Biochemistry and Clinical Pathology Enzymes

ENZYMES
Enzymes: Enzymes are the proteins which catalyse biochemical reactions in living organisms. They
increase the rate of reaction without altering its equilibrium. They are colloidal, thermolabile
and highly specific in the reaction that they catalyse. eg. Glucokinase catalyses conversion of
glucose to glucose 6- phosphate.
Intra cellular enzymes (Endoenzymes): The enzymes which are synthesized in the cell of a
particular tissue and are required by the same cell are called Intra cellular enzymes or
Endoenzymes. They generally catalyse the metabolic reactions of cell.
Extra cellular enzymes (exoenzymes): The enzymes produced by the cell of a tissue and are used
by the cell of other tissues are called extra cellular enzymes or Exoenzymes. The digestive
enzymes are exoenzymes.
Proenzymes/ Zymogens: Many enzymes are secreted as inactive precursors of the active enzyme.
These precursors on transformation or activation by selective proteolysis form the active
enzyme. Such precursors of enzymes are called proenzymes or zymogens. eg. digestive enzymes
pepsin, trypsin and chymotrypsin are secreted in form of their proenzymes pepsinogen,
trypsinogen and chymotrypsinogen respectively.
Proenzyme secretion is a protective mechanism to prevent autodigestion of the tissue of origin.
It also assures rapid availability of enzyme to a physiological demand since proenzymes are
stored as precursors.
Holoenzymes: Many enzymes require the presence of additional organic or inorganic substances for
their activity. Such enzymes along with the organic and inorganic activators are called
Holoenzymes.
The proteinaceous part of the holoenzyme is called Apoenzyme while the non protein organic
part is called Prosthetic group or Coenzyme and the inorganic part is called cofactor.
Coenzymes: Coenzymes are the heat stable, dialisable, non protein organic molecules required for
the activity of an enzyme. They are specific for the enzyme concerned and are essential for its
activity. Many coenzymes are derivatives of the water soluble vitamins of the vitamin B complex
group. Eg. NAD+, NADP+, FAD, FMN etc.
Co- factor: Cofactors are the inorganic ions or molecules required for the activity of an enzyme.
Many enzymes require metal cations like Ca+2, Mg+2, Zn+2, Fe+2 etc.
Marker enzymes: Marker enzymes are the enzymes whose increased level in body fluids like
Cerebrospinal Fluid, plasma, urine etc. in indicative of a particular disease or disorder. The
presence of marker enzymes is used as diagnostic tool in clinical laboratories. Eg. Lactate
dehydrogenase is marker enzyme of Myocardial infaction, acute asthma etc. SGOT and SGPT
are used as marker enzymes for Jaundice.
Nomenclature of enzymes (Enzyme coding): A systematic coding approach has been adopted for
nomenclature of enzymes. Each enzyme has a code number (EC) that characterizes the type of
reaction catalysed by the enzyme as to class (first digit) subclass (second digit) and sub sub class
(third digit). The fourth digit is for the specific enzyme. Thus EC 2.7.1.1 denotes class 2
(Transferases), subclass 7 (transfer of phosphate group) sub sub class 1 (alcohol as phosphate
acceptor). The last digit 1 denotes name of the enzyme Hexokinase. Other examples are;
1.1.1.1 Alcohol dehydrogenase
2.4.1.1 Phosphorylase
4.1.2.7 Aldoase

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Biochemistry and Clinical Pathology Enzymes

Classification of enzymes: According to the international union of biochemistry (IUB) system,

enzymes are classified into six major classes depending on the type and mechanism of reaction
that they catalyse.
1. Oxidoreductases: This class includes the enzymes which catalyse oxidation reduction reaction
between two substrates and require coenzymes like NAD, NADP+, FAD etc. common examples
are cytochrome oxidase, alcohol dehydrogenase, glutamate dehydrogenase, catalase,
peroxidase etc.
2. Transferases: This class includes the enzymes which catalyse transfer of a group other than
hydrogen from one substrate to another. the important examples are transaminases,
transmethylase, acyltransferase etc.
3. Hydrolases: This class includes the enzymes which catalyse hydrolysis of ester, ether, peptide
etc. bonds by addition of water molecule. Examples are lipase, pepsin, chymotrypsin, beta
galactosidase, pseudocholinesterase etc.
4. Lyases: This class includes the enzymes which catalyse removal of groups from substrate leaving
a double bond, by mechanism other than hydrolysis. common examples are fumarase, aldolase,
citrate lyase and various decarboxylases.
5. Isomerases: This class includes the enzymes which catalyse interconversion of optical,
geometrical or positional isomers of a substrate. examples are racemases, epimerases, cis trans
isomerases etc.
6. Ligases/ synthetases: This class includes the enzymes which catalyse the linking together of two
compounds. the energy required is derived from high energy phosphate bonds of Adenosine
triphosphate (ATP) and from pyrophosphate. examples are succinyl CoA synthetase, acetyl CoA
carboxylase etc.
Specificity of enzyme action: Enzymes are highly specific in the reaction that they catalyze. they
selectively catalyse the reaction of certain type of substrates or structurally similar compounds
or a specific optical or geometrical isomer. specificity of action is the most significant property
of enzymes. The specificity of enzyme action may be of following types;
1. Absolute specificity: certain enzymes catalyse reaction of only one substrate and not even
structurally similar compound or optical or geometrical isomer. such specificity is called
absolute specificity. eg. urease has absolute specificity for urea and catalase has absolute
specificity for hydrogen peroxide.
NH2CONH2 urease 2NH3 + CO2
2H2O2 Catalase 2H2O + O2
2. Relative specificity: Some enzymes catalyse reactions of structurally similar compounds. such
specificity of enzyme action in which several related substrates can be transformed by an
enzyme is called relative specificity. eg. Lactate Dehydrogenase catalyses interconversion of
pyruvic acid and lactic acid and some other structurally related compounds.
pyruvic acid lactate dehydrogenase lactic acid
Relative specificity may be group dependent eg. alcohol dehydrogenase acts on alcohols
similarly the specificity may be dependent on the type of bond eg. Glucosidase for glycosides
and pepsin, trypsin for peptide bonds.
3. Optical specificity: Certain enzymes catalyse the reaction of a particular optical isomer of a
substrate. this is called optical specificity. eg. Arginase acts only on L-arginine and not on the D
arginine.

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Biochemistry and Clinical Pathology Enzymes

4. Geometrical specificity: Some enzymes show specificity towards specific geometrical isomer of a
substrate. this is called geometrical specificity. eg. fumarases catalyse interconversion on
fumaric and maleic acid while it does not react with malic acid which is geometrical isomer of
fumaric acid.
Mechanism of enzyme action: Enzymes are biocatalysts. They increase the rate of reaction in low
concentration. In their native three dimensional concentration, they have a specific region
where the substrate binds and is acted upon. This portion of enzyme surface is called active site
or catalytic site of the enzyme.
Substrate binds to enzyme at the catalytic site and forms an intermediate enzyme substrate
complex [ES]. This complex undergoes biotransformation to enzyme product complex [EP]
which splits into product (P) and the enzyme (E). The enzyme released again participates in
catalysis and is recycled.

Most of the enzyme catalysed reactions are reversible in nature. Thus equilibrium of the
enzyme catalyzed reaction may be shifted to either direction depending on the concentration of
substrate and the product formed in the medium.
Theories of enzyme action: There are two main theories to explain mechanism of enzyme action.
They are Fischer’s lock and key theory and Koshland’s induced fit theory.
1. Lock and key theory: This theory was proposed by Fischer. According to this theory the active
site of enzyme already exists in proper conformation even in the absence of substrate and
provides a rigid preshaped template complementary to the size, shape and groups of the
substrate. The substrate binds to the active site of the enzyme as a key fits into a lock. The
enzyme catalyses reaction without any change in its three dimensional structure.

2. Induced fit theory: This theory was proposed by Koshland. According to this theory the
substrate induces conformational changes in the enzyme which align amino acid residues or
other groups on the enzyme in a correct special orientation for substrate binding and for the
reaction catalysis.
For substrate binding and for the reaction catalysis, the true substrate induces all the desired
conformational changes in the enzyme. The analogue of the substrate which induces some of
the desired conformational changes or partial change, also have affinity for the enzyme but the
enzyme does not catalyse reaction of such substrate analogues. This theory explains enzyme
saturation, competitive inhibition and allosteric inhibition of enzymes.

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Biochemistry and Clinical Pathology Enzymes

Enzyme inhibition: the phenomenon in which a compound adversely affects or stops an enzyme
catalysed reaction is called enzyme inhibition. The compounds or agents which cause
enzyme inhibition are called enzyme inhibitors. Enzyme inhibition can be classified as
follows;
a. Competitive enzyme inhibition:
b. Non competitive and uncompetitive enzyme inhibition:
c. Allosteric or feed back inhibition:
Competitive enzyme inhibition: In competitive enzyme inhibition the inhibitor competes with the
substrate for active site of the enzyme. The inhibitor has physicochemical and
stereochemical resemblance with the substrate and binds to the active site and prevents
availability of free enzyme and thus inhibits the reaction. The competitive inhibition can be
reversed by increasing substrate concentration.

Eg. Malonate is competitive inhibitor of succinate dehydrogenase enzyme.

Non competitive enzyme inhibition: In non competitive enzyme inhibition the inhibitor binds to the
enzyme at a site other than the active site and decreases the rate of reaction. The non
competitive inhibitor may not be having any structural resemblance with the substrate. In
this type of inhibition, increasing concentration of substrate does not affect the rate of
enzyme catalysed reaction. Hence it is called non competitive inhibition. Eg. Heavy metals
inhibit Urease enzyme.
Uncompetitive enzyme inhibition: Certain inhibitors bind to the enzyme substrate complex rather
than the free enzyme. This type of inhibition is called uncompetitive inhibition.
Allosteric/ feedback inhibition: In allosteric inhibition, the product of an enzyme catalysed reaction
acts as an inhibitor for the reaction. The product binds to the enzyme at a site different form
the active site called allosteric site. This type of inhibition regulates an enzyme catalysed
reaction and hence it is also called feedback inhibition. Generally in multistep synthesis the
end product acts as allosteric inhibitor for biosynthesis of intermediate metabolites as
shown below;

Eg. Biosynthesis of isoleucine is regulated by feedback inhibitiojn.

Factors affecting rate of enzyme catalysed reactions: enzymes are biocatalyst and hence the
reactions that they catalyse are affected by various paratmeters like;
1. Concentration of substrate:
2. Concentration of enzyme:
3. Temperature of reaction medium:
4. pH of reaction medium:
5. Concentration of product:
6. Activators and inhibitors:
Effect of substrate concentration: In an enzyme catalysed reaction in which concentration of
enzyme, temperature and pH are kept constant, the rate of reaction increases with increase
in substrate concentration till it reaches a constant value. At this stage the enzyme is
saturated with the substrate and reaction velocity is maximum (Vmax). A graph of reaction
velocity against substrate concentration give a hyperbolic curve.

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Biochemistry and Clinical Pathology Enzymes

Michaelis Menten correlated velocity of an enzyme catalysed reaction with the substrate
concentration by an equation given below;

This equation is called Michaelis Menten equation whrere;

V= Velocity of reaction
Vmax = Maximum velocity of the reaction
Km = Michaelis Menten constant
[S] = Concentration of substrate
Michalis Menten constant Km is define as the concentration of substrate [S] at which
velocity of reaction is half the maximum velocity (V = ½ Vmax)
Lineweaver Burke equation: Inverting the michaelis menten equation, we get;

This equation is called Lineweaver Burke equation. In this equation, Km/ Vmax and 1/ Vmax
are constant and the equation is of a straight line like y=mx + c. Thus a graph of 1/V versus
1/[S] gives a straight line with a slope Km/Vmax and Y intercept at 1/Vmax.

Effect of enzyme concentration:

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Biochemistry and Clinical Pathology Enzymes

In an enzyme catalysed reaction in which concentration of substrate, temperature and pH

are kept constant, the rate of reaction increases steadily provided that concentration of
substrate is high. On plotting reaction velocity against enzyme concentration a linear graph
is produced.
Effect of temperature: In an enzyme catalysed reaction in which concentration of substrate,
concentration of enzyme and pH are kept constant, with increase in temperature, the
velocity of reaction increases gradually until it reaches a maximum. Further rise in
temperature causes gradual decrease in velocity of reaction till it reaches zero due to
denaturation of the enzyme at elevated temperature.

The temperature at which the rate of an enzyme catalysed reaction is maximum is called
optimum temperature of the enzyme. Different enzymes have different optimum
temperature. Below 10 °C most of the enzymes are inactive and majority of enzymes in
human body have optimum temperature between 37 to 40 °C.
Effect of pH: In an enzyme catalysed reaction in which concentration of substrate, concentration of
enzyme and temperature are kept constant, with increase in pH, the velocity of reaction
increases gradually until it reaches a maximum. Further rise in pH causes decrease in
velocity of reaction. The pH at which the rate of an enzyme catalysed reaction is maximum is
called optimum pH of the enzyme. Different enzymes have different optimum pH. Certain
enzymes have optimum pH in acidic range eg. optimum pH of Pepsin is 1.5 and certain
enzymes have optimum pH in alkaline range eg. Alkaline phosphase has optimum pH 9.8.
Most of the enzymes have optimum pH at physiological pH ie. 7.4.

Applications of enzymes: the applications of enzymes can be divided into three groups;
a. Therapeutic applications
b. Pharmaceutical applications
c. Diagnostic applications
Therapeutic applications: Enzymes have significant role in treatment of various disease and
disorders. Some of the examples are given below;

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Biochemistry and Clinical Pathology Enzymes

1. Trypsin is used in treatment of thromboplebitis.

2. Streptokinase and Urokinase are used as anticoagulant in thrombosis and embolism.
3. Pepsin is used in gastric achylia.
4. Pancreatin is used in replacement therapy in its deficiency states like pancreatitis etc.
5. Fibrinolysin is used in venous thrombosis.
6. Lysozyme is used in eye infections.
Pharmaceutical applications:
1. The enzyme Penicillin acylase is used for production of 6-amino penicillanic acid (6-APA)
from Penicillihn G. 6APA is used in synthesis of semisynthetic penicillin.
2. The enzyme Glucose isomerase is used in production of high fructose corn syrup.
3. Various enzymes like Papain, Pepsin, Trypsin etc. are used in digestive preparations.
Diagnostic applications: Alterations in the levels of certain enzymes in plasma and other body fluids
is characteristic of some diseases and disorders. Hence, level of these enzymes in body fluids
is used as diagnostic tool for various diseases and disorders.
1. Elevated serum levels of Creatinine Phosphokinase is characteristic of myocardial infarction
and muscle disorders.
2. Elevated serum levels of Lactate dehydrogenase is characteristic of myocardial infarction.
3. Elevated serum levels of Aspartate aminotransferase (AST) also called Serum glutamate
oxaloacetate transaminase (SGOT) is characteristic of myocardial infarction.
4. Elevated serum levels of Alanine aminotransferase (ALT) also called Serum glutamate
pyruate transaminase (SGPT) is characteristic of viral hepatitis.
5. Elevated serum levels of γ-glutamyl transpeptidase is characteristic of various liver diseases.
6. Elevated serum levels of Alkaline phosphatase is characteristic of bone diseases and
obstructive liver diseases.
7. Elevated serum levels of Ceruloplasmin is characteristic of Wilsons disease.
8. Elevated serum levels of Amylase and Lipase is characteristic of Pancreatitis.