ENZYMES

A protein with catalytic properties due to its power of

specific activation
• Almost all processes in a biological cell need enzymes in order to
occur at significant rates.

• Since enzymes are extremely selective for their substrates and speed
up only a few reactions from among many possibilities, the set of
enzymes made in a cell determines which metabolic pathways occur in
that cell.
Mitochondria: TCA cycle
Cytosol: Glycolysis
Fatty acid synthesis
HMP pathway
Oxidation of Pyruvate
Fatty acid synthesis
Nucleus: DNA and RNA synthesis Lysosome: Degradation of complex molecules
Enzymes Lower a Reaction’s Activation Energy
 Competencies
• Explain fundamental concepts of enzyme, isoenzyme, apoenzyme, coenzyme
& co-factors. Enumerate the main classes of IUBMB nomenclature.
• Observe the estimation of SGOT & SGPT
• Describe and explain the basic principles of enzyme activity
• Describe and discuss enzyme inhibitors as poisons and drugs and as
therapeutic enzymes
• Describe and discuss the clinical utility of various serum enzymes as markers
of pathological conditions.
• Discuss use of enzymes in laboratory investigations (Enzyme-based assays)
• Interpret laboratory results of enzyme activities & describe the clinical utility
of various enzymes as markers of pathological conditions.
Specific Learning Objectives
The learner will be able to:
• Classify enzymes
• Explain the basic concepts and mode of action of enzymes
• Describe the active center of enzymes and specificity of enzymes
• Outline Michaelis-Menten theory, Fischer’s template theory and
Koshland’s induced fit theory
• Define Michaelis constant, Km value, Vmax
• Enumerate factors influencing enzyme activity
• Classify the coenzymes and cofactors
• Describe different types of enzyme inhibition with examples
• Explain the mechanisms of regulation, covalent modification,
repression, and induction
• Definition
• Properties
• Active center of enzymes and specificity of enzymes
• Coenzymes and cofactors
• Classification
• Nomenclature
 Enzymes are biological catalyst produced by living tissues.

 They are proteins with the exception of few classes of RNA

molecules called ribozyme.

 They accelerate specific chemical reactions without being

consumed in the process.

 They function in aqueous solutions under very mild conditions

of temperature and pH.
 The catalytic activity of enzyme depends on their native protein
conformation. If an enzyme is denatured or dissociated into its
subunits, catalytic activity is usually lost.

 Thus, the primary, secondary, tertiary, and quaternary structures

of protein enzymes are essential to their catalytic activity
 Definition
• Enzymes are biological material with catalytic properties i.e. they
increase the rate of chemical reaction in biological and in-vitro system.
i.e. they are Biocatalysts.
• Colloidal
• Thermolabile
• Protein
• Specific
Endoenzymes: Most of the enzymes, produced by the cell, function
within that cell and hence are called endoenzymes.
Ex. Enzymes of glycolytic pathway, Kreb’s cycle etc.
Exoenzymes: Some enzymes are liberated by living cells and catalyze
reactions in the cell’s environment i.e. out side the cell. Such enzymes
are known as exoenzymes.
Ex. Enzymes of digestive process like Amylases, lipoprotein lipase,
pectinase, pepsin, trypsin etc.
Zymogen or proenzymes:
• Proteolytic enzymes found in blood or the digestive tract are present in an
inactive ( precursor) form, called zymogen or proenzyme.
• Inactive or zymogen form of enzyme prevent them from catalyzing
reactions in the cell where they are synthesized.
Ex. Chymotrypsin is secreted by the pancreas as Chymotrypsinogen. Which
is then activated by trypsin, with cleaves off small peptide from its N-
terminal end.

To name these enzymes

Prefix “pro” like prothrombin, proelastase etc. or
Suffix “ogen” like Chymotrypsinogen, pepsinogen, trypsinogen etc.
Site of synthesis Zymogen Active form of enzyme

Stomach Pepsinogen Pepsin

Pancreas Chymotrypsinogen Chymotrypsin
Trypsinogen Trypsin
Procarboxypeptidase Carboxypeptidase
Liver Prothrombin Thrombin
 Properties:
Chemical Nature- Almost all enzymes are proteins except Ribozymes which
are RNA molecules. Enzymes are excellent catalysts, speeding up reactions
108 to 1020 fold. They speed up reactions without being used up.
Specificity-
Types
1 Sterio specificity
2 Reaction specificity
3 Substrate specificity
A) Absolute specificity
B) Relative specificity Group
Bond
1 Steriospecificity: Some enzymes are extremely specific in the
production of stereoisomers. (Isomers which have same structural formula but different
arrangement of groups or atoms around the carbon are known as stereoisomers)
Ex. Amylase hydrolyzes α- glycosidic bonds
Cellulase cleaves β- glycosidic bonds

2 Reaction specificity: Specific for every reaction

Aspartate
a s e
Citrat ox yl
Citrate e r b
syntha e c a
se Oxaloacetate D
a t e na se Tran
l e sa m i
Ma drog nase
Malate eh y Pyruvate
D
Example of reaction specificity.
3. Substrate specificity:
A. Absolute :
Urea Urease NH3 + CO2 /Glucokinase
H2O
B. Relative :
a) Group dependent –
• Trypsin hydrolyses peptide linkage involving lysine and
arginine.
• Chymotrypsin hydrolyses peptide linkage involving
aromatic amino acids
b) Bond dependent –
• Proteolytic enzymes hydrolyses peptide bonds
• Glycosidase hydrolyses glycosidic bonds
• Lipase hydrolyses ester bonds
C. Broad substrate specificity
• Enzyme acts on more than one structurally related substrates.
• Hexokinase catalyzes the phosphorylation of more than one
kind of hexoses such as glucose, fructose and mannose.
Protein Nature of Enzymes:
Some enzymes are simple protein in nature i.e. they contain only
amino acids.
Some are conjugated proteins contains protein part called
apoenzymes and non protein part known as prosthetic group or
coenzyme.
Coenzyme + Apoenzyme = Holoenzyme
Some enzymes require an additional chemical component for its catalytic
activity. These are either one or more inorganic ions, such as Fe, Mg, Mn,
Zn etc. called as cofactors or they require complex organic molecules
called coenzymes.

Coenzyme: The non protein part of an enzyme which is required for the
catalytic activity of the enzyme, is called coenzyme. They act as transient
carriers of specific functional groups. Generally these are vitamins.
Characteristics of coenzymes
1. They are heat stable
2. It is low molecular weight organic substances
3. Generally coenzymes loosely bind with enzymes. They are
separated easily by dialysis
4. Sometimes when reaction completed, the coenzyme released
from one enzyme and take part in other reaction.
Co-enzymes may be divided into two groups
• Those taking part in reactions catalyzed by oxidoreductases by
donating or accepting hydrogen atoms or electrons.
Ex. NAD, NADP, FMN, FAD
• Those co-enzymes taking part in reactions transferring groups
other than hydrogen
Ex.
Vitamins Coenzyme Reaction
Thiamine TPP Oxidative decarboxylation and
transketolase reactions
Riboflavin FAD, FMN Dehydrogenation
Pyridoxine (B6) Pyridoxal phosphate Transamination, deamination,
(PLP) decarboxylation of amino acids
Niacin NAD+, NADP Dehydrogenation
Biotin Biotin Decarboxylation
Pantothenic acid Coenzyme-A (CoA-SH) Acyl transferase
Folic acid Tetrahydrofolate (THF) Take part in One carbon group transfer
reactions
Coenzymes derived from nonvitamin precursors

• Lipoate function as a carrier of acyl groups and electrons

Lipoate is a cofactor for two enzymes of citric acid cycle: pyruvate
dehydrogenase complex and α- ketoglutarate dehydrogenase.
• Coenzyme-Q acts as hydrogen acceptor from flavoproteins
like NADH dehydrogenase and succinate dehydrogenase.
• Tetrahydrobiopterin serves as source of hydrogen
Metalloenzymes : These are enzymes, which require certain metal ions
for their activity. These metals are cofactors of that enzymes.
In some cases,
ex. Copper is tightly bound with Tyrosinase
In other cases, even without the metal ion, enzyme may be active
but when the metal ion is added the activity is enhanced. They are called
ion activated enzymes, like calcium ions will activate pancreatic lipase.
Metal Enzymes containing metals
Zinc Carbonic anhydrase, carboxypeptidase,
alcohol dehydrogenase
Magnesium Hexokinase, phosphofructokinase, enolase,
glucose-6-phosphatase
Manganese Phosphoglucomutase, hexokinase, enolase,
glycosyltransferase
Copper Tyrosinase, cytochrome oxidase, lysyl oxidase,
superoxide dismutase
Iron Cytochrome oxidase, catalase, peroxidase, xanthine oxidase
Calcium Lecithinase, lipase
Molybdenum Xanthine oxidase
The substrate
The substance on which an enzyme acts, is called the substrate.
• The substrate of an enzyme are the reactants that are activated by the
enzyme
• Enzymes are specific to their substrats
• The specificity is determined by the active site
Active site:
• Enzymes are bigger in size as compared to substrate.
• Only small portion of this come in contact with substrate while
formation of products.
• The small cleft like portion of an enzyme where the substrate (s) binds
and catalysis occurs is known as the active site or active center.
• The shape and the chemical environment inside the active site
permits a chemical reaction to proceed more easily
 Characteristics of active site :
1. Most of the enzymes, the active site contain substrate site and
catalytic site. The substrate binds at former site and reaction takes
place at latter site.
2. Coenzymes or cofactors bounds to catalytic site.
3. The active site is not rigid in conformation and shape but is flexible.
This is to ensure effective binding of substrate to the enzyme.
4. The active site is largely responsible for the substrate specificity of an
enzyme.
5. Serine is the most commonly occurring amino acid in the active
site of most of the enzymes.
6. Substrates are bound to enzymes at active sites by weak non
covalent bonds like hydrogen bonds, hydrophobic interactions or
van der Waals forces. This type of enzyme substrate binding is
usually reversible.

7. The binding of substrate to active site depends on the alignment of

specific groups or atoms at active site.
During the binding, these groups may rearrange themselves to provide
the unique conformational orientation so as to promote exact fitting of
substrate to the active site
 Enzyme Classification
 Enzymes are classified according to the type of reaction they catalyse.
 All enzymes have formal ‘EC’ (Enzyme Commission) number and names,
and most have trivial names.
 According to the International Union of Biochemistry and Molecular
Biology (IUBMB) system, enzymes are classified into seven major
classes
 Each enzyme is assigned a four-digit ‘EC’ (Enzyme Commission) number,
the first three digits of which define the reaction catalyzed and the fourth
of which is a unique identifier (serial number).
• EC 1 Oxidoreductases: catalyze oxidation/ reduction reactions .
• EC 2 Transferases: transfer a functional group (e.g. a methyl or
phosphate group).
• EC 3 Hydrolases: catalyze the hydrolysis of various bonds.
• EC 4 Lyases: cleave various bonds by means other than hydrolysis and
oxidation.
• EC 5 Isomerases: catalyze isomerization changes within a single
molecule.
• EC 6 Ligases: join two molecules with covalent bonds.
• EC-7 Translocases: movement of ions or molecules across membranes.

OTHLIL T
 EC-1 Oxidoreductases Catalyzes oxidation-reduction reactions.

This group of enzymes will catalase oxidation of one substrate with

simultaneous reduction of another substrate or co-enzyme.
Enzymes in this category include :
 Dehydrogenases
 Reductases
 Oxidases
 Oxygenases
 Peroxidases
1. Dehydrogenases
• Transfer of hydrogen from one substrate to another in a coupled
oxidation-reduction reaction.
• Transfer of electrons in respiratory chain of electron transport from
substrate to oxygenase.

Alcohol dehydrogenase
Alcohol + NAD+ Aldehyde + NADH + H+

Lactate dehydrogenase
Pyruvate Lactate
NAD NADH + H+
2. Oxidases: If the reaction involves a direct participation of oxygen, the
enzymes are termed as oxidases

Xanthine Oxidase
Xanthine Uric Acid
H2O + O2 H2O2

3. Oxygenases : When both oxygen atoms are added it is called as

oxygenases.
O2
Fe 2+
L tryptophan Tryptophan oxygenase
L Formylkynurenine

Niacin Acetyl CoA

 4. Reductases
Glutathione reductase
2GSH GS-SG
(Reduced) NAD NADH+ H+ (Oxidised)
 5. Hydroperoxidases
a. Peroxidases: Reduces peroxides by using various electron acceptors

Glutathione Peroxidase
2GSH+ H2O2 GSSG + 2H2O

b. Catalases : Use H2O2 as electron donor or acceptor

H2O2 2H2O + O2
Catalases
Dehydrogenase: NAD/FAD is electron acceptor in redox reaction
Oxidase: O2 is acceptor but oxygen atoms are not incorporated into substrate
Oxygenase: One or both oxygen atoms are incorporated
EC-2 Transferases
Catalyses the transfer of a group such as, amino, carboxyl, methyl or phosphate,
etc. from one molecule to another

Enzymes in this category include :

 Amino transferase or transaminase

 Kinase: catalyzes the transfer of phosphate groups
 Transmethylase: catalyzes the transfer of methyl groups
1. Amino transferase or transaminase

Alanine + α keto glutarate Alanine transaminase Pyruvate + Glutamic acid

Aspatrate + α keto glutarate Aspartate transaminase Oxaloacetate + Glutamic acid

2. Kinase: catalyzes the transfer of phosphate groups

Glucose Hexokinase/glucokinase Glucose-6 phosphate

ATP ADP + Pi
3. Transmethylase:

S-Adenosylmethionine S-Adenosylhomocysteine
Methyl group acceptor Methylated product
(Norepinephrine) Transmethylation (Epinephrine)
EC-3 Hydrolases
Catalyze the cleavage of C-O, C-N, C-C and some other bonds with
the addition of water.
 Enzymes in this category are:

 All digestive enzymes like:

- α-amylase (Starch Dextrins, Isomaltose, Maltose)
- pepsin (Proteins aminoacids)
- trypsin (Chymotrypsinogen Chymotrypsin)
- chymotrypsin ( Polypeptides Oligopeptides)

 Acid phosphatase

Orthophosphoric monoester + H2O Alcohol + H3PO4

Acidic pH
EC-4 Lyases

Catalyze the cleavage of C-O, C-C and C-N bonds by means other than
hydrolysis or oxidation, giving rise to compound with double bonds or
catalyze the reverse reaction, by the addition of group to a double bond.

In cases where reverse reaction is important, then synthase, (not synthetase

of group EC-6) is used in the name.
Synthase: No ATP required
Synthetase: required ATP
 Glycolysis
Aldolase
Fructose 1-6 bisphosphate Glyceraldehyde -3-phosphate +
Dihydroxyacetone phosphate
TCA Cycle
Fumarase
Fumarate Malate
H2O
Urea cycle
Argininosuccinase
Argininosuccinate Arginine
Fumarate
EC-5 Isomerases
Catalyze intramolecular struc­tural rearrangement in a molecule. They are
called epimerases, isomerases or mutases, depending on the type of
isomerism involved.

Glycolysis
Phosphohexose isomerase
Glu. 6 phosphate Fru. 6 phosphate

Phosphoglycerate Mutase
3 Phosphoglycerate 2 Phosphoglycerate
 Odd-chain fatty acid oxidation
Methylmalonyl CoA
L Methylmalonyl CoA Mutase Succinyl CoA
Vit. B12

HMP shunt pathway

se
Isomera Ribose 5 phosphate
Ribulose 5 phosphate
Epim
er as e Xylulose 5 phosphate
EC-6 Ligases (Synthetases)
Catalyze the joining of two molecules coupled with the hydrolysis of ATP.

DNA Ligase: During replication at some points in the DNA backbone

breaks in the phosphodiester bonds occurs. These breaks called nicks
which are sealed by the DNA ligase. This process require cleaves of ATPs
(EC.6.5.1.1)

Acetyl CoA carboxylase

Acetyl CoA + ATP+ CO2 Malonyl CoA+ ADP+ Pi
Glutamine synthetase:
Glutamine is produced by glutamate by glutamine synthase.
It is the major transport form of ammonia.

α-ketoglutarate

Transamination
NH4 NH4 Urea
Mg-ATP L-glutamate

Glutamine Glutaminase
synthetase

Mg-ADP + Pi
Glutamine
Cells Liver
EC-7: Translocases (A new EC Class)
Translocases catalyze the movement of ions or molecules across membranes
or their separation within membranes. Ex.ATPase
ATP synthase
Examples are:
 Enzymes catalyzing the translocation of:
Hydrons (H+), inorganic cations, inorganic anions, amino acids and
peptides, and carbohydrates and their derivatives.
 Enzymes of the reaction that provided the driving
force for the translocation linked to:
Oxidoreductase reactions, hydrolysis of a nucleoside triphosphate,
hydrolysis of a diphosphate, and decarboxylation reaction.
Dehydrogenase: NAD/FAD is electron acceptor in redox reaction
Oxidase: O2 is acceptor but oxygen atoms are not incorporated into substrate
Oxygenase: One or both oxygen atoms are incorporated
Synthase: No ATP required
Synthetase: Required ATP
Phosphatase: Use water to remove phosphoryl group
Phosphorylase: Uses Pi to break a bond and generate a phosphorylated
compound
Kinases: catalyze the transfer of Pi from high energy compound (ATP) to
specific substrate
 Nomenclature of Enzymes
• In early days, enzymes when discovered where given trivial names
such as pepsin, trypsin, chymotrypsin and so on which are still in use
• The name assigned to enzymes in the beginning were very vague and
uninformative
• As more and more enzymes were discovered and added to list, these
enzymes were named by adding the suffix “ase”
ex. Lactase acts on lactose
Lipase on lipids
Urease on urea etc.
IUBMB classification-to make the names precise, fully informative
and unambiguous, The International Union of Biochemistry and
Molecular Biology have developed a nomenclature for enzymes, the
EC numbers; each enzyme is described by a sequence of four numbers
preceded by "EC".
The first number: Functional class
Second number: Subclass
Third number: Sub subclass
Fourth number: Particular enzyme.
• As per this nomenclature, each enzyme is given a specific name
indicating its substrate, coenzyme and the type of reaction
catalysed by it.

Ex. ATP: glucose phosphotransferase (EC No. 2.7.1.1.) is the systemic

name of enzyme hexokinase (trivial name).

• Alcohol dehydrogenase / alcohol-NAD oxidoreductase (EC. 1.1.1.1)

• Lipase/ Triacylglycerol acyl hydrolase (EC.3.1.1.3)
• Glutamine synthetase/ L-glutamate ammonia ligase/ (EC. 6.3.1.2)
 Alcohol dehydrogenase ( EC 1.1.1.1) Aldolase (EC. 4.1.2.13)
1 oxidoreductase 4 lyases
1 action on primary alcohol 1 breaks C-C bond
1 indicate acceptor NAD/NADP 2 Aldehyde lyase
1 Individual no. of alcohol dehydrogenase 13 individual no.

SGOT (EC 2.6.1.1)

ALP (EC 3.1.3.1)
2 Transferase
3 Hydrolases
6 Nitrogenus group
1 acts on ester linkage
1 Trasaminase
3 Phosphoric monoester hydrolase
1 individual no.
1 Individual no.
SGPT (EC 2.6.1.2)
 glutathione synthase
ATP + g-L-glutamyl-L-cysteine + glycine
ADP + phosphate + glutathione

Systematic name for above enzyme is g-L-glutamyl-L-cysteine:glycine

ligase (ADP-forming)/ EC 6.3.2.3

Explorenz: The enzyme database IUBMB

http://www.enzyme-database.org
1. Translocase as per IUBMB system is classified as:
A) EC-1 B) EC-3 C) EC-6 D) EC-7
2. The digestive enzymes belong to
A) Isomerase B) Transferase C) Hydrolases D) Ligases
3. Zymogen is
A) Vitamin B) Modulator C) Hormone D) Enzyme precursor
4. Enzyme which cleave C-C bond
A) Lyase B) Ligase C) Oxidoreductase D) Isomerase

5. All of the following enzymes involved in oxidation- reduction reactions EXCEPT:

A) Dehydrogenase B) oxygenase C) Hydrolases D) Peroxidases

6. Transaminases belongs to the class of

A) Hydrolases B) Transferase C) Oxidoreductase D) Isomerase
7. Which of the following coenzymes is not derived from vitamins?
A) CoA-SH B) TPP C) Pyridoxal phosphate(PLP) D) Coenzyme Q

8. Pyridoxal phosphate is a coenzyme for the reactions, EXCEPT:

B) Transamination B) Deamination C) Decarboxylation D) oxidation-reduction

9. Biotin is involved in:

C) Oxidation – reduction B) Carboxylation
C) Decarboxylation D) Dehydrogenation

10. Thiamin pyrophosphate is required for the following enzymatic activity:

D) Hexokinase B) Transketolase C) Transaldolase D) Glu. 6 phosphatase

11. Which of following vitamins provides the coenzyme for transamination of amino acids
E) Thiamin B) Vit. B6 C) Niacin D) Folic acid
12. Which of following vitamins acts as a coenzyme for transfer of one-carbon units?
A)Niacin B) Thiamin C) vit. B6 D) Folic acid

13. For the synthesis of fatty acids which of following vitamin as a coenzyme required:
B) Biotin B) Riboflavin C) Folic acid D) Thiamin

14. Coenzyme form of Pyridoxine is:

C) ADP B) NAD C) PLP D) FAD

15. Transfer of one carbon unit requires:

A) Retinol B) Folic acid C) Niacin D) Riboflavin
Case: Pei, a twenty-six year old girl, had been experiencing
occasional discomfort after meals. The discomfort reached a
new peak last Thursday evening about an hour after eating a
cheeseburger and a large chocolate milk shake. Pei spent
much of that night in pain. She had abdominal cramps and
diarrhea. She went to the clinic and saw a doctor the next day.

Disorder: Lactose intolerance

Deficiency: Lactase