.2 Common Terms Used 9.6 Structure Activity Relationships

(SAR)
9.3 Medicines and Drugs
9.7 Enzymes as Drug Targets
9.4 Processes in Drug Action
9.8 Summary
Pharmacodynamics and
Pharmacokinetics 9.9 Terminal questions
9.5 Mechanism of Drug Action 9.10 Answers

9.1 INTRODUCTION
In the previous two units you have learnt about enzymes, the naturally
occurring biocatalysts, as to how they bring about changes in the biological
functions or physiological processes. You know that enzymes are constituted
of the biomolecules, proteins specifically. Drugs are another class of chemical
substances or molecules when administered inside the body, bring about
changes in the biological functions or the physiological processes. The drugs
are of two types; those with beneficial effects and the others with toxic effects.
The beneficial and toxic effects of drugs result from their interactions with
molecules in the human body. You can easily relate the former to your day-to-
day life by the medicines prescribed to you by a doctor when you fall ill. In fact,
the prehistoric man also recognised the beneficial or toxic effects of many
plants and animal materials. Early written records list remedies of many types,
including a few that are still recognised as useful drugs. The question which
you may ask here is: what is the relationship between the enzymes and the
drugs especially the beneficial category?

The intake of chemical substances or drugs affects the action of enzymes

directly or indirectly. How? You know that enzymes can catalyze multistep
chemical reactions and achieve phenomenal rate accelerations by matching
protein and substrate chemical groups in the transition state. Some of the
most potent and effective drugs take advantage of these chemical interactions
188 and act as inhibitors. The catalytic chemistry of enzymes is the key to
Unit 9 Enzymes and Drug Action

designing potent inhibitors and makes them a special class of drug targets. In
this unit you will study how enzymes are good drug targets. We would start
with defining some of the terms that you might not have studied earlier in the
course and are very important to the study of drugs. We would explain the two
very closely related terms often used interchangeably. These are drugs and
medicine. This is followed by the action of drugs. You will be introduced to the
concept of receptor and how drugs bind with these receptors to produce
changes in the biological functions and physiological processes. The structure
activity relationship of drugs would also be explained to understand how these
help in drug discovery.

Expected Learning Outcomes

After studying this unit, you should be able to:

 define and differentiate between drug and medicine;

 describe the drug receptor theory;

 Explain the structure activity relationships of drugs;

 Discuss enzymes as drug targets.

9.2 COMMON TERMS USED

Let us first get familiar with some of the terms that are essential to understand
the concepts involved in the study of drug interaction in human beings. These
are explained in brief below.

Dosage form: It is defined as the physical form of a dose of a compound used

as a drug intended for administration or consumption.

Dose: The amount of a medicine given at any one time.

Drug formulation: the process of designing by mixing other required

substances to the drug and producing the drugs that are eventually sold.

Excipient: inert ingredient added in the drug formulation to protect, support or

enhance stability of drug.

Active Pharmaceutical Ingredient: active ingredient in a drug responsible for

its effect is called the API.

Pharmacodynamics: The action of drugs on the body with reference to the

site and action is called pharmacodynamics

Pharmacokinetics: What the body does with the drug is called

pharmacokinetics. It involves absorption, distribution, metabolism and
excretion.

Receptor is part of cell or organism which interacts with a drug and produces
therapeutic or toxic effects.

Effector is a small molecule like protein that selectively binds to a protein and
regulates its biological activity.
189
Block 3 Enzymes

Now let us study another two important terms that are different from each
other but are used commonly interchangeably.

9.3 MEDICINES AND DRUGS

Most of us may use the terms medicine and drugs interchangeably but
scientifically they have different meanings. Medicine is originated from the
Latin “Medicus” meaning “healing, or physician” and drug is originated from
the French word ‘drogue’ means a dry herb and Greek “Pharmacon” meaning
“Drug”. Medicine is the formulated form of the drug having definite dose and
dosage form. While a drug can be defined as a medicine or other substance
which has a physiological effect when ingested or otherwise introduced into
the body. We can say that every medicine is a drug but not all drugs are
medicines. It can be said that drug is the active pharmaceutical ingredient (API)
and does not have a dosage form and dose. On the other hand when the drug
is formulated it becomes medicine and with dosage form and dose. As the
drug does not have a dosage form it cannot be directly used for treatment.
Drugs can be obtained from plant, animal, microorganisms, minerals, synthetic
source, semisynthetic source and recombinant DNA technology. While the
source of medicine is drug with or without excipient.

Let us suppose there are three drugs with three different therapeutic
properties - analgesic (relieves from pain), anti-inflammatory (reduces
inflammation or swelling) and antipyretic (decreases body temperature in
fever). Let us name these A, B and C. As these have therapeutic properties,
these can be classified as drugs. Drug A, though has therapeutic effect, also
has potential side effects due to which A cannot be used for treatment, cure
and management of any disease, so can’t be classified as medicine. Drug B is
good but is addictive so it can’t be used as medicine. Drug C has beneficial
effect and has no harmful side effects so can be used for treatment of
diseases and is classified as medicine. The difference between drugs and
medicines is summarized in Table 9.1.

Table 9.1: Differences between drugs and medicines

Drug Medicine

The word ‘Drug”is derived from Greek The word “Medicine” is

“Pharmacon” meaning “Drug”. derived from Latin “Medicus”
meaning “healing, or
physician”.

A Drug is any chemical substance A medicine is the formulated

which acts on the living body alter the form of drug having definite
physiological process and is used for dose and dosages form
prevention, diagnosis, control, and which isused for prevention,
treatment of diseases. diagnosis, control, and
treatment of disease.

A drug is only active pharmaceutical A medicine is the formulation

ingredients (API). of API with excipients or
without excipients.

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Unit 9 Enzymes and Drug Action

Drug has no appropriate dosage form Medicine has an appropriate

and dose. dosage form and dose.
Generally, a drug is not using directly A medicine is used for
for treatment because it needs to be treatment directly.
designed in suitable dosage from and
dose.
Sources of drugs are plants, animals, Source of medicine is drug
microorganisms, minerals, synthetic and excipient.
sources, semisynthetic sources,
recombinant DNA technology.
All medicines are drugs. All drugs are not medicines.
Examples: paracetamol, also known as Examples: Paracetamol
Acetaminophen (analgesic and tablet, Paracetamol syrup,
antipyretic); Morphine (analgesic); Paracetamol elixir.
Ampicillin (antibiotic).

You can proceed to learning the process of drug action after answering the
following SAQ.

SAQ 1
Write (T) for true and (F) for false statement(s) in the following.

(a) All drugs are medicines. .

(b) Drugs alter the physiological processes on entering the body.

(c) Drugs have appropriate dosage form.

(d) Medicine is only active pharmaceutical ingredient.

9.4 Processes in Drug Action

As mentioned earlier drug is a chemical substance that when introduced inside
the body alters the physiological processes. Once the drug enters the body
two processes take place. Firstly, the drug acts on the body and secondly the
body metabolises the drug. These two processes are explained by two terms
viz. pharmacodynamics and pharmacokinetics, respectively. These terms
have been defined also in Section 9.2. let us understand the two terms in
detail in the following subsection.

9.4.1 Pharmacodynamics and Pharmacokinetics

Pharmacodynamics is the study of the biochemical and physiological effects
of drugs in the body. It shows the interaction of the drug with tissue receptors
which are located on the cell membranes or in the intracellular fluid.

Pharmacokinetics is the study that determines the rapidity and concentration

of the drug in the body and its duration of appearance at the target organ, i.e.
onset, time of peak action and duration of action. It also determines the
route(s) and frequency of administration of drug. The two processes can be
depicted pictorially as given in Fig. 9.1. 191
Block 3 Enzymes

Fig 9.1: Pharmacokinetics and pharmacodynamics of a drug.

When you take a medicine, your body releases the drug to produce a final
medicinal product.Then the drug or substance is absorbed and enters the
blood. The circulatory system distributes or disperses the drug or substances
throughout the fluids and tissues of the body. The drug is then metabolized by
the organism and the irreversible transformation of initial compounds to
metabolites takes place. The metabolites are then excreted or removed from
the body.

Pharmacodynamics plays an important role in drug-response relationship i.e.,

the relationship between the drug concentration and its effect. It helps in
determining the minimum effective dose (MED) and the maximal tolerable
dose (MTD). MED is defined as the dose above which sufficient desired
effect is being achieved. The maximal tolerable dose (MTD) is defined as the
dose above which intolerable side effects can be observed. The dosage gap
between the MED and MTD is termed the therapeutic window. The drug-
response curve is illustrated in Fig. 9.2 (a) indicating the desired and the side
effects. As can be seen the drug concentration is on the x-axis and the y-axis
shows the biological effect. The desired biological effect is shown by the purple
line and detrimental side effects by the green line. The pharmacokinetic profile
of the curve is illustrated in Fig. 9.2 (b). As you would note here time is on the
x-axis and drug concentration on the y-axis. Also, you can see the unbroken
line that specifies the drug concentration and multiple applications of the drug
are demonstrated by the multiple peaks which decay with time.

(a) (b)

Fig. 9.2: (a) Illustration of typical dose response curves for desired and side effects.
(b) Illustration of a dose scheduling pharmacokinetic profile

Let us now study the important aspects of drug action in the next section.
192
Unit 9 Enzymes and Drug Action

9.5 MECHANISM OF DRUG ACTION

Drugs (except those gene based) like enzymes, just alter the rate of the
ongoing activities. They do not cause or produce any new functions to any
system, organ or cell. Very few drugs act because of their simple physical or
chemical property. Some drugs act due to physical mass e.g., bulk laxatives
(isabgal), due to physical form (petroleum jelly), due to adsorptive property
(activated charcoal), etc. The drug action can be broadly described as:

(a) Stimulation,is an increase in the selective activity of specialized cells,

which increases the secretion from a gland.

(b) Depressionis a reduction/decrease in the activity of specialized cells. For

example barbiturates depress central nervous system.

(c) Irritation: noxious effect, is the effect of drugs on the growth, nutrition and
morphology of living tissues which induce a gross change in cellular
function.

(d) Replacement,includes certain drugs, which are used to replace some

missing endogenous component of the body or when the production of
endogenous components is reduced. These are given in deficiency
diseases, e.g. insulin in diabetes mellitus.

(e) Bactericidal and Cytotoxic action is for killing parasites or cancer cells.
Bactericidal activity, which is bacterial death, is induced by certain types
of antibiotics. Cytotoxic action is selective for invading cancer cells and
altering them without affecting the host cells.

The drugs produce their effects mainly by interacting with a discrete target
biomolecules, which usually are proteins. These proteins are the targets of
drug action and grouped into four major categories, viz. enzymes, ion
channels, transporters and receptors. We will discuss in detail about the
receptors in the following subsection.

9.5.1 Drug Receptors

You by now know that the beneficial (therapeutic) and toxic effects of drugs Ligand (Latin: ligare—
result from their interactions with molecules in the patient. Most drugs act by to bind) Any molecule
associating with specific macromolecules in the membrane or inside the which attaches
selectively to a
cell and alter the biological function. Such macromolecules are called
particular receptor or
receptors. Thus, receptor is defined as a macromolecule or binding site site.
located on the surface or inside the effector cell that serves to recognize the
signal molecule/drug and initiate the response to it, but itself has no other
function. The function of receptor is to propagate regulatory signals from
outside to the effector cell, amplify the signal, integrate various extracellular
and intracellular regulatory signals and adapt to short term and long term Signal molecules are
changes. The overall scheme of drug action through receptors is depicted in also called ligands,
Fig. 9.3. the molecules that
bind specifically to
The binding of a drug to receptor depends on types of chemical bonds that other molecules like
receptors.
can be established between drug and receptor. The drug receptor interaction
is similar to a ligand binding that forms a complex with metal ions. The
interactions are of the following types: 193
Block 3 Enzymes

Agonist: When a drug binds with a receptor and produces an effect similar to
that of the physiological signal molecule as given in Figure 9.3 (b).

Inverse agonist : When a drug activates the receptor but produces an effect
in the opposite to that of a physiological signal molecule.

Antagonist: when a drug prevents the action of an agonist on a receptor or

the subsequent response but does not have any effect of its own as shown in
Fig. 9.3 (c).

Partial agonist: A drug which activates a receptor to produce submaximal

effect but antagonises the action of a full agonist.

The term only indicates affinity or binding without regard to functional change;
agonists and competitive antagonists are both ligands of the same receptor.

(a) (b) (c)

Fig. 9.3: Drug action through receptors: a) Natural action b) Agonist drug C)
Antagonist drug.

Receptors have dual functions, i.e., they recognise a specific ligand molecule
and then cause transduction/transmission/conversion of the signal into a
response. As only a handful of transducer pathways are shared by a large
number of receptors, the cell is able to generate an integrated response
reflecting the sum total of diverse signal input. The transducer mechanisms
can be grouped into 4 major categories:
Ligand-gated ion (i) Cell surface receptors: These are also called ligand-gated ion channels
channels (LGICs) are
(LGICs). These are integral membrane proteins that have ion selective
integral membrane
proteins that contain a channels (for Na+, K+, Ca2+ or Cl¯) across the plasma membranes. When
pore which allow the an agonist binds, it opens the channel and causes changes in cytosolic
regulated flow of ionic composition, depending on the ion that flows through.
+
selected ions like Na ,
+ 2+ ¯
K , Ca or Cl across
(ii) G-protein coupled receptors (GPCR): These are a large family of cell
the plasma
membrane. membrane receptors which are linked to the effector (enzyme)channel-
carrier protein) through one or more GTP-activated proteins(G-proteins)
for response.

(iii) Enzyme-linked receptors: These have a subunit with enzymatic property

or bind a Janus-Kinase (JAK) on activation. JAK are intracellular, non-
receptor tyrosine enzyme kinases.

(iv) Receptors regulating gene expression (Transcription factors) are

194 intracellular cytoplasmic or nuclear) soluble proteins.
Unit 9 Enzymes and Drug Action

All the four types of receptors have been described along with their schematic
representation is given in Table 9.2.

Table 9.2: Four types of receptors

Type-1 Type-2 G-protein Type-3 Type-4

Ligand-gated coupled receptor Enzyme-linked Nuclear
ion channels receptor receptor
Location Membrane Membrane Membrane Intracellular
Effector Channel Channel or Enzyme Gene
enzyme transcription
Coupling Direct G-protein Direct Via DNA
Examples Nicotinic Muscarinic Insulin receptor, Steroid/Thyro
Receptor, receptor, Growth factors, id receptors
GABAA R Adrenoceptors Cytokine R
Time scale for Milliseconds Seconds Hours Hours/Days
cellular effects

The mechanism of drug action can be well understood by drug-receptor theory

detailed in subsection 9.5.2. Before you study the receptor theory attempt the
following SAQ.

SAQ 2
Match the contents of column (A) with those of column (B) in the following:
(A) (B)
a) Pharmcokinetics 1) gene transcription
b) Pharmacodynamics 2) produces similar effect
c) Agonist 3) what the body does to the drug
d) Nuclear receptor 4) what the drug does to the body

9.5.2 Drug-Receptor Theory

The biological activity of a drug is related to the stability of drug-receptor
complex i.e., affinity of a drug for its receptor. Stability of drug receptor The strength of drug
complex is measured by the difficulty in dissociation of the drug-receptor receptor complex is
complex. The more the difficulty the more strong is the interaction and more is because of interaction
between drug and
the drug effect. These interactions are due to Van der waals, hydrophobic receptor.
and ionic interactions along with hydrogen and covalent bonding.There
have been several major theories that have been proposed to provide a
theoretical basis for understanding, modeling, and thereby predicting the drug
response. Three of the most widely known of these are described as follows.

 Occupancy theory: Clark (1937) propounded a theory of drug action

based on the occupation of receptors. The idea being that a response
emnates from a receptor only when it is occupied by an appropriate ligand
i.e. the drug. When the drug binds with the receptor it stimulates it and
produces a biological effect. The biological effect remains till the drug
occupies or binds the receptor. Once the drug dissociates from the receptor 195
Block 3 Enzymes

the drug action is lost. So we can say that the proportion of occupied
receptors is related to the effect of the drug. For example, for full agonists
and a linear relationship, 100% occupancy = 100% effect. The interaction
between the two molecular species, viz. the drug (D) and the receptor (R)
and the effect (E) to be a direct function of the drug-receptor complex (DR)
formed can be given by Clark’s equation as given below.
K1
D + R DR E ... (9.1)
K2

Later on it was observed that occupation of the receptor is essential but

not itself sufficient to elicit a response; the agonist must also be able to
activate i.e., induce a conformational change in the receptor. The ability to
bind with the receptor designated as affinity and the capacity to induce a
functional change in the receptor designated as intrinsic activity (IA) or
efficacy are independent properties. For example antagonists occupy the
receptor but do not activate it. So, a theoretical quantity denoting strength
of stimulus (S) imparted to the cell was interposed in the Clark’s equation
given below.
K1
D + R DR S E ... (9.2)
K2

Depending on the agonist, DR can generate a stronger or weaker S,

probably as a function of the conformational change brought about by the
agonist in the receptor.

 Rate Theory: It is based on the idea that a response emanates from a

receptor in proportion to the kinetic rate of onset and offset of drug binding
to the receptor.

 Operational Model: It is a modified, semi-empirical approach that is

based on occupancy theory modified to incorporate a factor relating
agonist-receptor complex and response.

Now that you are familiar with the interactions of a drug with the biological
system, it is also important to understand the effect of structure of a drug on its
effect in that system. Let us study in brief in the next section.

Structure activity
9.6 STRUCTURE ACTIVITY RELATIONSHIPS (SAR)
relationships can be
used to predict “Structure of the drug determines its biological function” was first stated by
biological activity from Crum-Brown and Fraser in 1868. Determination of structure and function
the structure of the
lead compound and is
relationship helps the medicinal chemists to synthesise new drugs. The
a means by which the biological effects of a new chemical compound can often be predicted from its
effect of a drug or molecular structure using data of other similar compounds. This is because
toxic chemical on an
similar compounds may have similar physical and biological properties. There
animal, plant or the
environment can be is a relationship between molecular structures and their biological activity, and
related to its this principle is referred to as Structure Activity Relationship (SAR). This
molecular structure. concept is used in drug discovery to characterise existing molecules and guide
the synthesis of compounds with increased activity, a different activity from the
196 existing drug and fewer side effects. It is used to determine the
Unit 9 Enzymes and Drug Action

pharmacophore and unwanted side effects. It also helps to know how the
minor changes in the molecule would affect the pharmacological activities. Pharmacophore is
part of a molecular
SAR is located at the intersection of biology, chemistry, and statistics structure that is
responsible for a
asdepicted in Fig. 9.4. The focused interlinking of these disciplines has particular biological or
broughtabout the development of a research activity resembling the “science pharmacological
of SAR”. interaction that it
undergoes.

Fig.9.4: SAR located at the intersection of biology, chemistry, and statistics.

The SAR is useful to study the effects of modifications of drug analogues i.e.,
how modifications will affect transport across membrane, water solubility,
receptor binding and pharmacokinetic properties. These modifications can be:
change in size and structure of parent compound, stereochemistry of lead
molecule (L or D) and nature and degree of substitution. The effect of change
in size and structure of parent compound is explained below.

Change in size and structure of parent compound: It is well known that a

drug when taken has to be absorbed i.e., cross a biological membrane,
transported to the target site and then attach to a receptor to produce its
pharmacological effects. For this whole process the structure of the
compound/drug is very important as the drug will bind to the receptor only if
the structure fits the receptor. If a group is added or removed it not only
changes the structure of compound but changes the function also by either
enhancing the effect of drug or decreasing the effect of drug. The effect of
addition of a few groups is explained in the following paragraphs.

 Addition of methyl group: causes an increase in lipophilicity which

causes and increases in transport across biological membrane. You have
learnt in Unit 1 that biological membranes are a lipid bilayer therefore,
increased lipophilicity will increase transport. Increase in transport across
biological membrane will increase its biological function. However,
increase in lipophilicity will cause a decrease in its property of water
solubility which might decrease its activity.

 Addition of hydroxyl group: causes an increase in hydrophilicity i.e.,

increase its ability of solubility. It also causes increase in hydrogen
bonding at the target site thus increases the drug activity.

 Addition of basic groups e.g., amines, amides: The basic groups when
in free form help drug to transport and in salt form helps the bonding of
drug with receptor by hydrogen bonds. 197
Block 3 Enzymes

SAQ 3
Match the following given in group (A) with that in group (B).
(A) (B)
a) Clark 1) Lipophilic
b) OH group 2) Hydrophilicity
c) Crum-Brown 3) SAR
d) Methyl group 4) Occupancy theory

9.7 ENZYMES AS DRUG TARGETS

You have read in previous units that enzymes catalyze multistep chemical
reactions and achieve phenomenal rate accelerations by matching protein and
substrate chemical groups in the transition state. You have also learnt that
enzymes act optimally and their activity cannot be either increased or
decreased. However, drugs can either increase or decrease the rate of
enzymatically mediated reactions. In fact, drugs can act by increasing or
decreasing the affinity of enzymes for its substrates. The activity of enzymes
can be altered by stimulation of an enzyme which increases its affinity for the
substrate and the rate constant (Km) of the reaction is lowered. Enzyme activity
can also be altered by enzyme induction i.e., increasing the synthesis of more
enzyme protein. Inhibition of enzymes is another common mode of drug
action. The catalytic chemistry of enzymes is the key to designing potent
inhibitors and makes them a special class of drug target. The effect of enzyme
stimulation, induction and inhibition on the kinetics of enzyme reaction is
depicted graphically in Fig. 9.5. In the graph Vmax is the maximum velocity of
reaction,Vmax(s) of stimulated enzyme,Vmax(i)in presence of noncompetitive
inhibitor,Kmrate constant of the reaction,Km (s)of stimulated enzyme,Km (i)in presence
of competitive inhibitor. Enzyme induction and noncompetitive inhibition
donot change the affinity of the enzyme i.e., Km is unaltered, whereas
enzyme stimulation and competitive inhibition respectively decrease and
increase the Km respectively.

Fig.9.5: Effect of enzyme stimulation,inductionand inhibition on kinetics of

enzyme reaction.

Inhibition of enzymes can occur in different ways. These are explained in the
following paragraphs.
 Nonspecific inhibition: As enzymes are proteins many drugs alter the
198 tertiary structure of a enzyme with which they come in contact and
Unit 9 Enzymes and Drug Action

denature them and thus enzymes get inhibited. Enzyme activity is also
inhibited by several factors, such as unfavourable pH conditions, rise (or
sometimes fall) in temperature or presence of protein precipitants, such as
alcohol, acetone and trichloroacetic acid. Inhibitory action, caused by
these agents is of a general nature and nonspecific. Examples of
chemical which inhibit enzymes non specifically are heavy metal salts,
strong acids and alkalies, alcohol, formaldehyde, phenol. As these
inhibitors cause damage rampantly they cannot be used systemically.

 Specific inhibition: Many drugs inhibit a particular enzyme without

affecting others. Such inhibitionscan be either competitive or
noncompetitive in nature. These are explained below.

 Competitive inhibition: Competitive inhibitors have a structure

resembling that of the substrate. The structural similarity between the
substrate and the inhibitory compound enables the inhibitors to
compete with the substrate for binding to the active side of the enzyme.
This prevents the access of the substrate to the active site and the
formation of ES complex. Since the substrates of enzymes are
generally metabolities, participating in metabolic transformations in the
cell, competitive inhibitors are known as antimetabolites. One of the
best known examples of a competitive inhibitor is sulphanilamide. This
is a competitive inhibitor of p-amino benzoate, a compound utilised in
the synthesis of folic acid coenzymes essential for the transfer of one
carbon fragments. It is, therefore, easy to understand why
sulphanilamide is successfully used as a drug, inhibiting the growth of
bacteria. In fact many other drugs also work by acting as competitive
inhibitors in enzymatic reactions necessary for the growth and survival
of a large number of bacteria.
NH2 NH2

COOH SO 2NH2
p - Amino benzoic acid Sulphanilamide

Another well known competitive inhibitor is malonic acid, which inhibits

the oxidation of succinic acid to fumaric acid by the enzyme succinic
dehydrogenase. The competitive action of malonic acid against
succinic acid can be appreciated by a comparison of their structures,
which are closely similar.
COOH

COOH CH2

CH2 CH2

COOH COOH

Malonic acid Succinic acid 199

Block 3 Enzymes

It is possible that the active site of the enzyme mistakenly accepts

malonic acid instead of succinic acid is its substrate, causing
competitive inhibition. You will find a diagrammatic representation of
competitive inhibition in Fig. 9.6 (b).

As the product is not formed or nonfunctional product is formed as

shown in Fig. 9.5 a new equilibrium is achieved in the presence of the
drug that is why it is also called equilibrium type inhibition. A
characteristic feature of competitive inhibitions is that it can be
reversed by increasing the substrate concentration. We can detect
competitive inhibition by a study of the rate of enzymatic reaction in the
presence, and in the absence of a competitive inhibitor. You can
observe from Fig. 9.5 that Vmax remains unchanged in the presence of
a competitive inhibitor, whereas Km is increased, since a higher
substrate concentration would be needed to out-compete with the
inhibitor and to achieve the saturation of half of the enzyme molecules.

A nonequilibrium type of enzyme inhibition can also occur with drugs

which react with the same catalytic site of the enzyme but either form
strong covalent bonds or have such high affinity for the enzyme that
the normal substrate is not able to displace the inhibitor. In these
situations, Km is increased and Vmax is reduced.

 Noncompetitive inhibition: Noncompetitive inhibitorsinhibit enzyme

action by combining with a group essential for the activity of the
enzyme or by removing a metal ion involved in the activity of the
enzyme. These compounds act by converting the enzyme into an
inactive or less active from. Such inhibitors cannot be displaced by
excess substrate and hence are called noncompetitive inhibitors. Since
their action does not involve displacement of the substrate, they have
no effect on the Km of the substrate. The inhibitor does not attach at the
catalytic site but reacts with an adjacent site and alters the enzyme in
such a way that it loses its catalytic property as shown in Fig. 9.6 (c).
Thus, Km is unchanged but Vmax is reduced as shown in Fig. 9.5.

(a) (b) (c)

Fig.9.6: Distinction between a Competitive and a Noncompetitive Inhibitor(a)

enzyme-substrate complex; (b) a competitive inhibitor binds at the
active site and thus prevents the substrate from binding; (c) a
noncompetitive inhibitor does not prevent the substrate from binding.
200
Unit 9 Enzymes and Drug Action

SAQ 4
Fill in the blanks in the following sentences:
(a) Drugs alter the tertiary structure of any enzyme with which they come in
contact and denature them such inhibition is called _________________
(b) _________ enzyme inhibitors increase the Km but the Vmax remains
same.
(c) In noncompetitive inhibition, Km is unchanged but Vmax is ______.

9.8 SUMMARY
 Medicine is the formulated form of the drug having definite dose and
disease. While a drug can be defined as a medicine or other substance
which has a physiological effect when ingested or otherwise introduced
into the body.

 Pharmacodynamics is the study of the biochemical and physiological

effects of drugs in the body. It shows the interaction of the drug with tissue
receptors which are located at the cell membranes or in the intracellular
fluid.

 Pharmacokinetics is the study that determines the rapidity and

concentration of the drug in the body and its duration of appearance
at the target organ, i.e. onset, time of peak action and duration of
action. It also determines the route(s) and frequency of
administration of drug.

 Drugs (except those gene based) like enzymes, just alter the rate of the
ongoing activities.The drug action can be broadly described as
Stimulation, Depression, Irritation, Replacement, and Bactericidal and
Cytotoxic action.

 Most drugs act by associating with specific macromolecules in the

membrane or inside the cell and alter the biological function. Such
macromolecules are called receptors.

 The drug receptor interactions are agonist, inverse agonist, antagonist,

and partial agonist. Agonist: When a drug binds with a receptor and
produces an effect similar to that of the physiological signal molecule.
Inverse agonist : When a drug activates the receptor but produces an
effect in the opposite to that of a physiological signal molecule.
Antagonist: when a drug prevents the action of an agonist on a receptor or
the subsequent response but does not have any effect of its own. Partial
agonist A drug which activates a receptor to produce submaximal effect
but antagonises the action of a full agonist.

 Occupancy theory suggests thatwhen the drug binds with the receptor it
stimulates it and produces a biological effect. The biological effect remains
till the drug occupies or binds the receptor. Once the drug dissociates
from the receptor the drug action is lost. 201
Block 3 Enzymes

 There is a relationship between molecular structures and their biological

activity, and this principle is referred to as Structure Activity Relationship
(SAR). This concept is used in drug discovery to characterise existing
molecules and guide the synthesis of compounds with increased activity,
a different activity from the existing drug and fewer side effects.

 Enzyme activity can be also be altered by enzyme induction i.e.,

increasing the synthesis of more enzyme protein. Inhibition of enzymes is
another common mode of drug action.

 Inhibition of enzymes can occur in non-specific and specific inhibition.

Specific inhibition is either competitive or noncompetitive.

9.9 TERMINAL QUESTIONS

1. Differentiate between drug and medicine.

2. What do you understand by pharmcodynamics?

3. Describe in brief the drug action.

4. Describe in brief the drug-receptor interaction.

5. How does change in structure affect the SAR?

6. Why are enzymes good drug targets?

9.10 ANSWERS
Self-Assessment Questions
1. a) F b) T c) F d) F

2. a) -3 b) -1 c) -2 d) -4

3. a) - 4 b) -2 c) - 3 d) - 1

4. a) Nonspecific inhibition b) Competitive c) reduced

Terminal Questions
1. Refer Table 4.2.

2. Pharmacodynamics is the study of the biochemical and physiological

effects of drugs in the body. It shows the interaction of the drug with tissue
receptors which are located at the cell membranes or in the intracellular
fluid.

3. The drug action can be broadly described as:

(a) Stimulation, is an increase in the selective activity of specialized cells,

which increases the secretion from a gland.

(b) Depression is a reduction/decrease in the activity of specialized cells.

For example barbiturates depress central nervous system.
202
Unit 9 Enzymes and Drug Action

(c) Irritation: noxious effect, is the effect of drugs on the growth, nutrition
and morphology of living tissues which induce a gross change in
cellular function.

(d) Replacement, includes certain drugs, which are used to replace some
missing endogenous component of the body or when the production
of endogenous components is reduced. These are given in deficiency
diseases, e.g. insulin in diabetes mellitus.

(e) Bactericidal and Cytotoxic action is for killing parasites or cancer

cells. Bactericidal activity, which is bacterial death,

4. The drug-receptor interactions are of the following types:

Agonist: When a drug binds with a receptor and produces an effect similar
to that of the physiological signal molecule.

Inverse agonist : When a drug activates the receptor but produces an

effect in the opposite to that of a physiological signal molecule.

Antagonist: when a drug prevents the action of an agonist on a receptor or

the subsequent response but does not have any effect of its own.

Partial agonist: A drug which activates a receptor to produce submaximal

effect but antagonises the action of a full agonist.

5. The structure of the compound/drug is very important as the drug will bind
to the receptor only if the structure fits the receptor. If a group is added or
removed it not only changes the structure of compound but changes the
function also by either enhancing the effect of drug or decreasing the
effect of drug. For example, methyl group is added it causes an increase
in lipophilicity which causes and increase in transport across biological
membrane. Addition of hydroxyl group causes an increase in
hydrophilicity. It also causes increase in hydrogen bonding at the target
site thus increases the drug activity. Addition of basic groups when in free
form help drug to transport and in salt form helps the bonding of drug with
receptor by hydrogen bonds.

6. Enzymes catalyse multistep chemical reactions and achieve phenomenal

rate accelerations by matching protein and substrate chemical groups in
the transition state. Inhibitors that take advantage of these chemical
interactions are among the most potent and effective drugs known. The
catalytic chemistry of enzymes is the key to designing potent inhibitors
and makes them a special class of drug target. The majority of drugs
which act on enzymes act as inhibitors and most of these are competitive.
Some inhibitors are non-competitive, binding away from the substrate
binding domain, competing for cofactor/coenzyme binding, or causing an
allosteric conformational change in the 3-dimensional protein structure
that prevents substrate interaction. Yet other inhibitors are irreversible and
these covalently bind to the enzyme, permanently inactivating catalytic
function (these are also known as suicide inhibitors).

203