ENZYMES

Lectures handouts
Dr. Adnan Riaz
HISTORY OF ENZYMES
Around late 17th and early 18th centuries, the digestion of meat by stomach secretions and the conversion of starch to
sugars by plant extracts and saliva were known. However, the mechanism by which this occurred had not been identified.

In 1833, French chemist Anselme Payen discovered the first enzyme, diastase (amylase). In 1877, German physiologist
Wilhelm Kühne first used the term enzyme, which is Greek for "in leaven", to describe this process. It was later used to
refer to nonliving substances, such as pepsin.

Introduction and Definition of Enzymes

 What are Enzymes?

 Almost every body knows that:

 Enzymes are the biological catalysts which speed up the chemical reactions
 This is the most simple but technically insufficient definition of enzymes

 Biochemical or complete Definition of Enzymes

 Enzymes are the organic macromolecules, Majorly* made up of proteins

 Which accelerate a chemical/metabolic reaction in the living systems by decreasing the energy of activation
 And results in the conversion of substrates in to the products
 Furthermore, they (enzymes) do not appear in the final products of the reaction

* Ribozymes are not proteins but they act as Enzymes (Imp. Viva Q)

 Some important technical points”

SUBSTRATE
These are the compounds or molecules which are acted upon by a specific enzyme Also called Reactant

PRODUCT
A product is something "manufactured" by an enzyme from its substrate.

CATALYST
A substance that lowers activation energy of a reaction so the reaction occurs more quickly but, in the end, is NOT used up
by the reaction is called a catalyst.

INTERNATIONAL UNIT (IU) OF ENZYME ACTIVITY:

It is defined as the amount of enzyme causing transformation of 1.0 µmol of substrate/ min at 250 C under optimal
condition of measurement.

KATAL – CATALYTIC UNIT :

It is defined as the amount of enzymes which catalyze a reaction at 1 mole of substrate/second. It is abbreviated as kat or k.

SPECIFIC ACTIVITY
It is the number of enzyme unit/ mg of total protein. The specific activity is a measure of enzyme purity.
ENERGY OF ACTIVATION
It is the minimal amount of energy required to start any given chemical reactions.In other words a reaction cannot take
place until unless the substrates gain this specific amount of Energy

SIMPLE ENZYMES
Enzymes composed wholly of protein are known as simple enzymes. They are the purely proteins in nature

HOLO-ENZYME
Complex enzymes are also known as Holo-Enzyme. They are made up of proteins plus any other non-protein (organic or
inorganic substance)

APO-ENZYME
The protein part of a Holo-enzyme is called Apo-Enzyme. They are purely proteins in nature

CO-FACTOR
Non-protein component is known as the Co-Factor. It is further subdivided on the basis of its chemical nature i.e. organic or
inorganic and its combination with apoenzyme i.e. covalent bonding or tightly bound and non-covalently or loosely bound.
CO-FACTORS include: coenzymes, Prosthetic Groups, Metals and their ions (See flow chart for classification of a co factor)
 Important metals which acts as a cofactor
Metal Enzyme
Zn++ Carbonic anhydrase
Zn++ Alcohol dehydrogenase
Zn++ Carboxypeptidase
Fe+++ or Fe++ Cytochromes
Cu++ or Cu+ Cytochrome oxidase
K+ Propionyl CoA carboxylase
Mg++ Hexokinase
Mn++ Superoxide dismutase
Se Glutathione peroxidase
Mo Xanthine oxidase
Ni++ Urease

COENZYMES
Heat stable, low mol wt organic compounds non-covalently linked with enzymes can be separated.
If covalently Linked to apoenzymes = prosthetic group Coenzymes serve as recyclable shuttles—or group transfer agents—
that transport many substrates from their point of generation to their point of utilization. The water-soluble B vitamins
supply important components of numerous coenzymes
+
Nicitinamide Adenine Dinucleotide (NAD )
Its formula can be represented as Nicitinamide – Ribose – Phosphate – phosphate – Ribose – Adenine
Functional group of molecule is only Nicitinamide part where as rest of the molecule is represent as (R) The oxidized form
can be written as NAD+. The reduced form can be written as NADH+H+. NADH+H+ is primarily used in ATP generation

Nicotinamide Adenine Dinucleotide Phosphate (NADP+)

It has same structure NAD+ except that it has one additional Phosphoric Acid residue at carbon no 2 of D – Ribose
molecule. The oxidized form can be written as NADP+. The reduced form can be written as NADPH+H+. NADPH+H+ is
primarily used in biosynthetic pathways that require reduction e.g fatty acid and cholesterol synthesis.
 Flavin Mono-nucleotide (FMN)
Its formula can be represented as Riboflavin–Phosphate. The oxidized form can be written as FMN. The reduced form can
be written as FMNH2

Flavin Adenine Dinucleotide (FAD)

Its formula can be represented as Riboflavin–Phosphate – phosphate – Ribose – Adenine. Functional group of molecule is
only the dimethylisoalloxazine part where as rest of the molecule is represent as (R) The oxidized form can be written as
FAD . The reduced form can be written as FADH2

Some important coenzymes

Coenzyme Vitamin Present Associated Enzyme Reaction Catalyzed

Nicotinamide Adenine Nicotinamide Malate Dehydrogenase L- Malate Oxaloacetate

Dinucleotide (NAD+)
Nicotinamide Adenine Nicotinamide Glucose-6- Phosphate Glucose-6- Phosphate
Dinucleotide Phosphate (NADP+) Dehydrogenase
6-Phosphogluconolactone
Thiamine Pyrophosphate Thiamine Pyruvate Dehydrogenase Pyruvate Acetyl CoA
Complex
Flavin Mono-nucleotide (FMN) Riboflavin NADH+H+ Dehydrogenase NADH+H+ + FMN NAD++ FMNH2
complex
Flavin Adenine Dinucleotide Riboflavin Succinate Succinate Fumarate
(FAD) Dehydrogenase
Coenzyme A (CoA) Pantothenic Acid Pyruvate Dehydrogenase Pyruvate Acetyl CoA
Complex
Tetrahydrofolic Acid Folic Acid Hydroxymetylase Glycine Serine
 General Mechanism of Action of Enzymes
Enzymes are catalysts and increase the speed of a chemical reaction without themselves undergoing any permanent
chemical change. They are neither used up in the reaction nor do they appear as reaction products.

The basic enzymatic reaction can be represented as follows

Where E represents the enzyme catalyzing the reaction, S the substrate, the substance being changed, and P the product of
the reaction.

Enzymes employ multiple mechanisms to facilitate catalysis. The mechanism of action of enzymes can be explained by
three perspectives-

1) Enzyme substrate binding

2) Thermodynamic changes

3) Processes at the active site

Active site – Characteristics of active site

The active site of an enzyme is the region that binds the substrates (and the cofactor, if any). It also contains the residues
that directly participate in the making and breaking of bonds. These residues are called the catalytic groups. Although
enzymes differ widely in structure, specificity, and mode of catalysis, a number of generalizations concerning their active
sites can be stated:

Characteristics of active site

1) The active site takes the form of a cleft or pocket which is formed by groups that come from different parts of the amino
acid sequences.

2) It takes up a relatively small part of the total volume of an enzyme. Most of the amino acid residues in an enzyme are not
in contact with the substrate. Nearly all enzymes are made up of more than 100 amino acid residues, some amino acids
serve as a scaffold to create the three-dimensional active site. The remaining amino acids also constitute regulatory sites,
sites of interaction with other proteins, or channels to bring the substrates to the active sites.

3) Substrates are bound to enzymes by multiple weak attractions. The noncovalent interactions in Enzyme Substrate (ES)
complex are much weaker than covalent bonds.The electrostatic interactions, hydrogen bonds, van der Waals forces, and
hydrophobic interactions mediate reversible interactions of biomolecules.

4) The specificity of binding depends on the precisely defined arrangement of atoms in an active site. Because the enzyme
and the substrate interact by means of short-range forces that require close contact, a substrate must have a matching
shape to fit into the site.

1) Enzyme substrate binding

Two models have been proposed to explain how an enzyme binds its substrate: the lock-and –key model and the induced-
fit model.
1) Lock-and-Key Model of Enzyme-Substrate Binding- In this model, the active site of the unbound enzyme is
complementary in shape to the substrate. “lock and key model” accounted for the exquisite specificity of enzyme-substrate
interactions, the implied rigidity of the enzyme’s active site failed to account for the dynamic changes that accompany
catalysis.

2) Induced-Fit Model of Enzyme-Substrate Binding In this model, the enzyme changes shape on substrate binding. The
active site forms a shape complementary to the substrate only after the substrate has been bound.When a substrate
approaches and binds to an enzyme they induce a conformational change, a change analogous to placing a hand (substrate)
into a glove (enzyme).

The induced fit model has been amply confirmed by biophysical studies of enzyme motion during substrate binding.
Besides explaining the specificity, it also explains the regulation of enzyme activity and the dynamic changes occurring at
the active site

2) Thermodynamic changes
2) Thermodynamic changes - All enzymes accelerate reaction rates by providing transition states with a lowered G for
formation of the transition states. However, they may differ in the way this is achieved.
Transition states: it is intermediate molecular event in which bond breakage, bond formation, and charge are not similar to
substrate or product
A chemical reaction of substrate S to form product P goes through a transition state S‡ that has a higher free energy than
does either S or P. (The double dagger denotes a thermodynamic property of the transition state).
The difference in free energy between the transition state and the substrate is called the Gibbs free energy of activation or
simply the activation energy, symbolized by ∆G‡

The activation energy barrier suggests how enzymes enhance reaction rate without altering ∆G of the reaction: enzymes
function to lower the activation energy, or, in other words, enzymes facilitate the formation of the transition state. The
combination of substrate and enzyme creates a new reaction pathway whose transition-state energy is lower than that of
the reaction in the absence of enzyme. The lower activation energy means that more molecules have the required energy
to reach the transition state. Decreasing the activation barrier is analogous to lowering the height of a high-jump bar; more
athletes will be able to clear the bar. The essence of catalysis is specific binding of the transition state.
 3) Processes at the active site
2) Processes at the active site-Enzymes use various combinations of four general mechanisms to achieve dramatic catalytic
enhancement of the rates of chemical reactions. These are as follows-

a) Catalysis by Bond Strain: In this form of catalysis, the induced structural rearrangements that take place with the binding
of substrate and enzyme ultimately produce strained substrate bonds, which more easily attain the transition state.
Enzymes that catalyze lytic reactions that involve breaking a covalent bond typically bind their substrates in a conformation
slightly unfavorable for the bond that will undergo cleavage. The resulting strain stretches or distorts the targeted bond,
weakening it and making it more vulnerable to cleavage.

b) Catalysis by Proximity and Orientation: For molecules to react, they must come within bond-forming distance of one
another. The higher their concentration, the more frequently they will encounter one another and the greater will be the
rate of their reaction. When an enzyme binds substrate molecules at its active site, it creates a region of high local
substrate concentration. Enzyme-substrate interactions orient reactive groups and bring them into proximity with one
another.

c) Catalysis Involving Proton Donors (Acids) and Acceptors (Bases): Other mechanisms also contribute significantly to the
completion of catalytic event, for example, the use of glutamate as a general acid catalyst (proton donor). The ionizable
functional groups of aminoacyl side chains and (where present) of prosthetic groups contribute to catalysis by acting as
acids or bases. the rate of reaction is sensitive to changes in the concentrations of acids (proton donors) or bases (proton
acceptors) present at the active site

d) Covalent Catalysis: In catalysis that takes place by covalent mechanisms, the substrate is oriented to active sites on the
enzymes in such a way that a covalent intermediate forms between the enzyme or coenzyme and the substrate. One of the
best-known examples of this mechanism is serine proteases, which include both digestive enzymes (trypsin, chymotrypsin,
and elastase) and several enzymes of the blood clotting cascade. These proteases contain an active site serine whose R
group hydroxyl forms a covalent bond with a carbonyl carbon of a peptide bond. In Covalent catalysis activation energy is
lower and therefore reaction is faster. The chemical modification of the enzyme is, however, transient. On completion of
the reaction, the enzyme returns to its original unmodified state. Its role thus remains catalytic.

Classification of Enzymes
Classification of enzymes- More than 2000 different enzymes are currently known. The commonly used names for most
enzymes describe the type of reaction catalyzed, followed by the suffix -ase. For example, dehydrogenases remove
hydrogen atoms, proteases hydrolyze proteins, and isomerases catalyze rearrangements in configuration. Modifiers may
precede the name to indicate the substrate (xanthine oxidase), the source of the enzyme (pancreatic ribonuclease), its
regulation (hormone-sensitive lipase) etc.
To address ambiguities, the International Union of Biochemists (IUB) developed an unambiguous system of enzyme
nomenclature in which each enzyme has a unique name and code number that identify the type of reaction catalyzed and
the substrates involved. For instance, hexokinase has systemic name: ATP, D–Glucos –6–Phosphotransferases and
hexokinase has the classfication number EC 2.7.1.1.It is a transferase, so it is in subgroup 2;It transfers a phosphate group,
so it is in subgroup 7;It has an alcohol group as an acceptor, so it is in subsubgroup 1;It is the first such enzyme to be
classified. However, it is still called as hexokinase.

Enzymes are grouped into six classes:

1) The oxidoreductases (class 1) catalyze the oxidation-reduction
2) The transferases (class 2) catalyze the transfer of other groups from one molecule to another
3) The hydrolases (class 3) catalyze hydrolytic cleavage
4) Lyases (class 4, often also referred to as“synthases”) catalyze reactions involving either the cleavage or formation of
double bonds
5) The isomerases (class 5) move groups within a molecule causing geometric or structural changes within a molecule
6) The ligation reactions catalyzed by ligases (“synthetases,” class 6) joining together of two molecules along with
braekdown of ATP
 Oxidoreductases
Catalysing oxidation reduction reaction where one substrate is oxidized and other is reduced
Oxidases. Catalyzing oxidation of the substrate and atomic oxygen acts as recipient of hydrogen e.g. Glucose
oxidase, Cytochrome oxidase, Xanthine oxidase

Hypoxanthine+ O2 Xanthine oxidase Xanthine + H2O2

Glucose+ O2 Glucose oxidase Gluconolactone + H2O2

Dehydrogenases. Catalyzing oxidation of the substrate and coenzymes act as recipients of hydrogen e.g Lactate
Dehydrogenase with NAD and Glucose 6 phosphate dehydrogenase with NADP

Lactate + NAD Lactate dehydrogenase Pyruvate+NADH–H+

Glucose-6-P + NADP Glucose-6-P- dehydrogenase 6-Phosphogluconolactone +NADPH–H+

Oxygenases . Catalyzing the reactions in which oxygen is added to the substrate e.g Homogentisate oxygenase,
L -Tryptophan dioxygenase, Phenylalanine Hydroxylase

Phenylalanine+NADPH–H++O2 Phenylalanine Hydroxylase Tyrosine+ NADP +H2O

TRANSFERASES
These enzymes transfer alkyl, acyl, aldehyde, amino, phosphate groups from one substrate to another.
Transaminases. Catalyzing transfer of amino group between an amino acid and a ketoacid e.g. Aspartate
transaminase (AST), Alanine transaminase (ALT)

Glutamic acid + Oxaloacetic acid Aspartate transaminase (AST)  ketoglutaric acid + Aspartic acid
Glutamic acid + Pyruvic acid Alanine transaminase (ALT)  ketoglutaric acid + Alanine
Transmethylases. Catalyzing transfer of methyl group between to substrates e.g. COMT

Noradrenalin + CH3 Catechol O methyltransferase (COMT) Adrenaline

Transpeptidases. Catalyzing transfer of amino acids to substrates e.g. Benzyl-CoA transpeptidase

Benzyl-CoA + Glycine Benzyl-CoA transpeptidase Hippuric acid

Phosphotransferases. Catalyzing transfer of phosphate group to substrates e.g. Hexokinase, glucokinase

ATP + Glucose Hexokinase ADP + D-Glucose –6-P

Acetyltransferase. Catalyzing transfer of acetyl group to substrates e.g. choline acetyltransferase

Acetyl-CoA+ Choline choline acetyltransferase CoA + Acetyl- Choline

HYDROLASES
Catalyzing hydrolytic breakdown of different bonds e.g C-C,C-O, C-N and P-O. Most of the GIT enzymes belong to
this class
Enzymes hydrolyzing carbohydrates
Polysaccharidases
Starch Amylase Maltose, maltotrios, dextrins

Disacharidases
Maltose, Lactose, Sucrose Disacharidases monosaccharides
Enzymes Acting on Peptide Bonds
Exopeptidases
Exopeptidases amino acids
These are further subdivided into
1. Polypeptidases: 1) aminopeptidase
2) carboxypeptidase
2. Tripeptidases
3. Dipeptidases
Endopeptidase

Endopeptidase Pepsin smaller peptides

These include pepsin, trypsin and elastase

Enzymes Hydrolysing Lipids

These are Lipases, cholestrol esterases and phospholipases

Triacyl glycerol lipase diglycerol + F.F.A
Cholesterol ester cholesterol esterase Free cholesterol + FFA

Phosphatases
i. Phosphomonoesterases:
Glucose-6-P + H2O G-6-Phosphatase Glucose +Pi

ii. Phosphodiesterases:
Removal of one phosphate Group of diesters

LYASES
Catalysing reactions in which NH3, H2O, CO2 are removed without hydrolysis leaving a double bond or by adding
these groups to already existing double bonds

Fumarate + H2O Fumerase Malate

ISOMERASES
Thes enzymes catalyzes the structural changes within a single molecule by transfer of groups within it resulting in
the formation of an isomeric form of the substrate.
Glucose 6. P Phosphoglucomutase Glucose I.P

Glucose 6.P Phosphohexose Isomerase Fructose 6.P

All trans retinene Retinene Isomerase 11-Cis retinene

 LIGASES
These enzymes catalyze reactions in which two molecules linked together by forming C–O, C–N, C–S and C–C
bonds along with the breakdown of a high energy phosphate bonds like ATP, GTP

Acetate + CoA +ATP Acetyl CoA Synthetase Acetyl CoA+AMP+PP

Pyrovate+HCO3+ATP Pyrovate Carboxylase Oxaloacetate+ADP+Pi

General Properties of Enzymes

I. Chemical Nature
II. Catalytic Efficiency
III. Specificity
IV. Proenzymes Or Zymogens.
V. Location Within The Cell
VI. Direction of Enzyme Reaction
VII. Induction and Repression of Enzymes
VIII. Isoenzymes Or Isozymes
IX. Rate Limiting Reactions

Chemical Nature
Enzymes are majorly made up proteins except Ribozymes which are made up of RNA molecule. As they are protein in
nature they have isoelectric PH, they are heat labile and can be denatured. They have special pockets or clefts which is
usually takes up a relatively small part of the total volume of an enzyme these clefts are known as Active Site which is the
site for substrate binding
Catalytic Efficiency
Enzymes are said to be efficient If they can speed up the reaction 103 to 108 times faster than uncatalyzed reaction.
In other words: One mol of an efficient enzyme can convert 100 to 1000 Molecule of substrate into the products Per
second

Enzyme Specificity
In general, there are two distinct types of specificity:
 Absolute specificity - the enzyme will catalyze only one reaction.
  Relative specificity - it can be subdivided into fallowing subgroups.
1. Group specificity - the enzyme will act only on molecules that have specific functional groups, such as amino,
phosphate and methyl groups

2. Linkage specificity - the enzyme will act on a particular type of chemical bond regardless of the rest of the
molecular structure

3. Stereo chemical specificity - the enzyme will act on a particular steric or optical isomer.

proenzymes or zymogens
Certain enzymes are synthesized and secreted as inactive precursor enzymes these enzymes are termed proenzymes or
zymogens. Selective proteolysis converts a proenzymes by one or more successive proteolytic "clips" to an active form that
exhibits the characteristic activity of the mature enzymes, e.g the digestive enzymes pepsin, trypsin, and chymotrypsin
(proenzymes = pepsinogen, trypsinogen, and chymotrypsinogen, respectively), several factors of the blood clotting and
blood clot dissolution cascades, complement and Kinin system are examples of Zymogen activation.

Location within the cell

Many enzymes are localized in specific organelles within the cell, this compartmentalization helps in the study of
enzyme activity, regulates the certain pathways which are confined to certain portions of cells i.e. cytosol or mitochondria.
Furthermore this property of enzymes also helps in confirming the clinical diagnosis by performing the Lab. Assay of certain
enzymes
Direction of Enzyme Reaction
Majority of the reaction catalyzed by enzymes are reversible in the presence of same enzyme.

A+B C+D

The direction of the reaction is determined by

1. Active mass of reactant
2. Availability of the free energy
For example reaction catalyzed by Carbonic anhydrase

H2O+CO2 H2CO3

In muscles: the active mass of H2O+CO2 is graeter than H2CO3 so reaction will continue to formH2CO3 while In lungs: the
active mass of H2O+CO2 is less than H2CO3 so reaction will start in reverse direction to form H2O+CO2

Expired Out

H2CO3 Carbonic anhydrase H2O+CO2

In Lungs

H2CO3 Carbonic anhydrase H2O+CO2

In muscles
However in the living body many metabolic reaction are carried out in one direction

A+B C+D

It is due to number of reason other than these two main factors in these cases the product of the enzyme activity are
immediately acted upon by the next enzyme of the metabolic pathway. The rapid removal of these products does not allow
the reaction in the reverse direction.

Induction and Repression of enzyme synthesis

There are certain factors which either increase or decrease synthesis of a particular enzyme by effecting their synthesis at
gene level on DNA in nucleolus, according the requirements. The substances which cause the increase of enzyme
concentration are called inducers while the substances which causes the decrease of enzyme concentration are called
repressors.
Induction of enzyme synthesis
The enzymes which were previously absent or present only in traces can be induced by certain substances known as
inducers, e.g . Escherichia coli grown on glucose do not have a β-galactosidase but if these E. Coli are grown in a medium
containing Lactose they start to synthesis β-galactosidase. Thus lactose is inducer and β-galactosidase is inducible enzyme.
Similarly penicillinase is induced by penicillin.
Inducible enzymes of humans include: enzymes of gluconeogenesis induced by Cortisol while insulin induces enzymes of
glycolysis and fatty acid synthesis.
some drugs also act as inducers like phenobarbitone which induces many hepatic enzymes including bilirubin glucuronosyl
transferase used in patient of hyperbilirubinemia.

Repression of enzyme synthesis

It is the reverse of induction The synthesis of certain enzymes can be stopped by certain substances known as
repressor, e.g . Escherichia coli make the enzyme tryptophan synthetase only when the medium in which they are grown
does not contain tryptophan but when the tryptophan is added to the medium the synthesis of this enzyme stops. Similarly
leucine synthetase is repressd by Leucine
In humans the insulin repress the enzymes of gluconeogenesis.
Enzymes included by insulin and repressed by glucagon are HMG-CoA reductase, Glucokinase and pyruvate kinase
Isoenzymes
Isoenzymes are different forms of an enzyme that catalyze the same reaction in different tissues in the body and they have
slight variations in the amino acid sequences of the subunits of their structure
For example, lactate dehydrogenase (LDH), which converts lactate to pyruvate, consists of five isoenzymes

Factors affecting enzyme activity

Another example is Isoenzymes of creatine phosphokinase(CPK) or creatine kinase (CK). it catalyse phosphocreatine to
creatine .CPK exists in 3 isoenzymes and has 2 subunits M (muscle) , B (brain) or both

CPK1 - BB - brain

CPK2 - MB - heart

CPK3 - MM - skeletal muscle

Rate Limiting Reactions

Rate Limiting Reactions is reaction catalyzed by an enzyme whose catalytic efficiency determine the effiency of entire
metabolic pathway.

For example: HMG – CoA reductase is the enzyme for rate limiting reaction in the cholesterol biosynthesis. Inhibition of this
enzyme by statin drugs decreases cholesterol synthesis and decreases level of cholesterol in blood.

Enzyme Kinetics
Enzyme kinetics is the field of biochemistry concerned with the quantitative measurement of the rates of enzyme-
catalyzed reactions and the systematic study of factors that affect these rates.

The rate of a chemical reaction is described by the number of molecules of reactant(s) that are converted into
product(s) in a specified time period.

Michaelis – Menten Equtaion

Reaction Model

Michaelis and Menten proposed a simple model for most of the features of enzyme-catalyzed reactions. In this
model, the enzyme reversibly combines with its substrate to form an ES complex that subsequently yields product,
regenerating the free enzyme.

where S is the substrate

E is the enzyme
ES is the enzyme–substrate complex
P is the product
k1, k-1, and k2 are rate constants

Michaelis – Menten Equtaion describes the relationship between reaction velocity and the substrate Concentration
it is represented as:

where vo = initial reaction velocity

Vmax = maximal velocity
Km = Michaelis constant = (k-1 + k2)/k1
[S] = substrate concentration
 1. Km—the Michaelis constant:

Km reflects the affinity of the enzyme for that substrate. Km is numerically equal to the substrate concentration at
which the reaction velocity is equal to 1⁄2Vmax. Km does not vary with the concentration of enzyme.

a. Small Km:

A numerically small (low) Km reflects a high affinity of the enzyme for substrate, because a low concentration of
substrate is needed to half-saturate the enzyme—that is, to reach a velocity that is 1⁄2Vmax

b. Large Km:

A numerically large (high) Km reflects a low affinity of enzyme for substrate because a high concentration of
substrate is needed to half-saturate the enzyme.

It is observed that Most enzymes fallows the Michaelis-Menten kinetics but not all enzymes fallows these kinetics.

Those enzyme reactions which fallows these kinetics shows hyperbolic curve (similar to the oxygen-dissociation
curve of myoglobin) when initial reaction velocity (Vo) is plotted against substrate concentration (S)

While those which do not follow Michaelis-Menton kinetics (e.g allosteric enzymes) show a sigmoidal curve that
is similar in shape to the oxygen dissociation curve of hemoglobin
 The following assumptions are made in the Michaelis-Menten equation:
1. Relative concentrations of E and S:

The concentration of substrate is much greater than the concentration of enzyme, so only small amount of substrate
is bound to the enzyme at any one time.

2. Steady-state assumption:

The [ES] is said to be in steady state when the rate of formation of ES is equal to that of the breakdown of ES, that
is, [ES] does not change with time.

3. Initial velocity:

Only initial reaction velocities (Vo) are used in the analysis of enzyme reactions. This means that the rate of the
reaction is measured as soon as enzyme and substrate are mixed. At that time, the concentration of product is very
small and, therefore, the rate of the back reaction from P to S can be ignored.

Lineweaver-Burk plot
When [Vo] is plotted against [S] it is not always possible to determine when Vmax has been achieved, because of
the gradual upward slope of the hyperbolic curve at high substrate concentrations. However, if 1/vo is plotted
versus 1/[S], a straight line is obtained.

This plot, the Lineweaver-Burk plot (also called a double-reciprocal plot) can be used to calculate Km and Vmax,
as well as to determine the mechanism of action of enzyme inhibitors.

The equation describing the Lineweaver-Burk plot is:

where the intercept on the x-axis is equal to −1/Km, and the intercept on the y-axis is equal to 1/Vmax.
 Factors affecting Enzyme activity
Numerous factors affect the reaction rate

1. Effect of enzyme concentration

2. Effect of substrate concentration
3. Effect of Temperature
4. Effect of PH

Effect of enzyme concentration

As the amount of enzyme is increased, the rate of reaction increases. i.e The rate of the reaction is directly
proportional to the enzyme concentration. For example, if the enzyme concentration is half, the initial rate of the
reaction (Vo), as well as that of Vmax, are reduced to half that of the original.

Effect of substrate concentration

Initially at the start of reaction As the amount of substrate is increased, the rate of reaction increases. i.e The rate of
the reaction is directly proportional to the substrate concentration.(first order reaction) At lower concentrations of
substrate the active sites on most of the enzymes are not filled because there is not much substrate.

The rate of an enzyme-catalyzed reaction increases with increase of substrate concentration until a maximal
velocity (Vmax) is reached. Increased substrate concentration after this point will not increase the rate of
reaction.(zero order reaction) This shows the active sites on most of the enzymes are now filled and saturation
occurred with substrate.

Order of reaction
1. First order reaction

When concentration of Substrate is less than Km, the velocity of the reaction is proportional to the substrate
concentration. This is the first order reaction.

2. Zero order reaction

When concentration of Substrate is greater than Km, the velocity of the reaction is constant and equal to Vmax.
And independent of substrate concentration. This is the zero order reaction.
 Effect of Temperature
Initially the reaction rate increases with temperature to an optimum level, this increase is the result of the increased
number of molecules having sufficient energy to pass over the energy barrier.

Then Further elevation of the temperature results in a abrupt decline in reaction velocity as a result of temperature-
induced denaturation of the enzyme

Most animal enzymes rapidly become denatured at temperatures above 40oC

The optimal temperatures of the enzymes in higher organisms rarely exceed 50 °C.
Some thermophilic bacteria have optimum temperatures of 70°C.
In human and other mammals changes in enzyme reaction rates with temperature can be observed in fever or
hypothermia.

The Q10, or temperature coefficient, is the factor by which the rate of a reaction increases for a 10 °C increase in
temperature. Majority of enzyme have Q10 value of 3 or lower which means that rise of 10 °C increases the
reaction rate 3 times
 Effect of PH
The rate of almost all enzyme-catalyzed reactions exhibits a significant dependence on hydrogen ion concentration
There is also an optimum PH at which enzyme works at its maximum.
Most intracellular enzymes exhibit optimal activity at pH values between 3 and 9.

The changes in PH of the medium act by 2 ways

1. By denaturation of enzymes
2. By decreasing or increasing the ionized or unionized state of enzyme or substrates or both

Enzyme Inhibition
Inhibitors are chemicals that reduce the rate of enzymic reactions.They are usually specific and they work at
low concentrations.They block the enzyme but they do not usually destroy it. Many drugs and poisons are
inhibitors of enzymes in the nervous system

Inhibitors of the catalytic activities of enzymes provide both pharmacologic agents and research tools for study
of the mechanism of enzyme action.

Types of Enzyme inhibition

There are 2 categories of enzyme inhibition

1. Irreversible inhibition
2. Reversible inhibition
 Irreversible inhibitors: Combine with the enzymes irreversibly. irreversible Inhibitors bind through
covalent interactions.

 Reversible inhibitors: These can combine with the enzymes reversibly. Reversible Inhibitors bind through
non-covalent interactions.
1. Competitive Enzyme Inhibition
2. Non Competitive Enzyme Inhibition
3. Uncompetitive Enzyme Inhibition
4. Allosteric Enzyme Inhibition
 Competitive Enzyme Inhibition
A competitive inhibitor

 Has a structure similar to substrate(Structural Analog)

 Occupies active site
 Competes with substrate for active site
 Has effect reversed by increasing substrate concentration
 Vmax remains same but Km is increased
 Competitive Inhibitors look like substrate

COO OOC H
CH2 succinate dehydrogenase
CH2
H COO
COO
Fumarate
Succinate

COO
CH2 succinate dehydrogenase
No reaction
COO

Malonate
 Non Competitive Enzyme Inhibition
 Noncompetitive inhibitors bind enzymes at sites distinct from the substrate-binding site
 Generally bear little or no structural resemblance to the substrate
 Binding of the inhibitor does not affect binding of substrate
 Effect is not reversed by adding substrate.Formation of both EI and EIS complexes is therefore possible
 The enzyme-inhibitor complex can still bind substrate, its efficiency at transforming substrate to product,
reflected by Vmax, is decreased.

Examples of Non Competitive Enzyme Inhibitors

 Cyanide inhibits cytochrome oxidase
 Fluoride inhibits Enolase and hence glycolysis
 BAL ( British Anti Lewisite, dimercaprol) is used as an antidote for heavy metal poisoning
 EDTA (Ethylene Diamine Tetra Acetic acid) inhibits all enzymes which need calcium as a cofactor.

Uncompetitive Enzyme Inhibition

 In this type of inhibition the inhibitor does not bind with the free enzyme but Inhibitor binds to enzyme-
substrate complex forming enzyme- substrate- inhibitor complex
 Effect is not reversed by adding substrate
 Both Vmax and Km are decreased
 Usually seen in those reactions where two or more than two substrates are involve
 e.g ; Inhibition of placental alkaline phosphatase (Regan isoenzyme) by phenylalanine
 Regulation of Enzyme Activity
Reasons for regulation

 To complete reactions in a timely fashion and without wasting of resources

 Conservation of energy to consume just enough energy
 Rapid adjustment in response to environmental changes
 To coordinate numerous metabolic processes.

Regulation of Enzyme Activity

1. Allosteric regulation
2. Regulation by Covalent modification
3. Regulation by Gene Expression
4. Regulation by Calcium

Allosteric regulation

Allosteric enzymes are those whose activity can be adjusted by reversible, non-covalent binding of a specific
modulator to the regulatory sites, specific sites on the surface of enzymes.

The presence of an allosteric effecter can alter the affinity of the enzyme for its substrate, or modify the maximal
catalytic activity of the enzyme.

Effectors that inhibit enzyme activity are termed negative effectors, whereas those that increase enzyme activity
are called positive effectors.

Properties of allosteric enzymes

Allosteric enzymes are normally composed of multiple subunits which can be either identical or different.
The multiple subunits are
 catalytic subunits
 regulatory subunits

cAMP
R R
C
C cAMP
+ 4 cAMP +
C C cAMP
R R
cAMP

protein kinase protein kinase

(inactive) (active)

There are two conformational forms, T (tight)and R (relaxed), which are in equilibrium.
 R form: if substrates is bind to the enzyme
 T form: if inhibitors is bind to the enzyme

Homotropic effectors: When the substrate itself serves as an effector, the effect is said to be homotropic. Most
often, an allosteric substrate functions as a positive effecter. In such a case, the presence of a substrate molecule at
one site on the enzyme enhances the catalytic properties of the other substrate-binding sites—that is, their binding
sites exhibit cooperativity. These enzymes show a sigmoidal curve when reaction velocity (vo) is plotted against
substrate concentration ([S]),

Heterotropic effectors: The effector may be different from the substrate, in which case the effect is said to be
heterotropic.
 Allosteric curve
Kinetic plot of [V] versus [S] is sigmoidal shape.Demonstrating either positive or negative cooperative effect.

Allosteric activation

Allosteric enzyme

Allosteric represion

[S]
Feed Back Inhibition of enzymes: in some cases an allosteric enzyme is inhibited by the product of its own
catalytic action. When the product is formed in excess it bring about inhibition of enzyme activity thus decreasing
its own production.

Covalent modification

There are two types of regulation by covalent modification

1. Irreversible covalent modification

2. Reversible covalent modification

Irreversible covalent modification

Certain enzymes are synthesized and secreted as inactive precursor enzymes these enzymes are termed
proenzymes or zymogens.

Selective proteolysis converts a proenzymes by one or more successive proteolytic "clips" to a form that exhibits
the characteristic activity of the mature enzymes, e.g the digestive enzymes pepsin, trypsin, and chymotrypsin
(proenzymes = pepsinogen, trypsinogen, and chymotrypsinogen, respectively),

Reversible covalent modification

Activity of several enzymes are regulated by covalent attachment or detachment of specific groups such as
phosphate, methyl or nucleotide (most commonly phosphate) this attachment or detachment results in changed
shape of the enzyme and hence change its activity.

Common modifications

 phosphorylation - dephosphorylation
 adenylation - deadenylation
 methylation - demethylation
 acetylation - deacetylation
Phosphorylation and Dephosphorylation

Phosphorylation reactions are catalyzed by a family of enzymes called protein kinases that use adenosine
triphosphate (ATP) as a phosphate donor.
Dephosphorylation reactions are catalyzed by a family of enzymes called phosphoprotein phosphatases .
ATP ADP
2+
Mg
phosphorylation
protein
kinase
E-OH E-O-PO3H2

phosphatase
dephosphorylation

Pi H2O

Depending on the specific enzyme, the phosphorylated form may be more or less active than the unphosphorylated
enzyme. For example, phosphorylation of glycogen phosphorylase (an enzyme that degrades glycogen) increases
activity and dephosphorylation decreases its activity.
While on the other hand the addition of phosphate to glycogen synthase (an enzyme that synthesizes glycogen)
decreases activity and dephosphorylation increases its activity.
Regulation by Gene Expression

There are certain factors which either increase or decrease synthesis of a particular enzyme by effecting their
synthesis at gene level on DNA in nucleolus, according the requirements. The substances which causes the
increase of enzyme concentration are called inducers while the substances which causes the decrease of enzyme
concentration are called repressors.

Regulation By Calcium

Ca2+ is released into the cytoplasm in response to neural stimulation and binds to calmodulin which is calcium-
binding proteins.
The binding of four molecules of Ca2+ to calmodulin triggers a conformational change and activate Ca2+-
calmodulin complex which binds to and activates the enzymes—that are inactive in the absence of this complex.
Thus, calmodulin functions as an essential subunit of many enzyme. One such enzyme is phosphorylase kinase b ,
which is activated by the Ca2+-calmodulin complex.
 Clinical significance of enzymes
1) Enzymes can act as diagnostic markers of underlying diseases .
2) Enzymes also used for the treatment of different diseases

Possible mechanisms responsible for increased serum levels

1. Necrosis of cell
2. Increased permeability of cell without gross cellular damage
3. Increased production of enzyme within the cell resulting in increase in serum by overflow
4. Increase in tissue source of enzyme as in malignancy

Decreased serum levels

A. Decreased formation which may be

i. Genetic
ii. Acquired
B. Enzyme inhibition
C. Lack of cofactors

Enzyme estimations are helpful in the diagnosis of –

1) Myocardial Infarction
2) Liver diseases
3) Muscle diseases
4) Bone diseases
5) GI Tract diseases

Diagnosis of AMI
• The diagnosis of AMI is usually predicated on the WHO criteria of chest pain, ECG changes, and
increases in biochemical markers of myocardial injury.
• Biochemical markers have excellent sensitivity for diagnosing AMI. By using these markers, diagnostic
accuracy can be enhanced.

Enzyme assays routinely carried out for the diagnosis of Acute Myocardial Infarction are-

1)Creatine Phospho kinase,

2) Aspartate transaminase
3) Lactate dehydrogenase
** Cardiac troponins T and I
1) Creatine Kinase (CK, CPK)
• It is an enzyme found primarily in the heart and skeletal muscles, and in the brain
• After myocardial infarction- serum value is found to increase within 3-6 hours, reaches a peak level in
24- 30 hours and returns to normal level in 2-4 days (usually in 72 hours).
• Normal Value- serum activity varies from 10-50 IU/L at 30°C.
• CK is a sensitive indicator in the early stages of myocardial ischemia

Isoenzymes of creatine phosphokinase(CPK) or creatine kinase (CK) exists in 3 isoenzymes and has 2 subunits M
(muscle) , B (brain) or both

CPK1 - BB - brain
CPK2 - MB - heart
CPK3 - MM - skeletal muscle
 2) Aspartate amino Transferase (AST)

• It is also called as Serum Glutamate Oxalo acetate Transaminase (SGOT).

• The level is significantly elevated in Acute MI.
• Normal Value- 0-41 IU/L at 37°C
• In acute MI- Serum activity rises sharply within the first 12 hours, with a peak level at 24 hours or over
and returns to normal within 3-5 days.
• Other diseases- The rise in activity is also observed in muscle and hepatic diseases. These can be well
differentiated from simultaneous estimations of other enzyme activities like SGPT etc, which do not show
and rise in activity in Acute MI.

3) Lactate dehydrogenase (LDH)

• Lactate dehydrogenase catalyzes the reversible conversion of pyruvate and lactate.

• Normal level- 55-140 IU/L at 30°C. The levels in the upper range are generally seen in children.
• In Acute MI-The serum activity rises within 12 to 24 hours, attains a peak at 48 hours (2 to 4 days)
reaching about 1000 IU/L and then returns gradually to normal from 8 th to 14 th day.
• Other diseases-The increase in serum activity of LDH is also seen in hemolytic anemias, hepatocellular
damage, muscular dystrophies, carcinoma, Leukemias, and any condition which causes necrosis of the
body cells.
Iso enzymes of LDH
• LDH enzyme is tetramer with 4 subunits.
• The subunit may be either H(Heart) or M(Muscle) polypeptide chains.
• These two chains are the product of 2 different genes.
• Although both of them have the same molecular weight, there are minor amino acid variations.
• There can be 5 different types of isoenzymes seen in all individuals.

4) Cardiac Troponins

• They are not enzymes; these are infect regulatory proteins involve in myocardial contractility however they
are accepted as markers of myocardial infarction.
• The Troponin complex consists of 3 components; Troponin C, Troponin I, and Troponin T.
• Troponin I is released in to the circulation within 4 hours of the onset of cardiac manifestations, peak is
observed at 14-24 hours and remains elevated for 3-5 days post infarction.
• Serum level of Troponin T increases within 6 hous of myocardial infarction, peaks at 72 hours and
then remains elevated up to 7-10 days. Cardiac troponin estimation is 100% sensitive index for
myocardial infarction.
 Serum enzymes in liver diseases
Serum enzyme tests can be grouped into two categories:
(1) enzymes whose elevation in serum reflects damage to hepatocytes
(2) enzymes whose elevation in serum reflects cholestasis.
(1) Enzymes that Reflect Damage to Hepatocytes

The aminotransferases (transaminases) are sensitive indicators of liver cell injury and are most helpful in
recognizing acute hepatocellular diseases such as hepatitis. These include-
1) Aspartate aminotransferase (AST) and
2) Alanine aminotransferase (ALT).
• AST is found in the liver, cardiac muscle, skeletal muscle, kidneys, brain, pancreas, lungs, leukocytes, and
erythrocytes in decreasing order of concentration.
• Normal level- 0-41 IU/L
• ALT is found primarily in the liver.
• Normal level-0-45 IU/L
• The aminotransferases are normally present in the serum in low concentrations. These are released into the
blood in when there is damage to the liver cell membrane resulting in increased permeability.
Diagnostic significance of Aminotransferases
• Levels of up to 300 U/L are nonspecific and may be found in any type of liver disorder.
• Striking elevations—i.e., aminotransferases > 1000 U/L—occur almost exclusively in disorders
associated with extensive hepatocellular injury such as (1) viral hepatitis, (2) ischemic liver injury (3)
toxin- or drug-induced liver injury.
• In most acute hepatocellular disorders, the ALT is higher than or equal to the AST.
• An AST:ALT ratio > 3:1 is highly suggestive of alcoholic liver disease.
• In obstructive jaundice the aminotransferases are usually not greatly elevated.

(2) Enzymes that reflect Cholestasis

1)Alkaline phosphatase,
2) γ-Glutamyl transpeptidase (GGT)—
These are usually elevated in cholestasis. Alkaline phosphatase and GGT is located in the in bile ducts.
Alkaline phosphatase
The normal serum alkaline phosphatase consists of many distinct isoenzymes found in the liver, bone, placenta,
and, less commonly, small intestine.
Physiological Variations-
• Patients over age 60 can have a mildly elevated alkaline phosphatase.
• It is also nonpathologically elevated in children and adolescents undergoing rapid bone growth, because of
bone alkaline phosphatase.
Pathological variations-
• cholestasis- Very high values are found in obstructive jaundice due to cancer, common duct stone,
sclerosing cholangitis, or bile duct stricture.
• The level of serum alkaline phosphatase elevation is not helpful in distinguishing between intrahepatic and
extrahepatic cholestasis.
- Glutamyl transferase ( GT)
 Found mainly in biliary ducts of the liver, kidney and pancreas.
 Enzyme activity is induced by a number of drugs and in particular alcohol.
 -GT increased in liver diseases especially in obstructive jaundice.
 -GT levels are used as a marker of alcohol induced liver disease and in liver cirrhosis.
3) Serum enzymes in Bone diseases
1) Alkaline Phosphatase-Rises in Rickets, osteomalacia, hyperparathyroidism and in Paget’s disease. Also
rises in primary and secondary malignancies of bones.
2) Acid Phosphatase-Highly increased in bony metastasis of carcinoma prostate
 4) Serum Enzymes in muscle disease
1. Aldolase-Moderate increase in Dermatomyositis, muscular dystrophies, highest values are seen in
Deuchenne type of muscular dystrophies
2. CPK(MM)- Elevated in neurogenic muscular dystrophies, highest values are seen in Deuchenne type of
muscular dystrophies
5) Serum enzymes in GI tract diseases
Amylase-Serum activity > 1000 units is seen within 24 hours in acute Pancreatitis, values are diagnostic. A raised
serum activity is also seen in inflammation of salivary glands.

Lipase-High Levels are seen in acute pancreatitis. Also reported high in perforated duodenal and peptic ulcers and
intestinal obstruction.
Enzymes as therapeutic agents
Enzyme Therapeutic Application

Streptokinase/Urokinase Acute MI, Pulmonary embolism, DVT(Deep vein thrombosis)

Trypsin, lipase and amylase Pancreatic insufficiency

Asparaginase/Glutaminase Acute lymphoblastic leukemias

Hyaluronidase Enhanced local anesthesia and for easy diffusion of fluids

Chymotrypsin Pain killer and Anti inflammatory

Alpha- 1 Antitrypsin Emphysema

Enzyme inhibitor as therapeutic agents

Name of Drug Enzyme Inhibited Used in Treatment of

Captopril Angiotensin Converting Enzyme (ACE) Hypertension, Cardiac Faliure

Cabidopa DOPA decarboxylase Parkinsonisim

Allopurinol Xanthine Oxidase Gout

Physostigmine Acetylcholine estrase Myasthenia gravis

Acetazolamide Carbonic anhydrase Glaucoma

Fenistride 5 α reductase Benign prostatic hyperplasia, baldness

Aspirin and other NSAIDs Cyclo-oxygenase 1 and 2 Pain killer and Anti inflammatory

Phenelzine Monoamine oxidase (MAO) Mental depression

Levostatin + other statin HMG-CoA reductase Hypercholestremia

Omeprazole Proton pump in stomach Peptic ulcer

Cardiac glycosides Na+/K+ ATPase in heart Heart Failure