ENZYMES AND COENZYMES

Enzymes are protein catalysts that increase the rate of chemical reactions without themselves being
changed in the overall process. Example: Oxidase, Aldolase, Kinase etc. Zymogens are the inactive
precursors of proteolytic enzymes that become activated in a suitable environment.

Isoenzymes (or isozymes) are a group of enzymes that catalyze the same reaction but have different
enzyme forms and catalytic efficiencies. Isozymes are usually distinguished by their electrophoretic
mobilities.

Allosteric enzyme is a regulatory enzyme. The term allosteric derives from Greek word, allo means
other and steros means space or site. Allosteric enzymes are those having other site. Like all
enzymes, allosteric enzymes have active site for binding of the substrate but they also have one or
more regulatory (or allosteric) sites for binding regulatory metabolites which is called modulator.

• Allosteric enzymes may be inhibited or stimulated by their modulators

• Modulators that inhibit enzyme activity are termed negative modulators. Whereas those that
increase enzyme activity are called positive modulators.

• Just as the active site of an enzyme is specific for its substrate, the allosteric site is specific for its
modulators. Allosteric enzymes are generally larger and more complex than those of simple
enzymes.

Structure

Enzymes are a linear chain of amino acids, which give rise to a three-dimensional structure. The
sequence of amino acids specifies the structure, which in turn identifies the catalytic activity of the
enzyme. Upon heating, enzyme’s structure denatures, resulting in a loss of enzyme activity, that
typically is associated with temperature.

Compared to its substrates, enzymes are typically large with varying sizes, ranging from 62 amino acid
residues to an average of 2500 residues found in fatty acid synthase. Only a small section of the
structure is involved in catalysis and is situated next to the binding sites. The catalytic site and binding
site together constitute the enzyme’s active site. A small number of ribozymes exist which serve as an
RNA-based biological catalyst. It reacts in complex with proteins.

Enzymes may require a non-peptide component as a cofactor. The peptide component is called the
apoenzyme, the cofactor is called as the coenzyme and the combined functional unit is the
holoenzyme.

Cofactors that are tightly bound to the polypeptide are called prosthetic groups. Such proteins are
called as complex or conjugated proteins. Proteins without prostetic groups are simple proteins

Common coenzymes are vitamins and metal ions

 Structure of an enzyme

Functions of Enzymes
The enzymes perform a number of functions in our bodies. These include:
1. Enzymes help in signal transduction. The most common enzyme used in the process includes
protein kinase that catalyzes the phosphorylation of proteins.
2. They break down large molecules into smaller substances that can be easily absorbed by the
body.
3. They help in generating energy in the body. ATP synthase is the enzymes involved in the
synthesis of energy.
4. Enzymes are responsible for the movement of ions across the plasma membrane.
5. Enzymes perform a number of biochemical reactions, including oxidation, reduction,
hydrolysis, etc. to eliminate the non-nutritive substances from the body.
6. They function to reorganize the internal structure of the cell to regulate cellular activities.
7. The digestive system – enzymes help the body break down larger complex molecules into
smaller molecules, such as glucose, so that the body can use them as fuel.
8. DNA replication – each cell in your body contains DNA. Each time a cell divides, that DNA
needs to be copied. Enzymes help in this process by unwinding the DNA coils and copying the
information.
9. Liver enzymes – the liver breaks down toxins in the body. To do this, it uses a range of
enzymes.

Properties of enzymes:

o Enzymes are made up of proteins.

o They have molecular weights ranging from 10,000 to 2,000,000.
o Enzymes are heat liable.
o It is not dialyzable.
o Enzyme remain unchanged after reaction.
o Enzymes are specific, each enzyme usually catalyses only one reaction.
o Many enzymes need cofactors in order to function.

Classification of Enzyme:
The classification of enzyme was described in 1961 by enzyme commission of the International
Union of Biochemistry (IUB). According to this classification, each enzyme is characterized by a code
number called enzyme code number or ‘EC’ number, consisting of four digits.
According to the IUB system, enzymes are classified
into six major classes as follows:
1. EC-1 : Oxidoreductases- Involved in oxidation-reduction reactions Examples: Lactate
dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G6PD)

2. EC-2 : Transferases: Transfer functional groups from one substrate to another Examples: Aspartate
transaminase or aspartate aminotransferase (AST), Alanine transaminase or alanine
aminotransferase (ALT)

3. EC-3 : Hydrolases: Catalyze the hydrolysis of various bonds Examples: acid phophatase, lipase

4. EC-4 : Lyases: Catalyze removal of groups from substrates without hydrolysis, product has double
bond. Examples: aldolase, decarboxylase

5. EC-5 : Isomerases: Involved in molecular rearrangements Examples: glucose phosphate isomerase

6. EC-6 : Ligases: Catabolism reactions with cleavage of ATP Example: DNA ligase

Example:
EC-1 Oxidoreductases
Those enzymes that catalyze oxidation-reduction reactions, are included in this class which can be
illustrated schematically as follows:

Mechanism of action of enzyme:

Step 1: Enzyme (E) molecule first combine with the substrate (S) to form transient enzyme- substrate
complex
(EX). E + S ⇄ ES Enzyme Substrate Complex
Step 2: Then conversion of the substrate (S) to product (P) occur. ES ⇆ EP Enzyme product complex
Step 3: At last product (P) release from the enzyme (E). EP ⇄ E + P

Enzyme-Substrate Interactions:
Enzyme substrate complex is formed through two methods namely:
• Lock-and-Key Model
• Induced Fit Model
The favourable model of enzyme-substrate interaction is called the induced-fit model. This model
states that the interaction between substrate and enzyme is weak, and these weak interactions
induce conformational changes rapidly and strengthen binding and bring catalytic sites close enough
to substrate bonds.

There are four possible major mechanisms of catalysis:

Catalysis by Bond Strain
The induced structural rearrangements in this type of catalysis produce strained substrate bonds
that attain transition state more easily. The new conformation forces substrate atoms and catalytic
groups like aspartate into conformations that strain substrate bonds.

Covalent Catalysis
The substrate is oriented to active place on the enzymes in such a manner that a covalent
intermediate develops between the enzyme and the substrate, in catalysis that occurs by covalent
mechanisms. The best example of this involves proteolysis by serine proteases that have both
digestive enzymes and various enzymes of the blood clotting cascade. These proteases possess an
active site serine whose R group hydroxyl generates a covalent bond with a carbonyl carbon of a
peptide bond and results in the hydrolysis of the peptide bond.

Catalysis Involving Acids and Bases

Other mechanisms add to the completion of catalytic events which are launched by strain
mechanisms such as the usage of glutamate as a general acid catalyst.
Catalysis by Orientation and Proximity
Enzyme-substrate interactions induce reactive groups into proximity with one another. Also, groups
like aspartate are chemically reactive, and their proximity towards the substrate favours their
involvement in catalysis.
Enzyme Activation:
Enzyme is activated by an activator which is called enzyme activator. Enzyme activators are
molecules that bind to enzymes and increase their activity.
Example. An example of an enzyme activator working in this way is fructose 2,6-bisphosphate, which
activates phosphofructokinase 1 and increases the rate of glycolysis in response to the hormone
insulin.

Enzyme Inhibition: Substances or compounds that decrease the rate of an enzyme-catalyzed

reaction are known as inhibitors and the phenomenon is described as enzyme-inhibition.
Enzyme inhibition can be classified into two types-
• Competitive inhibition
• Non- Competitive inhibition
Coenzymes
Cofactors are non-proteinous substances that associate with enzymes. A cofactor is essential for the
functioning of an enzyme. An enzyme without a cofactor is called an apoenzyme. An enzyme and its
cofactor together constitute the holoenzyme.

There are three kinds of cofactors present in enzymes:

Prosthetic groups: These are cofactors tightly bound to an enzyme at all times. A fad is a prosthetic
group present in many enzymes.
Coenzyme: A coenzyme binds to an enzyme only during catalysis. At all other times, it is detached
from the enzyme. NAD+ is a common coenzyme.
Metal ions: For the catalysis of certain enzymes, a metal ion is required at the active site to form
coordinate bonds. Zn2+ is a metal ion cofactor used by a number of enzymes.
Coenzymes are organic non-protein molecules that bind with the protein molecule (apoenzyme) to
form the active enzyme (holoenzyme).

Salient features of coenzyme:

• Coenzymes are heat stable.
• They are low-molecular weight substances.
• The coenzymes combine loosely with the enzyme molecules and so, the coenzyme can be
separated easily by dialysis.
 • When the reaction is completed, the coenzyme is released from the apo-enzyme, and goes
to some other reaction site
Examples of coenzymes
Thiamine pyrophosphate: Thiamine pyrophosphate (TPP), or thiamine diphosphate (ThDP), is a
thiamine derivative, is also known as vitamin B1. Biological Function of Thiamine pyrophosphate: It
maintains:
• The normal function of the heart;
• Normal carbohydrate and energy-yielding metabolism;
• The normal function of the nervous system;
• Normal neurological development and function;
• Normal psychological functions. Fig: Thiamine pyrophosphate

Thiamine pyrophosphate

Flavin Coenzymes:
• Flavin is the common name for a group of organic compounds based on pteridine,
• The biochemical source is the riboflavin.
• It is commonly known as Vitamin B2.
• Flavin is often attached with an adenosine diphosphate to form flavin adenine dinucleotide
(FAD), and, in other circumstances, is found as flavin mononucleotide (FMN).

Biological Functions and Importance:

• Biological Functions of Flavin coenzymes are:
• Energy-yielding metabolism;
• Metabolism of iron in the body;
• The maintenance of normal skin and mucous membranes;
• The maintenance of normal red blood cells;
• The maintenance of normal vision;
• The maintenance of the normal function of the nervous system.
 flavine adenine dinucleotide

flavine mononucleotide

Factors affecting enzyme-catalysed reactions

Typically, enzyme activities are accelerated with increasing temperatures. As enzymes are functional
in cells, the feasible conditions for nearly all enzymes are temperatures that are moderate. At higher
temperatures, given a specific point, there is a drastic decrease in the activity with the denaturation
of enzymes. In diluted solutions, purified enzymes denature quickly compared to enzymes in crude
extracts. Denaturation of enzymes can also take place when enzymes are incubated for long durations.
More appropriate is to utilize a shorter time duration when it comes to incubation time to gauge the
starting velocities of such enzyme reactions.

The International Union of Biochemistry suggests the standard assay temperature to be 30 °C.
Almost all enzymes are extremely sensitive to pH change. Just some enzymes feasibly operate with
pH above 9 and below 5. Most enzymes have their pH – optimum near to neutrality. Any alteration
of pH causes the ionic state of amino acid residues to change in the whole protein and in the active
site. The modifications in the ionic state can modify catalysis and substrate binding. The preference
of substrate concentration is critical as at lower concentrations, the rate is driven by concentration,
however, at high concentrations, the rate does not depend on any increase in the concentration of
the substrate.

Active site

Enzymatic catalysis depends upon the activity of amino acid side chains assembled in the active centre.
Enzymes bind the substrate into a region of the active site in an intermediate conformation.

Often, the active site is a cleft or a pocket produced by the amino acids which take part in catalysis
and substrate binding. Amino acids forming an enzyme’s active site is not contiguous to the other
along the sequence of primary amino acid. The active site amino acids are assembled to the cluster in
the right conformation by the 3-dimensional folding of the primary amino acid sequence. The most
frequent active site amino acid residues out of the 20 amino acids forming the protein are polar amino
acids, aspartate, cysteine, glutamate, histidine, Serine, and lysine. Typically, only 2-3 essential amino
acid residues are involved directly in the bond causing the formation of the product. Glutamate,
Aspartate, and histidine are the amino acid residues which also serve as a proton acceptor or donor.

Temperature and pH

Enzymes require an optimum temperature and pH for their action. The temperature or pH at which a
compound shows its maximum activity is called optimum temperature or optimum pH, respectively.
As mentioned earlier, enzymes are protein compounds. A temperature or pH more than optimum may
alter the molecular structure of the enzymes. Generally, an optimum pH for enzymes is considered to
be ranging between 5 and 7.

CLINICAL SIGNIFICANCE OF ENZYMES

Certain enzymes are used:

• For the diagnosis of the disease

• As therapeutic agents

• As analytical reagents.