ENZYMES

Prepared by
Debajit Dewan
M. pharm
 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.

The term enzyme was coined by Wilhelm Friedrich

Kuhne in 1876 from a Greek word meaning 'in yeast’.
In 1897 the German chemist Eduard Buchner
accidentally discovered the first free enzyme zymase
from yeast, used to preparation of alcohol from sugar.
They are made up of combined amino acids. The
molecules on which enzymes act are called substrates
and reacted with the substrates to form products of
different molecules.
Proenzyme or zymogens are inactive enzyme produced
naturally but become active when required. Ex.
Pepsinogen (Pepsinogen + H+---------Pepsin).
Properties of Enzyme
PROPERTIES OF ENZYMES
1. Enzymes are dynamic protein macromolecules.
2. They are organic biocatalysts.
3. They are soluble in water and dilute alcohol but form precipitation with concentrated alcohol.
4. They are colloidal in nature and globular protein.
5. They require small quantity for biological action.
6. Their activity depends on temperature and changes in hydrogen ion concentration (pH).
7. They easily desaturated by heat and other chemical.
8. They act only one type of specific substrate (Lock & Key model).
9. They are reusable.
10. Some enzymes are required as cofactors for proper functioning. Cofactors are non-protein part derived
from niacin, riboflavin etc. which are bound within the enzyme molecules.
11. They lower the activation of energy, so reactions are occurred at mild temperature in all living cells.
12. Many enzymes are stereo-specific based on the isomerism of substrate, like L-amino acid oxidase and D-
amino acid oxidase act only on L- and D-amino acids, respectively.
Composition of enzyme
Though enzymes are proteinaceous in nature but depend on presence or
absence of non-protein components. Enzymes are divided into simple
enzymes and holo enzymes.
• Simple enzyme is made up of only protein molecules Example:
Pancreatic ribonuclease.
• Holo enzyme is made up of protein group along with nonprotein
component.
• The protein component of this holo-enzyme is known as apoenzyme
• Non-protein part is known as cofactor. Cofactors are of two types-
organic and inorganic.
• Organic cofactors are known as coenzymes whereas inorganic cofactors
are known as activators.
• When the cofactor/coenzyme is bound tightly with the apoenzyme then
it is known as prosthetic group.
➢ Prosthetic group is the active site for catalytic activity, Like,
aminotransferases require pyridoxal phosphate. Prosthetic groups are
small molecules which are covalently attached at the active site.
Holoenzymes are fully active enzymes.
Holoenzyme = apoenzyme + prosthetic group
Nomenclature of Enzyme
• The nomenclature of first discovered enzymes was named according to their sources. like Pepsin,
Trypsin etc.
Name of enzyme + suffix (-in); Pepsin is found in stomach. (Theodor Schwann named the enzyme
pepsin in 1836. The name comes from the Greek word Ttewic (pepsis), which means "digestion".)
• Enzymes were named according to their substrate.
They are named by adding suff (-ase) to the substrate. e.g. Lactase acts on lactose (Substrate). Lipase
catalyzes the hydrolysis of lipids. Urease catalyzes the hydrolysis of urea. Sucrase catalyzes the
hydrolysis of sucrose, etc.
• Enzymes are classified as per their functions
like oxidases catalyze oxidation reaction
• Sometimes some names of enzymes are described by both the substrate and function,
like alcohol dehydrogenase oxidize alcohol.
❖ Based on Reaction-
❖ This Nomenclature is similar like IUB-MB Classification of enzyme.
IUB-MB Classification of enzyme
The International Union of Biochemistry (I.U.B.) Initiated standards of enzyme nomenclature in
1961. Further, the International Union of Biochemistry and Molecular Biology (IUBMB) was
renamed in 1991.
As per them the enzyme names indicate both the substrate acted upon and the type of reaction
catalyzed.
Based on their action they are divided into 6 major classes.
Each enzyme is assigned a 4 Digit code number (that was approved in 1992 by the last
commission)
These code numbers are prefixed by EC (Enzymes commission).
This system is now widely used. e.g. Lactate dehydrogenase (EC number of 1.1.1.27).
Each digit represents as follows:
(a) The first number shows the main class of enzyme to which it belongs,
(b) The second number indicates the sub class,
(c) The third number indicates sub-sub class.
(d) The fourth number indicates the serial number of an enzyme of sub-sub class
The six major classes of enzymes are shortly known as "OTHLIL"
Oxido-Reductases (EC1): This category of enzymes are involved in Oxidation-Reduction
reactions. Examples: Alcohol dehydrogenase, cytochrome oxidase, D-Amino acid oxidases.

Transferases (EC2):Transferases are enzymes that catalyze the transfer of a functional group
from a donor molecule to an acceptor molecule. Common transferase examples-
Acyltransferase, Peptidyl transferase.

Hydrolases (EC3):These are enzymes which bring about hydrolysis of various compounds.
Examples: Lipase, Cholinesterase, Pepsin, Urease.

Lyases (EC4):These enzymes are cleaved C-C, C-O, C-N, and other bonds by elimination,
double bonds or adding groups to double bonds. Examples: Aldolase, Fumarase, Histidase.

Isomerases (EC5):These enzymes are involved in all isomerization reactions. Examples:

Phosphotriose isomerase, Triose phosphate isomerase, Retinol isomerase.

Ligases (EC6): These enzyme catalyze synthetic reaction where two molecules are joined
together by hydrolysis of a diphosphate bond in ATP. Ex.- Succinate thiokinase, acetyl CoA Carboxylase
Mechanism of enzyme action

Proposed by EMIL FISCHER in 1894. Proposed by DANIAL KOSH LAND in 1958.

Lock and key hypothesis assumes the active site of an enzymes According to this , an enzyme to substrate cause a change in
are rigid in its shape. There is no change in the active site enzyme, which causes the active site to change it’s shape to allow
before and after a chemical reaction. enzyme and substrate to bind.
 Enzyme kinetics
✓The primary function of enzymes is to enhance rates of reactions so that they are
compatible with the needs of the organism.
✓To understand how enzymes function, we need a kinetic description of their activity.
✓For many enzymes, the rate of catalysis Vo, which is defined as the number of
moles of product formed per second, varies with the substrate concentration[S].
✓Different theories and plots have been proposed by various scientists some of
which are Michaelis -Menton plot, Line weaver plot, Hill's plot and so on.
Factor affecting Enzyme Activity

1. Concentration of Enzyme- The correlation between enzyme

concentration and velocity of reaction is directly
proportional.
2. Concentration of Substrate- The rate of velocity of the
reaction is also directly proportional with the concentration
of substrate, the velocity of the reaction also increased.
3. Effect of temperature- With increased temp., velocity of
reaction increase at a certain level and after some time it
decreased. Increased in temp. results in faster enzyme
substrate reaction to form products. So, the rate of reaction
gets faster due to activation energy of molecules
4. Effect of pH- Enzyme activity increases with increased
hydrogen ion concentration at certain level and then decreases.
All enzymes act on alkaline and acidic pH media. Maximum
biological enzymes has optimum pH of 7.4. Some examples like
pepsin has optimum pH 2.0, acid phosphatase active at pH 4-5,
alkaline phosphatase active at pH 10-11, enzymes from fungi are
active at pH 4-6, trypsin is active at PH 8-9.

5. Effect of Time- Under ideal and optimum pH and temperature,

less time is required for enzyme reaction.

6. Effect of Activators- Some enzymes require certain inorganic

metallic cations like Mg+2, Mn+2, Zn+2, Ca+2 Co+2, Na+2, K+ etc. for
optimum activity. Metals function as activators of enzyme velocity
through various mechanisms. Some metal activators are ATPase
(Mg+2, Ca+2), Enolase (Mg+2) etc.

7. Effect of Light- Certain enzymes are inactivated with the

exposure of UV light, beta and gamma rays, X-ray etc. due to the
formation of peroxides.
Enzyme Inhibitor (Michaelis plot, Line
Weaver Burke plot)
Enzyme inhibitors are molecules that reduce the catalytic activity of enzymes.
Reducing of effective enzymatic activity or complete blocking of enzyme of enzyme
may cause either complete death of cell either modifications in the pathways.
Michaelis-Menten kinetics equation
Michaelis-Menten kinetics plot represent the graph that shows the relationship between
the concentration of a substrate and the rate of the corresponding enzyme-controlled
reaction. Michaelis-Menten kinetics equation is-

Vmax- As the enzyme's active sites become saturated with substrate, the reaction rate reaches its maximum.
Michaelis constant Km – this term refers to the term where it describes the concentration of the substrate at
which 50% of the maximum rate of reaction occurs.
As Km measures the affinity of enzymes for their substrates, the lower the value of Km, the more efficient are the
enzymes in carrying out their functions while working with a lower substrate concentration

An ES concentration is considered to remain constant where a

steady-state is assumed in the reaction.
•With an increase in the substrate concentration (1st order
kinetics), the rate linearly increases.
•As the enzyme active sites have been saturated with the
substrate (0 order kinetics), the increasing amount of substrate
concentration will not increase the velocity of the reaction.
Line Weaver Burke plot
Lineweaver-Burke plot represent the graph by taking the inverse of the
reaction rate (1/r) against the inverse of substrate concentration (1/[S]).
This plot was generated using the equation:
Enzyme Inhibitors
Specific Inhibitor: They exert their effect on a single enzyme. Based on the mechanism this are
two types-
(a) Irreversible/Non-specific Inhibitors-
They form strong covalent bond with an enzyme and permanently to inactivate the enzymes.
They act on some specific group of enzymes.
Example- Diisopropyl flurophosphate inhibits the active site of acetylcholine esterase by
reacting with the hydroxyl group.
(b) Reversible Inhibitors:
• Reversible inhibitors attach to enzymes with non-covalent interactions like H-bonds,
hydrophobic interactions and ionic bonds. During binding with an enzyme they do not undergo
any chemical reactions.
• Based on the effect of varying the concentration of the enzyme and substrate on the inhibitor,
they are classified as competitive, non-competitive, uncompetitive Allosteric Inhibition.
1. Competitive Inhibition:
This type of inhibition is based on the affinity of inhibitors towards the active site of the
enzyme where the substrate also can bind

Example- Cyanide acts as competitive inhibitor to the enzyme

Cytochrome c oxidase. It inhibits the working of electron-transport chain
2. Uncompetitive Inhibition:
The inhibitor binds to the enzyme-substrate complex rather than the free enzyme to give
active enzyme-inhibitor complex. This type of inhibition causes decrease of Vmax and Km.
and provides higher binding affinity. The inhibitor "locks" the substrate in the enzyme here by
preventing the enzyme from catalyzing the substrate and resulted decrease in amount of free
enzyme. Changing both Km and Vmax leads to double reciprocal plots, in which intercepts
on vertical and horizontal axis are proportionately changed.
Example: The L-isomer of the amino acid phenylalanine is an uncompetitive inhibitor of
alkaline phosphatase.
3. Non-competitive Inhibition:
In this type of inhibition, the inhibitor binds to the free enzyme or enzyme-substrate complex
and reduces its activity but does not affect the binding of substrate. During binding, Inhibitor
deforms the shape of enzyme. The inhibition depends only on the concentration of the
inhibitor. Vmax will decrease due to the inability for the reaction to proceed as efficiently and
Km will remain the same as the actual binding of the substrate will still function properly.

Example- Alanine inhibits the enzyme pyruvate kinase

which is non-competative type.
 4. Allosteric Inhibition:
Allosteric inhibition is the enzyme inhibition process in which
an allosteric inhibitor inhibits enzyme activity by binding to
the allosteric site. This causes a conformational change in the
active site for the substrate, preventing binding.

Therapeutic application of enzyme:

1. Enzymes are used for aiding digestion. e.g. Amylases, Proteases and Lipases.
2. They are used as deworming agents. e.g. Papain.
3. They act as anti-clotting agents like fibrinolytic and thrombolytic. e.g. Urokinase and
Streptokinase.
4. They act to treat atherosclerosis like serratopeptidase.
5. They are used to treat woundsand swellings. e.g. Trypsin, Chymotrypsin, Serratopeptidase.
6. They are used to assist metabolism like hyaluronidase.
7. They are used as surface disinfectants. e.g. Trypsin
Diagnostic application of Enzyme
 Pharmaceutical Application of Enzyme
1. The enzymes are playing important role in pharmaceutical industries. They can be used as drugs.
Insulin hormone is essential for controlling blood sugar. Diabetes mellitus is a disorder which results
due to deficiency of hormone insulin.
2. Enzyme based pharmaceutical product is protease capsule which provides therapeutic benefits by
enhancing circulatory and immune systems, and also provides quicker healing and better stamina.
3. Enzymes are also used as digestive aid where they are used to supplement digestive of enzymes like
amylase, lipase, and protease.
4. Penicillin acylases are a group of enzymes that cleave the acyl chain of penicillins to yield 6-amino
penicillanic acid (6-APA) and the corresponding organic acid.
5. A serum detoxifying enzyme, the human butyryl-cholinesterase has been shown to be effective in
the treatment of cocaine overdose.
6. There are some enzymes that have been shown to have cellular detoxification function such as
superoxide dismutase and catalase. Both these enzymes work in conjunction to detoxify the body.
7. The superoxide dismutase transforms the highly toxic superoxide anion to moderately toxic
hydrogen peroxide. The catalase then converts hydrogen peroxide to harmless water and oxygen.
 Isoenzymes and their Clinical Significance
Isoenzymes (also known as isozymes) are enzymes that differ in amino acid sequence but
catalyze the same chemical reaction. Isoenzymes are useful biochemical markers and can be
measured in the bloodstream to diagnose medical conditions. Some iso enzyme is Lactate
dehydrogenase (LDH), Creatine Kinase (CK)

Type Composition Location Diagnosis Importance

LDH1 HHHH Heart muscle Myocardial Infection
LDH2 HHHM RBC Megaloblastic anemia
LDH3 HHMM Brain Leukemia, Malignancy
LDH4 HMMM Liver Pulmonary infraction
LDH5 MMMM Skeletal Muscles Liver disease, Muscle damage
CK1 BB Brain Neurological injury
CK2 MB Heart muscle Myocardial Infraction
CK3 MM Skeletal muscle Muscular dystropnies and myopatnies
Coenzyme:
Coenzyme is an organic non-protein compound, binds with an enzyme to catalyze a reaction. They are also known as
cofactors but are chemically different. They attach to the active site of an enzyme.
The structure and function of various coenzymes are discussed below-
NAD/NADP: Nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) are most
important coenzymes in the cell. They are derivatives of vitaminB3 (Nicotinic acid or Niacin).
NAD has various essential roles in metabolism:
1. It is a cofactor in redox reactions.
2. It is a donor of ADP-ribose moieties in ADP-ribosylation reactions.
3. It is a precursor of the second messenger molecule cyclic ADP-ribose.
4. It has an important extracellular role as adenine dinucleotide.
FMN and FAD: Flavin mononucleotide and Flavin adenine dinucleotide are known as
Flavoproteins which contain vitamin-B and act as redox cofactors. FAD is derived
from vitamin B2 (riboflavin). They are hydrogen transferring coenzymes associated
with hydrogenases.
Functions:
1. Both FAD and FMN act as electron carriers in redox reactions.
2. They are responsible for energy production.
3. FMN and FAD both are capable of reversibly accepting two hydrogen atoms and
form FMNH2 and FADH2 respectively.
Coenzyme A (CoA): It consists of complex structure containing an adenosine triphosphate. Coenzyme A is synthesized from
pantothenate (vitamin B5) by five-step process that requires four molecules of ATP and cysteine.
Functions:
1. Coenzyme A acts as acyl group carrier because it is chemically thiol. It reacts with carboxylic acids to form thioesters.
2. It helps in transferring fatty acids from cytoplasm to mitochondria.
3. Pantothenic acid is an essential part for coenzyme A.
4. It is essential for heme formation in hemoglobin.

Thiamine Pyrophosphate (TPP):

This coenzyme is involved in transfer of aldehydes like acetaldehyde and glycol aldehyde.
It is synthesized from thiamine (Vitamin B1) which is produced by the enzyme thiamine diphosphokinase enzyme.
It has pyrimidine ring that is connected to a thiazole ring and diphosphate functional group.
Functions:
1. It is essential cofactor for enzymes that catalyze the oxidative decarboxylation of alpha-ketoglutaric acid to
form acetyl CoA.
2. It plays role in nerve conduction.
3. It phosphorylates a chloride channel in the nerve membrane.
4. It is required for normal brain and CNS functions.
5. It supports normal appetite.
Thank You