MAKING PHARMACY EASIER PVT LTD.

Biochemistry notes
KARTIKAY PRAKASH AND SANA
PARVEZ(FOUNDER & C.E.O)
MAKING PHARMACY EASIER PVT LTD.

UNIT-5TH ENZYME
B.Pharma 2nd Sem

Enzymes Introduction, properties, nomenclature and IUB cl Enzyme kinetics (Michaelis plot, Line
Weaver Burke plot) Enzyme inhibitors with examples. Regulation of enzymes: enzyme induction and
repression, allosteric enzymes regulation. Therapeutic and diagnostic applications of enzymes and
Coenzymes –Structure and biochemical functions
 TOPIC:-ENZYME B.Pharma
Biochemistry notes 2nd Sem

Enzyme
“Enzymes can be defined as biological polymers that catalyze
biochemical reactions.”

INTRODUCTION
 Enzyme, a substance that acts as a catalyst in living organisms, regulating
the rate at which chemical reactions proceed without itself being altered
in the process.
 Enzymes are biological molecules (proteins) that act as catalysts and help
complex reactions occur everywhere in life.

Enzyme Structure
 Enzymes are a linear chain of amino acids that generate the three-
dimensional structure.
 The sequence of amino acids enumerates the structure, which in turn
identifies the catalytic activity of the enzyme.

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 The structure of the enzyme denatures when heated, leading to loss of

enzyme activity, which is typically connected to the temperature.
 Enzymes are larger than their substrates, and their size varies, which range
from sixty-two amino acid residues to an average of two thousand five
hundred residues present within fatty acid synthase.
 Only a small section of the structure is involved in catalysis and are situated
next to binding sites.
 The catalytic site and binding site together constitute the enzyme’s active
site.
 A small number of ribozymes exists which serves as an RNA-based biological
catalyst.
 It reacts in complex with proteins.

Properties of enzymes :
(1) Enzymes are complex macromolecules with high molecular weight.
(2) They catalyze biochemical reactions in a cell. They help in the breakdown of large
molecules into smaller molecules or bring together two smaller molecules to form a
larger molecule.
(3) Enzymes do not start a reaction. However, they help in accelerating it.
(4) Enzymes affect the rate of biochemical reaction and not the direction of the
reaction.
(5) Most of the enzymes have a high turnover number. Turnover number of an
enzyme is the number of molecules of a substance that is acted upon by an enzyme

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per minute under saturated substrate concentration. High turnover number of

enzymes increases the efficiency
ficiency of the reaction.
(6) Enzymes are specific in action.
(7) Enzymatic activity decreases with increase in temperature and all enzymes show
maximum activity at an optimum of 30
30-40˚C.
(8) They show maximum activity at an optimum pH of 6 – 8.
(9) The velocity
locity of enzyme increases with increase in substrate concentration and
then, ultimately reaches maximum velocity.

Enzymes Classification

According to the International Union of Biochemists (I U B),

 enzymes are divided into six functional classes and are classified based on
the type of reaction in which they are used to catalyze.
 The six types of enzymes are oxidoreductases, hydrolases, transferases,
lyases, isomerases, ligases
ligases.

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Types Biochemical Property

The enzyme Oxidoreductase catalyzes the oxidation reaction

Oxidoreductases where the electrons tend to travel from one form of a
molecule to the other.

The Transferases enzymes help in the transportation of the

Transferases
functional group among acceptors and donors molecules.

Hydrolases are hydrolytic enzymes, which catalyze the

Hydrolases hydrolysis reaction by adding water to cleave the bond and
hydrolyze it.

Adds water, carbon dioxide or ammonia across double bonds

Lyases
or eliminate these to create double bonds.

The Isomerases enzymes catalyze the structural shifts present

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

The Ligases enzymes are known to charge the catalysis of a

Ligases
ligation process.

Oxidoreductases
 These catalyze oxidation and reduction reactions,e.g. pyruvate dehydrogenase,
which catalyzes the oxidation of pyruvate to acetyl coenzyme A.

Transferases
 These catalyze the transfer of a chemical group from one compound to
another.
 An example is a transaminase, which transfers an amino group from one
molecule to another.

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Hydrolases
 They catalyze the hydrolysis of a bond. For example, the enzyme pepsin
hydrolyzes peptide bonds in proteins.

Lyases
 These catalyze the breakage of bonds without catalysis, e.g. aldolase (an
enzyme in glycolysis) catalyzes the splitting of fructose-1, 6-bisphosphate to
glyceraldehyde-3-phosphate and dihydroxyacetone phosphate.

Isomerases
 They catalyze the formation of an isomer of a compound. Example:
phosphoglucomutase catalyzes the conversion of glucose-1-phosphate to
glucose-6-phosphate (transfer of a phosphate group from one position to
another in the same compound) in glycogenolysis (conversion of glycogen to
glucose for quick release of energy.

Ligases
 Ligases catalyze the joining of two molecules.
 For example, DNA ligase catalyzes the joining of two fragments of DNA by
forming a phosphodiester bond.

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Enzyme kinetics and Km value :

 The enzyme (E) and substrate (S) combine with each other to form an unstable
enzyme-substrate complex (ES) for the formation of product (P).

 Here k1, k2 and k3 represent the velocity constants for the respective
reactions, as indicated by arrows.

 Km the Michaelis-Menten constant (or Brigrs and Haldane's

constant), is given by the formula.

 The following equation is obtained after suitable algebraic

manipulation.

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 Let us assume that the measured velocity (v) is equal to f Vrr". Then the
equation (1) may be substituted as follows:-

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 K stands for a constant and m stands for Michaelis (in Km.).

 Km or lhe Michaelis-Menten constant is defined as the substrate
concentration (expressed in moles/l) to produce half-maximum velocity in
an enzyme catalysed reaction.
 lt indicates that half of the enzyme molecules (i.e. 50%) are bound with the
substrate molecules when the substrate concentration equals the K. value.
 Km value is a constant and a characteristic feature of a given enzyme
(comparable to a thumb impression or signature).
 lt is a represertative for measuring the strength of ES complex.
 A low K^ value indicates a strong affinity between enzyme and substrate,
whereas a high K. value reflects a weak affinity between them.
 For majority of enzymes, the Km values are in the range of 10-s to 10-2
moles. lt may however, be noted that K. is not dependent on the
concentration of enzyme.

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Lineweaver-Burk
Burk double reciprocal plot :
 For the determination of K, value, the substrate saturation curve is not very
accurate since Vmax is approached asymptotically. By taking the
reciprocals
iprocals of the equation (1), a straight line graphic representation is
obtained.

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 The Lineweaver-Burk plot is shown in fig. lt is much easier to calculate the

Km. from the intercept on x-axis which is -(l/Km).
 Further, the double reciprocal plot is useful in understanding the effect of
various inhibitions.

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Enzyme inhibitors with examples.

Enzyme inhibitor is defined as a substance which binds with the enzyme and brings
about a decrease in catalyrtc activity of that enzyme. The inhibitor may be organic
or inorganic in nature.
There are three broad categories of enzyme inhibition
1 . Reversible inhibition.
2. Irreversible inhibition.
3. Allosteric inhibition.

l. Reversible inhibition

 The inhibiior binds non-covalently with enzyme and the enzyme inhibition
can be reversed if the inhibitor is removed.
 The reversible inhibition is further sub-divided into
l. Competitive inhibition
ll. Non-competitive inhibition

l. Competitive inhibition :
 The inhibitor (l) which closely resembles the real substrate (S) is regarded as
a substrate analogue.
 The inhibitor competes with substrate and binds at the active site of the
enzyme but does not undergo any catalysis.
 As long as the competitive inhibitor holds the active site, the enzyme is not
available for the substrate to bind.
 During the reaction, ES and El complexes are formed as shown below.

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ll. Non-competitive inhibition :

 The inhibitor binds at a site other than the active site on the enzyme
surface.
 This binding impairs the enzyme function.
 The inhibitor has no structural resemblance with the substrate.
 However, there usually exists a strong affinity for the inhibitor to bind at the
second site.
 In fact, the inhibitor does not interfere with the enzyme-substrate binding.
 But the catalysis is prevented, possibly due to a distortion in the enzyme
conformation.

 The inhibitor generally binds with the enzyme as well as the ES complex.

 The overall relation in non-competitive inhibition is represented below.

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2. lrreversible inhibition
 The inhibitors bind covalently with the enzymes and inactivate them, which
is irreversible. These inhibitors are usuallv toxrc poisonous substances.
 lodoacetate is an irreversible inhibitor of the enzymes like papain and
glyceraldehyde 3-phosphate dehydrogenase.
 lodoacetate combi nes with sulfhydryl (-SH) groups at the active site of
these enzvmes and makes them inactive.

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3. Allosteric inhibition
 The details of this type of inhibition are given under allosteric regulation as
a part of the regulation of enzyme activity in the living svstem

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Biochemistry notes 2nd Sem

Regulation of enzymes:.
Enzyme Induction-Repression
 Enzyme induction refers to the increase in the amount of enzyme protein as
a result of some stimulus, whereas enzyme repression refers to a decrease
in enzyme after a stimulus.
 While common in bacterial enzyme regulation, they are observed less often
in animal metabolism.
 The cholesterol pathway provides examples of enzyme induction and
repression.
 A lack of intracellular cholesterol leads to an increased synthesis
of hydroxymethylglutaryl-CoA (HMG-CoA) reductase, an example of enzyme
induction. Increased intracellular cholesterol leads to an increased synthesis
of acylcholesterol acyltransferase (ACAT).
 In contrast, increased intracellular cholesterol represses the synthesis of
hydroxymethylglutaryl-CoA (HMG-CoA) reductase.
 The regulation of gluconeogenesis is another example of a pathway
regulated by enzyme induction and repression.
 The activity of phosphoenolpyruvate carboxykinase (PEP carboxykinase) is
increased during fasting, starvation, and diabetes mellitus owing to enzyme
induction by glucagon.
 In contrast, enzyme synthesis is repressed by insulin.

Allosteric Regulation Mechanism

There are two types of allosteric regulation on the basis of substrate and effector
molecules:

Homotropic Regulation: Here, the substrate molecule acts as an effector also. It is

mostly enzyme activation and also called cooperativity, e.g. binding of oxygen to
haemoglobin.

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Biochemistry notes 2nd Sem

Heterotropic Regulation: When the substrate and effector are different. The
effector may activate or inhibit the enzyme, e.g. binding of CO 2 to haemoglobin.

On the basis of action performed by the regulator, allosteric regulation is of two

types, inhibition and activation.

Allosteric Inhibition: When an inhibitor binds to the enzyme, all the active sites of
the protein complex of the enzyme undergo conformational changes so that the
activity of the enzyme decreases.

Allosteric Activation: When an activator binds, it increases the function of active

sites and results in increased binding of substrate molecules.

There are two models proposed for the mechanism of regulation of allosteric
enzymes:

1. Simple Sequential Model

It was given by Koshland. In this model, the binding of substrate induces a change
in the conformation of the enzyme from T (tensed) to R (relaxed). The substrate
binds according to the induced fit theory. A conformational change in one unit
stimulates similar changes in other subunits. This explains the cooperative binding.

The same way inhibitors and activators bind, the T form is favoured, when the
inhibitor binds and R form is favoured, when the activator binds. The binding at
one subunit affects the conformation of other subunits.

The sequential model explains the negative cooperativity in enzymes, e.g. tyrosyl
tRNA synthetase, where the binding of substrate inhibits the binding of another
substrate.

2. Concerted or Symmetry Model

This model was proposed by Monad. According to this model, there is a

simultaneous change in all the subunits of an enzyme. All the subunits are either

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present in R form (active form) or T form (inactive form), that have more affinity
and less affinity to a substrate, respectively.

An inhibitor shifts the equilibrium of T ⇄ R, towards T, and activator shifts the

equilibrium towards R form and favours the binding. It explains the cooperative
regulation of activators as well as inhibitors.

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Therapeutic and diagnostic applications of enzymes

Enzymes as therapeutic agents

1. Streptokinase prepared from streptococcus is useful for clearing the blood

clots. Streptokinase activates plasma plasminogen to plasmin which, in
turn, attacks fibrin to convert into soluble product

2. .

Enzymes as analytical reagents

 Some enzymes are useful in the clinical laboratory for the measurement of
substrates, drugs, and even the activities of other enzymes.
 The biochemical compounds (e.g. glucose, urea, uric acid, cholesterol) can
be more accurately and specifically estimated by enzymatic procedures
compared to the conventional chemical methods.

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 A good example is the estimation of plasma glucose by glucose oxidase and

peroxidase method.

lmmobilized enzymes
 Enzymes can be used as catalytic agents in industrial and medical
applications.
 Some of these enzymes are immobilized by binding-.them to a solid,
insoluble matrix which will not affect the enzyme stability or its catalytic
activity.
 Beaded gels and cyanogen bromide activated sepharose are commonly
used for immobilization of enzymes.
 The bound enzymes can be preserved for long periods without loss of
activity.

diagnostic applications
Estimation of enzyme activities in biological fluids (particularly plasma/serum) is
of great clinical importance.

Enzymes in the circulation are divided into two Eroups - plasma functional and
plasma non-functional.

l. Plasma speeific or Plasma functional enzYmes

 Certain enzymes are normally present in the plasma and they have specific
functions to perform.
 Generally, these enzyme activities are higher in plasma than in the tissues.
 They are mostly synthesized in the liver and enter the circulation e.g.
lipoprotein lipase, plasmin, thrombin, choline esterase, ceruloplasmin etc.
 lmpairment in liver function or genetic disorders often leads to a fall in the
activities of plasma functional enzymes e.g' deficiency of ceruloplasmin in
Wilson's disease.

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2, ion-plasma specific or plasma non-functional enzymes

 These enzymes are either totally absent or oresent at a low concentration in
plasma compared to their levels found in the tissues.
 The digestive enzymes of the gastrointestinal tract (e.g. amylase, pepsin,
trypsin, lipase etc.) present in the plasma are known as secretory enzymes.
 All the other plasma enzymes associated with metabolism of the cell are
collectivefy referred to as consfitutive enzymes (e.g. lactate dehydrogenase,
transaminases, acid and alkaline phosphatases, creatine phosphoki nase).
 Estimation of the activities of non-plasma specific enzymes is very important
for the diagnosis and prognosis of several diseases.

Isoenzymes
 The multiple forms of an enzyme catalysing the same reaction are
isoenzymes or isozymes.
 They, however, differ in their physical and chemical properties which include
the structure, electrophoretic and immunological properties, Km and

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V.max" values, pH optimum, relative susceptibility to inhibitors .and degree

of denaturation.

Explanation for the existence of isoenzymes

 Many possible reasons are offered to explain the presence of isoenzymes
in the living systems. .

1 . lsoenzymes synthesized from different genes e.g. malate

dehydrogenase of cytosol is different from that found in
mitochondria.

2. Oligomeric enzymes consisting of more than one type of subunits

e.g. lactate dehydrogenase and creatine phosphokinase.

3. An enzyme may be active as monomer or oligomer e.g. glutamate

dehydrogenase.

4. In glycoprotein enzymes, differences in carbohydrate content may

be responsible for isoenzymes e.g. alkaline phosphatas.

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Coenzymes –Structure and biochemical functions

 The protein part of the enzyme, on its own, is not always adequate to
bring about the catalytic activity.
 Many enzymes require certain nonprotein small additional factors,
collectively referred to as cofactors for catalysis.
 The cofactors may be organic or inorganic in nature.
 The non-protein, organic, Iow molecular weight and dialysable substance
associated with enzyme function is known as coenzyme.
 The functional enzyme is referred to as holoenzyme which is made up of
a protein part (apoenzyme) and a non-protein part (coenzyme).
 The term prosthetic group is used when a non-protein moiety is tightly
bound to the enzyme which is not easily separable by dialysis.
 The term activator is referred to the inorganic cofactor (like Ca2+,
Mg2+, Mn2+ etc.; necessary to enhance enzyme activity.
 lt may, however, be noted that some authors make no distinction
between the terms cofactor, coenzyme and prosthetic group and use
them interchangeably.

Coenzymes are second substrates :

 Coenzymes are often regarded as the second substrates or co-substrafes,
since thev have affinity with the enzyme comparable with that of the
substrates.
 Coenzymes undergo alterations during the enzymatic reactions, which are
later regenerated.
 This is in contrast to the substrate which is converted to the product.
 Coenzymes participate in various reactions involving transfer of atoms or
groups like hydrogen, aldehyde, keto, amino, acyl, methyl, carbon dioxide
etc. Coenzymes play a decisive role in enzyme function
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Coenzymes from B-complex vitamins :

 Most of the coenzymes are the derivatives of water soluble B-complex
vitamins.
 In fact, the biochemical functions of B-complex vitamins are exerted
through their respective coenzymes.
 The chapter on vitamins gives the details of structure and function of the
coenzyme.

Non-vitamin coenzymes :
 Not all coenzymes are vitamin derivatives.
 There are some other organic substances, which have no relation with
vitamins but function as coenzymes.
 They may be considered as non-vitamin coenzymes e.g. ATP, CDP, UDP etc.

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