CONTENTS

Abstract
1. Introduction
2. Structures and Mechanisms
3. Specificity
4. “Lock and Key” Model
5. Mechanisms
6. Dynamics and Function
7. Allosteric Modulation
8. Cofactors and Coenzymes
8.1 Cofactors
8.2 Coenzymes
9. Inhibition
9.1 Competitive Inhibition
9.2 Uncompetitive Inhibition
9.3 Non-competitive Inhibition
9.4 Mixed Inhibition
10. Biological Function
11. Conclusions
References
 ABSTRACT

Enzymes are biological catalysts (also known as biocatalysts) that

speed up biochemical reactions in living organisms, and which can be
extracted from cells and then used to catalyse a wide range of
commercially important processes. This chapter covers the basic
principles of enzymology, such as classification, structure, kinetics and
inhibition, and also provides an overview of industrial applications. In
addition, techniques for the purification of enzymes are discussed.
1. Introduction

• A catalyst is a substance which alters to promote the reaction, and

a substance especially an enzyme that initiates or modifies the rate
of a chemical reaction in a living body is termed as biocatalyst.
• They are enzymes or microbes that initiates or accelerate chemical
reactions.

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1. Structures and Mechanisms

Ribbon diagram showing human carbonic anhydrase II. The grey

sphere is the zinc cofactor in the active site. Diagram drawn from PDB
1 MOO. Enzymes are generally globular proteins and range from just
62 amino acid residues in size to over 2,500 residues. A small number
of RNA-based biological catalysts exist, with the most common being
the ribosome; these are referred to as either RNA-enzymes or
ribozymes. The activities of enzymes are determined by their three-
dimensional structure. However, although structure does determine
function, predicting a novel enzyme's activity just from its structure is
a very difficult problem that has not yet been solved.

Most enzymes are much larger than the substrates they act on, and only
a small portion of the enzyme (around 3-4 amino acids) is directly
involved in catalysis. The region that contains these catalytic residues,
binds the substrate, and then carries out the reaction is known as the
active site. Enzymes can also contain sites that bind cofactors, which
are needed for catalysis. Some enzymes also have binding sites for
small molecules, which are often direct or indirect products or
substrates of the reaction catalysed. This binding can serve to increase
or decrease the enzyme's activity, providing a means for feedback
regulation.

Like all proteins, enzymes are long, linear chains of amino acids that
fold to produce a three-dimensional product. Each unique amino acid
sequence produces a specific structure, which has unique properties.
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Individual protein chains may sometimes group together to form a
protein complex. Most enzymes can be denatured that is, unfolded and
inactivated by heating or chemical denaturants, which disrupt the three-
dimensional structure of the protein. Depending on the enzyme,
denaturation may be reversible or irreversible.

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2. Specificity

Enzymes are usually very specific as to which reactions they catalyze

and the substrates that are involved in these reactions. Complementary
shape, charge and hydrophilic/hydrophobic characteristics of enzymes
and substrates are responsible for this specificity.

Some of the enzymes showing the highest specificity and accuracy are
involved in the copying and expression of the genome. These enzymes
have "proof-reading" mechanisms. Here, an enzyme such as DNA
polymerase catalyzes a reaction in a first step and then checks that the
product is correct in a second step. This two-step process results in
average error rates of less than 1 error in 100 million reactions in high-
fidelity mammalian polymerases. Similar proofreading mechanisms
are also found in RNA polymerase, aminoacyl tRNA synthetases and
ribosomes.

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3. "Lock and Key" Model

Enzymes are very specific, and it was suggested by the Nobel laureate
organic chemist Emil

Fischer in 1894 that this was because both the enzyme and the substrate
possess specific complementary geometric shapes that fit exactly into
one another. This is often referred to as "the lock and key" model.
However, while this Diagrams to show the induced fit hypothesis of
enzyme action.

In 1958, Daniel Koshland suggested a modification to the lock and key

model: since enzymes are rather flexible structures, the active site is
continually reshaped by interactions with the substrate as the substrate
interacts with the enzyme. In some cases, such as glycosidases, the
substrate molecule also changes shape slightly as it enters the active
site. The active site continues to change until the substrate is completely
bound, at which point the final shape and charge is determined.

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4. Mechanisms

Enzymes can act in several ways, all of which lower G+:

• Lowering the activation energy by creating an environment in

which the transition state is stabilized (e.g., straining the shape of
a substrate by binding the transition-state conformation of the
substrate/product molecules, the enzyme distorts the bound
substrate(s) into their transition state form, thereby reducing the
amount of energy required to complete the transition).
• Lowering the energy of the transition state, but without distorting
the substrate, by creating an environment with the opposite
charge distribution to that of the transition state.
• Providing an alternative pathway. For example, temporarily
reacting with the substrate to form an intermediate ES complex,
which would be impossible in the absence of the enzyme.
• Reducing the reaction entropy change by bringing substrates
together in the correct orientation to react. Considering alone
overlooks this effect.

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5. Dynamics and Function

The internal dynamics of enzymes is linked to their mechanism of

catalysis. Internal dynamics are the movement of parts of the enzyme's
structure, such as individual amino acid residues, a group of amino
acids, or even an entire protein domain. These movements occur at
various time-scales ranging from femtoseconds to seconds. Networks
of protein residues throughout an enzyme's structure can contribute to
catalysis through dynamic motions. However, although these
movements are important in binding and releasing substrates and
products, it is not clear if protein movements help to accelerate the
chemical steps in enzymatic reactions. These new insights also have
implications in understanding allosteric effects and developing new
drugs.

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6. Allosteric Modulation

Allosteric transition of an enzyme between R and T states, stabilized

by an agonist, an inhibitor and substrate (the MWC model) Allosteric
sites are sites on the enzyme that bind to molecules in the cellular
environment. The sites form weak, noncovalent bonds with these
molecules, causing a change in the conformation of the enzyme. This
change in conformation translates to the active site, which then affects
the reaction rate of the enzyme. Allosteric interactions can both inhibit
and activate enzymes and are a common way that enzymes are
controlled in the body.

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7. Cofactors and Coenzymes

7.1. Cofactors
Some enzymes do not need any additional components to show full
activity. However, others require non-protein molecules called
cofactors to be bound for activity. Cofactors can be either inorganic
(e.g., metal ions and iron-sulphur clusters) or organic compounds.
Organic cofactors can be either prosthetic groups, which are tightly
bound to an enzyme, or coenzymes, which are released from the
enzyme's active site during the reaction. Coenzymes include NADH,
NADPH and adenosine triphosphate. These molecules transfer
chemical groups between enzymes.

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7.2. Coenzymes
Space-filling model of the coenzyme NADH. Coenzymes are small
organic molecules that can be loosely or tightly bound to an enzyme.
Tightly bound coenzymes can be called allosteric groups. Coenzymes
transport chemical groups from one enzyme to another. The chemical
groups carried include the hydride ion (H-) carried by NAD or NADP+,
the phosphate group carried by adenosine triphosphate, the acetyl group
carried by coenzyme A, formyl, methenyl or methyl groups carried by
folic acid and the methyl group carried by S-adenosylmethionine.

Since coenzymes are chemically changed as a consequence of enzyme

action, it is useful to consider coenzymes to be a special class of
substrates, or second substrates, which are common to many different
enzymes. For example, about 700 enzymes are known to use the
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coenzyme NADH. Coenzymes are usually continuously regenerated
and their concentrations maintained at a steady level inside the cell: for
example, NADPH is regenerated through the pentose phosphate
pathway. This continuous regeneration means that even small amounts
of coenzymes are used very intensively. For example, the human body
turns over its own weight in ATP each day.

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8. Inhibition

Competitive inhibitors bind reversibly to the enzyme, preventing the

binding of substrate. On the other hand, binding of substrate prevents
binding of the inhibitor. Substrate and inhibitor compete for the
enzyme.

The coenzyme folic acid (left) and the anticancer drug methotrexate
(right) are very similar in structure. As a result, methotrexate is a
competitive inhibitor of many enzymes that use folates.

8.1. Competitive Inhibition

In competitive inhibition, the inhibitor and substrate compete for the
enzyme (i.e., they cannot bind at the same time). Often competitive
inhibitors strongly resemble the real substrate of the enzyme. For
example, methotrexate is a competitive inhibitor of the enzyme

dihydrofolate reductase, which catalyzes the reduction of dihydrofolate

to tetrahydrofolate. The similarity between the structures of folic acid
and this drug are shown in the figure.

8.2. Uncompetitive Inhibition

In uncompetitive inhibition the inhibitor cannot bind to the free
enzyme, but only to the ES-complex. The EIS-compIex thus formed is
enzymatically inactive. This type of inhibition is rare, but may occur in
multimeric enzymes.

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8.3. Non-competitive Inhibition
Non-competitive inhibitors can bind to the enzyme at the binding site
at the same time as the substrate, but not to the active site. Both the

El and EIS complexes are enzymatically inactive. Because the inhibitor

cannot be driven from the enzyme by higher substrate concentration (in
contrast to competitive inhibition), the apparent Vmax changes. But
because the substrate can still bind to the enzyme, the Km stays the
same.

8.4. Mixed Inhibition

This type of inhibition resembles the non-competitive, except that the
EIS-complex has residual enzymatic activity. This type of inhibitor
does not follow Michaelis-Menten equation.

In many organisms inhibitors may act as part of a feedback mechanism.

If an enzyme produces too much of one substance in the organism, that
substance may act as an inhibitor for the enzyme at the beginning of
the pathway that produces it, causing production of the substance to
slow down or stop when there is sufficient amount. This is a form of
negative feedback. Enzymes which are subject to this form of
regulation are often multimeric and have allosteric binding sites for
regulatory substances. Their substrate/velocity plots are not hyperbolar,
but sigmoidal (S-shaped).

Irreversible inhibitors react with the enzyme and form a covalent

adduct with the protein. The inactivation is irreversible. These
compounds include eflornithine a drug used to treat the parasitic
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disease sleeping sickness. Penicillin and Aspirin also act in this manner.
With these drugs, the compound is bound in the active site and the
enzyme then converts the inhibitor into an activated form that reacts
irreversibly with one or more amino acid residues.

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9. Biological Function

Enzymes serve a wide variety of functions inside living organisms.

They are indispensable for signal transduction and cell regulation, often
via kinases and phosphatases. They also generate movement, with
myosin hydrolysing ATP to generate muscle contraction and also
moving cargo around the cell as part of the cytoskeleton. Other
ATPases in the cell membrane are ion pumps involved in active
transport. Enzymes are also involved in more exotic functions, such as
luciferase generating light in fireflies. Viruses can also contain
enzymes for infecting cells, such as the HIV integrase and reverse
transcriptase, or for viral release from cells, like the influenza virus
neuraminidase.

An important function of enzymes is in the digestive systems of

animals. Enzymes such as amylases and proteases break down large
molecules (starch or proteins, respectively) into smaller ones, so they
can be absorbed by the intestines. Starch molecules, for example, are
too large to be absorbed from the intestine, but enzymes hydrolyse the
starch chains into smaller molecules such as maltose and eventually
glucose, which can then be absorbed. Different enzymes digest
different food substances. In ruminants which have herbivorous diets,
microorganisms in the gut produce another enzyme, cellulase to break
down the cellulose cell walls of plant fiber.

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Several enzymes can work together in a specific order, creating
metabolic pathways. In a metabolic pathway, one enzyme takes the
product of another enzyme as a substrate. After the catalytic reaction,
the product is then passed on to another enzyme. Sometimes more than
one enzyme can catalyze the same reaction in parallel, this can allow
more complex regulation: with for example a low constant activity
being provided by one enzyme but an inducible high activity from a
second enzyme.

Glycolytic enzymes and their functions in the metabolic pathway of

glycolysis

Enzymes determine what steps occur in these pathways. Without

enzymes, metabolism would neither progress through the same steps,
nor be fast enough to serve the needs of the cell. Indeed, a metabolic
pathway such as glycolysis could not exist independently of enzymes.

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Glucose, for example, can react directly with ATP to become
phosphorylated at one or more of its carbons. In the absence of
enzymes, this occurs so slowly as to be insignificant. However, if
hexokinase is added, these slow reactions continue to take place except
that phosphorylation at carbon 6 occurs so rapidly that if the mixture is
tested a short time later, glucose-6-phosphate is found to be the only
significant product. Consequently, the network of metabolic pathways
within each cell depends on the set of functional enzymes that are
present.

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10. Conclusions

Enzymes are used in the chemical industry and other industrial

applications when extremely specific catalysts are required. However,
enzymes in general are limited in the number of reactions they have
evolved to catalyze and also by their lack of stability in organic solvents
and at high temperatures. Consequently, protein engineering is an
active area of research and involves attempts to create new enzymes
with novel properties, either through rational design or in vitro
evolution. These efforts have begun to be successful, and a few
enzymes have now been designed "from scratch" to catalyze reactions
that do not occur in nature.

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 References

1. Comprehensive Laboratory Manual in Biology-XII

2. Biology Text for Class XII– NCERT
3. http://www.wikipedia.org

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