Enzyme mbbs

Enzymes- Biocatalyst
A catalyst is defined as a substance that increase the
velocity or rate of a chemical reaction without itself
undergoing any change in overall process.

Definition
Enzymes may be defined as “protein catalysts that
catalyse chemical reactions in biological systems”
Exceptions- Ribozymes(RNA molecules)
Enhance the rates of the corresponding non catalyzed
reaction by factors of at least 10 6

Enzymes are proteins that increase the rate of reaction by
lowering the energy of activation
They catalyze nearly all the chemical reactions taking
place in the cells of the body.
Not altered or consumed during reaction.
Reusable

Importance
Metabolism
Diagnostic
Therapeutic

Substrate- Reactant on which enzyme acts is known
as a substrate for that enzyme
Enzyme converts Substrate into Products
Enzyme(E)
Amylase(E)
Substrate Product
Starch Glucose

Active Site
A Cleft like region on Enzyme where substrate binds

Chemical Nature
Water soluble
Amphoteric
Non dialyzable
Isoelectric point
Mainly protein in nature ( exception ribozymes)

Source and distribution
Produced in living cells, but can act outside also
Distributed in cells, organs, fluids , blood
Intracellular distribution related to function of the cell

Location Function Examples
Cell membrane Transport Na-K ATPase
Mitochondria Energy TCA, Beta Oxidation
Lysosomes Hydrolysis Peptidases,Glycosidase
Nucleus Genetic information DNA and RNA
polymerase
Cytosol Accessory Pathway Glycolysis,HMP shunt
Microsomes Synthetic FA chain elongation

Molecular forms
Monomeric/Multimeric
Multifunctional ( FA synthesis- 7 enzymes)
( One polypeptide chain with different activity)
Multi –enzyme Complex
( Multiple Enzymes Same activity- PDH complex)

Proenzymes or Zymogens
Activated by proteolysis/ Covalent modification
Eg. Chymotrypsinogen---Chymotrypsin
Isoenzyme- physically distinct form of same enzyme
Different AA sequence, different location, same
function
Eg. Alkaline Phosphatase, LDH

Nomenclature
Earlier ‘ –ase’ to the name of the substrate
Eg. Urease acts on Urea
Arginase acts on Arginine

Classification of Enzyme
1. Oxidoreductases
2. Transferases
3. Hydrolases
4. Lyases
5. Isomerases
6. Ligases

1.Oxidoreductase
Oxidation reduction reactions of all types
Oxidation------------------> Reduction
Reduction -----------------> Oxidation
AH2 + B --------> A + BH2

Others..
Succinate Dehydrogenase
Glutamate Dehydrogenase
Alpha Keto Glutarate Dehydrogenase

2. Transferases
“Transfer various groups (Other than Hydrogen)”
Transaminase- Transfer of amino group
Methyltransferase- Transfer of one Carbon group
Kinase- Transfer of Phosphate group

Transferases
 A-X + B -------> A + B-X
 Group Transfer

Others…
Glucokinase
Aspartate Transaminase (AST)
Alanine Transaminase (ALT)

3.Hydrolases
These are hydrolytic enzymes which hydrolyse bonds
such as peptide, ester, glycosidic bonds etc by adding a
water molecule.
These enzymes are commonly found in the digestive
secretions and lysosomes
DIGESTIVE ENZYMES…

They hydrolyse carbohydrates, lipids, proteins
Examples are amylase, lipase, pepsin, ribonuclease,
sucrase, lactase, maltase

Hydrolase
Hydrolysis
A-B + H2O -----> AH + BOH

4.Lyases
Removal of Group (other than hydrolysis)
Breaks down C-C, C-O bond
Removal of water, amino group, carbon

Lyases
Addition ------> Elimination
A-B + X-Y -----> AX-BY

5.Isomerases
Interconversion of Isomers
Rearrangement of molecules
A------> A+

Others…
Triose phosphate Isomearse
Retinal Isomerase
Methyl malonyl CoA racemase

6. Ligases
Bond formation by linking two compounds with help
of ATP
Formation of C-O, C-C,C-N bond

Ligases
Condensation
A+B ---------> A-B
ATP-----> ADP + Pi

Others..
Acyl CoA Synthetase
Amino Acyl t-RNA synthetase

COFACTOR & COENZYME
Protein part of Enzyme ( Apoenzyme) is inactive,
It requires a non protein molecule for its catalytic
activity ( Co Enzyme)

Enzyme
(HoloEnzyme)
Non Protein
Part
(Co Factor)
Metal ions
(Prosthetic)
Co Enzymes
Protein
Part
Apoenzyme

Metal ions as a Cofactor
• Metal ions tightly bound to enzyme protein
• Metal ions cant be separated without
breakdown of enzyme
• Eg. Cytochrome Oxidase ( Fe++ and Cu++ )
• Carbonic Anhydrase ( Zn++)
Metallo
Enzyme
• Metal ions loosely bound to enzyme protein
• Metal ions easily separated without
breakdown of enzyme
• Eg Hexokinase ( Mg++)
• Amylase (Cl-)
Metal
Activated
enzyne

Coenzyme
“ Non protein, dialysable, low molecular and organic
substance associated with enzyme activity is known
as Co enzyme”

COENZYME Vitamin
Derivatives
Non Vitamin
Derivatives
Nucleotide
Derivatives

Vitamin Derivatives
Vitamin COENZYME
Vitamin B1(Thiamine) Thiamine Pyrophosphate(TPP)
Vitamin B2(Riboflavin) Flavin Adenine Dinucleotide (FAD)
Flavin Mono Nucleotide( FMN)
Vitamin B3 (Niacin) Nicotinamide Adenine Dinucleotide
(NAD)
NADP
Vitamin B6 (Pyridoxine) Pyridoxal phosphate

Non Vitamin Derivatives
Co Enzymes Function
ATP Give rise to ADP and AMP
UDP Glycogen Synthesis
CTP Phospholipid synthesis

Nucleotide Derivatives
(Class – 1, Oxidoreductase-
Transfers H+ groups)
NAD
NADP
FAD
FMN

Class-2 ( other than H+)
Co Enzymes Group Transfer
TPP Hydroxymethyl
Biotin CO2
PLP Aminogroup
Co A Acyl group
TetraHydro Folate One Carbon group
ATP phosphate

COENZYME
COENZYME can act as Co-Substrate
It does not affect substrate specificity of enzyme

Specificity- Property of
Active Site
Active Site??

• L-Amino acid oxidase
• Exceptions--IsomerasesStereo
• Peptidase,Amino &
Carboxypeptidase
• Glycosidase, Lipase, Hexokinase
Group/Bond
• Only one substrate
• Glucokinase,UreaseAbsolute

Reaction Specificity
Pyruvate
Oxaloacetate Acetyl CoA Alanine
Carboxylase Dehydrogenase Transaminase

Questions
1) Difference between Synthase and
Synthatase(3)
2) Vitamine derivative helps in
transfer of amino group(3)
3) Cleave the bond without help of
water(7)
4) Property of active site(11)
5) Enzyme with group specificity(10)
6) Non protein part of enzyme(8)
7) Metal require for cytochrome
oxidase(4)
8) Coenzyme of class 1(3)
9) I activate Xanthine Oxidase(10)
10) I am the current topic which you
are learning (6)

Mechanism of Action of Enzymes
“Enzymes increases reaction rate by
decreasing the Activation energy”
Enzyme-Substrate Interactions:
 Formation of Enzyme substrate
complex by:
 Lock-and-Key Model
 Induced Fit Model
 Substrain strain Theory

Lowering the activation
energy
Activation energy: energy
required to reach the
transition state
Transition state: state in
which there is a high
probability that a chemical
bond will be made or broken
to form a product
No change in ∆G and Keq of
reaction

Enzyme Catalyzed Reactions
• When a substrate (S) fits properly in an active site, an
enzyme-substrate (ES) complex is formed:
E + S ⇄ ES
• Within the active site of the ES complex, the reaction
occurs to convert substrate to product (P):
ES → E + P
• The products are then released, allowing another
substrate molecule to bind the enzyme
- this cycle can be repeated millions (or even more) times
per minute
• The overall reaction for the conversion of substrate to
product can be written as follows:
E + S ⇄ ES → E + P

Enzyme-substrate complex
• Step 1:
• Enzyme and substrate combine to form
complex
• E + S ES
• Enzyme Substrate Complex
+

Enzyme-product complex
• Step 2:
• An enzyme-product complex is formed.
• ES EP
ES EPtransition
state

Product
• The enzyme and product separate
• EP E + P
The product
is made
Enzyme is
ready
for
another
substrate.
EP

Fischer’s Template
theory
• Active site is rigid
• Does not explain
substrate specificity
• Not acceptable

Koshland’s Induced
fit theory
• Active site is not rigid
• Explain substrate
specificity
• Mostly acceptable

Substain strain
theory
• Enzyme produces strain
on substrate
• Widely acceptable in
association with induced
fit theory

FACTORS AFFECTING
ENZYME ACTIVITY

Factors
Temperature
pH
Enzyme Concentration
Substrate Concentration
Activators

Rate of Reaction(Velocity)
Defined as a rate of change from substrate to
product per unit of time ( V)
Maximum velocity ( Vmax) – 100 % enzyme
molecules binds to substrates
½ Vmax – 50%

Effect of Temperature
Optimum temperature- at which enzyme reaction is
fastest

 In the initial part of the curve, the number of
times the velocity increases when the
temperature rises by 10°C is known as the
temperature coefficient (Q10) of the enzyme
 For most of the enzymes, the temperature
coefficient is two
• This means that the velocity is doubled when
the temperatures rises by 10°C

Effect of pH
• Enzymes are proteins
• Share the same property
• Very High or Low pH
away from optimum pH
may denature an enzyme

Substrate Concentration and Reaction Rate
• The rate of reaction increases as substrate
concentration increases (at constant enzyme
concentration)
• Maximum activity occurs when the enzyme is
saturated (when all enzymes are binding substrate)

Km(Michaelis Menten
Constant)
“The concentration of substrate at which the rate of
reaction is equal to the half of the maximum rate of
reaction”
Represents the affinity of an enzyme towards an
substrate

Km and Affinity
Hexokinase (Km = 0.05 mmol/L) for glucose
Glucokinase (Km = 10 mmol/L) for glucose
Higher the Km lower the Affinity
Lower the Km Higher the Affinity

 The relationship between the velocity of the
reaction and the substrate concentration can be
expressed by Michaelis-Menten equation, which is:
[ ]
[ ]
maxV Sv
Km S
=
+
.

 When the substrate concentration is very low, the
sum of Km and [S] is nearly equal to Km as [S] is
negligible
 Therefore, the equation may be rewritten as:
[ ]maxV S
v
Km
= .
 Since both Vmax and Km are constant,
V  [S]
[ ]
[ ]
maxV Sv
Km S
=
+
.

 When the substrate concentration is very high,
the sum of Km and [S] is nearly equal to [S] as Km is
relatively negligible
 Therefore, the equation may be rewritten as:
or = Vmax
[ ]
[ ]
maxV Sv
S
= .
v
[ ]
[ ]
maxV Sv
Km S
=
+
.

 When the substrate concentration is exactly
equal to Km, the sum of Km and [S] may be taken
as 2 [S]
 The equation may be rewritten as:
Thus, when the substrate concentration is equal
to Km, the velocity is half of Vmax
=
[ ]
[ ]
max maxV S V
v
2 S 2
= .[ ]
[ ]
maxV Sv
Km S
=
+
.

Features of Km
Definition
50% of Enzyme molecules are occupied
Signature of an enzyme, for each substrate
Not dependent on enzyme concentration
Denotes Affinity

Effect of Activators
Metals
Zymogens
Trypsinogen-- Trypsin

Enzyme Inhibitor
“A substance that binds with the enzyme and
decreases the catalytic activity of that enzyme”

Classification
• Based on Reversibility
• Based on Competitiveness
• Allosteric Inhibition
Reversible
Irreversible
Competitive
Non-competitive

Competitive – Non competitive!!!

Competitive Inhibition
• competes with the for active site of
• Inhibitor is a structural analogue of the substrate
• Is concentration dependent ; reversible
I S E

E + S E-S complex E + P
E + I E-I Complex
+
Inhibitor
Product

Some competitive inhibitors used as drugs:
1. Amethopterin, Methotrexate (Folic Acid)
2. Allopurinol (Hypoxanthine)
3. Statins (HMG CoA)
4. Sulfonamide ( PABA )
5. Isoniazid ( Pyridoxal- Vitamin B6)

H N2 N
N
|
OH
1
2
3
4
N
5
6
7
8
N
9 10
CH — N —2 — C — N — CH
| |
H COOH
COOH
|
CH2
|
CH2
|
O
||
H
|
Pteridine para-Amino-
benzoic acid
Glutamic
acid
Pteroylgutamic acid (folic acid)H N2 N
N
|
OH
1
2
3
4
N
5
6
7
8
N
9 10
CH — N —2 — C — N — CH
| |
H COOH
COOH
|
CH2
|
CH2
|
O
||
H
|
Pteridine para-Amino-
benzoic acid
Glutamic
acid
C 3
Folic acid
Amethopterin
Amethopterin and aminopterin

Amethopterin and aminopterin (Contd.)
 These are competitive inhibitors of dihydrofolate
reductase
Dihydrofolate + NADPH + H
+
Tetrahydrofolate + NADP
+
Dihydrofolate
reductase
4
Folate + NADPH + H
Dihydrofolate
reductase
4
+

Amethopterin and aminopterin (Contd.)
 This reaction is important in the synthesis of purine
and thymine nucleotides
 Inhibition of this reaction decreases the availability of
nucleotides for DNA synthesis, and inhibits cell division
 Therefore, amethopterin and aminopterin are used as
anti-cancer drugs to suppress the unregulated cell division that
occurs in cancer

Allopurinol
 Allopurinol is a structural analogue of
hypoxanthine
 It is a competitive inhibitor of xanthine oxidase
N
HN
O
||
C
C
CHC
N
H
H
C
N
N
HN
O
||
C
C
CHC
N
H
N
CH
Hypoxanthine Allopurinol

Allopurinol (Contd.)
 Xanthine oxidase converts hypoxanthine into
xanthine, and xanthine into uric acid
 In gout, which results from overproduction of uric
acid, allopurinol is used to inhibit the formation
of uric acid
Hypoxanthine
Xanthine
oxidase
Uric acidXanthine
Xanthine
oxidase

Hypoxanthine
Xanthine
oxidase
Uric acidXanthine
Xanthine
oxidase
Allopurinol
Xanthine
oxidase
Alloxanthine
Its also known as Suicide
Inhibition…!!!
Uric acid

Examples
Succinate Fumarate
Methanol Formaldehyde
HMG CoA Mevalonate
Succinate
dehydrogenase
X
Malonate
Alcohol
dehydrogenase
X
Ethanol
HMG CoA Reductase
X
Statins

Let us Revise……
• Definition and types
 Substrate & inhibitor have structural similarity
 Compete for same active site of enzyme
 Reversible
 Km and Vmax remains same
 Reversed by increasing substrate conc.
Competitive inhibition

Non Competitive Inhibition
No competition between inhibitor and substrate to
bind with the enzyme..

NonCompetitive Inhibition
• does not competes with the for active site of
• Inhibitor is not a structural analogue of the substrate
• Is not concentration dependent ; Irreversible
I S E

Examples
Cyanide inhibits cytochrome oxidase
Fluoride inhibits Enolase
BAL in heavy metal poisoning

Examples of Non competitive inhibition includes use of
Disulfiram in treatment of Alcoholism.
Alcohol -------> Acetaldehyde ------->Acetic Acid
Alcohol
dehydrogenase
Acetaldehyde
dehydrogenase

COMPETITIVE NON COMPETITIVE
Acting on Active site May or May not
Structure of inhibitor Substrate analogue Not related
Inhibition Reversible Irreversible
Excess Substrate Relieved Inhibition No effect
Km Increased No change
Vmax No change Decreased
Significance Drug action Toxicological

Allosteric Regulation
Allosteric enzymes have two binding sites:
Catalytic site- Substrate binds
Allosteric site- Allosteric modulator binds
Controls the key enzymes of Metabolic pathway

Substrate----->A------>B------->C------->D
E1 E2 E3 E4

Covalent Modification
Addition or removal of a group may activate or
deactivate an enzyme
Under hormonal influence
Phosphorylation/ Dephosphorylation

ISO ENZYMES
Multiple form of an Enzyme that carries out same
reaction
Different in amino acid sequence
Difference in properties such as temp, pH,Km and
effect of inhibitor etc.

IsoEnzymes Subunits Tissue of origin
LDH1 H4 Heart
LDH2 H3M RBC
LDH3 H2M2 Brain
LDH4 HM3 Liver
LDH5 M4 Skeletal Muscle

Flipped Pattern
Normal person LDH-2 > LDH-1
Myocardial Infarction LDH-1 > LDH-2

Elevated LDH
Hemolytic Anemias
Liver disease
Muscular dystrophies
Leukamias
Myocardial Infarction

Creatine Kinase (CK)
Creatine--------CK--- Creatine Phosphate

IsoEnzymes Subunits Tissue of origin
CK1 BB Brain
CK2 MB Heart
CK3 MM Skeletal muscle

CK Total – Muscular dystrophies
CK MB – Myocardial Infarction

Alkaline Phosphatase
Bone disease
Liver disease
Lymphomas
Ulcerative Colitis

Application
of Enzymes
Diagnostic Therapeutic

The following plasma enzymes have become
established diagnostic tools:
1.Lactate dehydrogenase (LDH)
2. Transaminases (GOT and GPT)
3. Creatine kinase (CK)
4. Gamma glutamyl transpeptidase (GGT)
5. Alkaline phosphatase (ALP)
6. Acid phosphatase (ACP)
7. Amylase
8. Lipase

ALT AST
ALP GGT
Hepatic
Disease

CK-MB AST
LDH Troponins
Myocardial
Infarction

• CK
• AST
• Aldolase
Muscular
disease
• Alkaline PhosphataseBone
• Acid phosphatase
• Prostate Specific AntigenProstate

 This enzyme catalyses the interconversion of
pyruvate and lactate
 Its tissue distribution is very wide
 However, its concentration is much higher in
myocardium, muscles and liver than in other
tissues

 Therefore, plasma LDH rises in myocardial
infarction, viral hepatitis and muscle injuries
 In myocardial infarction, the rise begins 24 hours
after the episode of infarction, the peak value is
reached in about three days, and the level returns to
normal in about a week

 The two most important transaminases are
glutamate oxaloacetate transaminase (GOT)
and glutamate pyruvate transaminase (GPT)
 These are also known as aspartate
aminotransferase (AST) and alanine
aminotransferase (ALT) respectively
 These are present in high concentrations in
myocardium, liver and muscles

 Therefore, their plasma levels are raised in
myocardial infarction, viral hepatitis and
muscle injuries
 Concentration of SGOT is higher than that of
SGPT in myocardium while the situation is reverse in
liver
 Therefore, the rise in plasma SGOT is more
pronounced in myocardial infarction and that in
SGPT is more pronounced in viral hepatitis

 It is also known as creatine phosphokinase
(CPK), and catalyses the following reaction:
 CK is present in myocardium, muscles and brain
Creatine + ATP Creatine ~ P + ADP

 Plasma CK rises in myocardial infarction,
myopathies and muscle injuries
 Plasma CK is a more specific and early indicator
of myocardial infarction than LDH and GOT
 It begins to rise within 3-6 hours of occurrence of
infarction, reaches its peak in 24 hours, and returns to
normal in about three days

Marker Onset Peak Duration
CK MB 3-6 Hr 18-24 Hr 36-72 Hr
Troponins 4-10 Hr 18-24 Hr 8-14 Days
LDH 6-12 Hr 24-48Hr 6-8 Days
AST 24-36 Hr 4-5 Days 10-12 Days
Myoglobin 1-4 Hr 6-7 Hr 24 Hrs

1 2 3 4 5 6 70
Enzyme
level
Upper limit
of normal


Days
CK GOT LDH

Apart from LDH, GOT and CK, some non-enzyme
proteins are also released from myocardium after
myocardial infarction, and are useful in diagnosis
Begins to
rise in
Reaches
peak in
Returns to
normal in
Specificity
Myoglobin 1-3 hrs 4-6 hs 18-24 hrs Low
Cardiac troponin T 4-6 hrs 18-36 hs 5-15days Low
Cardiac troponin I 4-6 hrs 12-24 hs 5-10days High

 This enzyme catalyses the transfer of the
gamma-glutamyl residue of glutathione to
other substrates
 Its plasma level increases in most of the liver
diseases, and is an early indicator of alcoholic
hepatitis

 This is a group of enzymes that hydrolyse
organic phosphate esters at an alkaline pH
 ALP is released in circulation mainly from
bones and liver
 Smaller amounts come from intestines and
placenta
 Liver excretes ALP in bile

 The maximum elevation of plasma ALP occurs in
obstructive jaundice
 Smaller elevations occur in viral
hepatitis, rickets,hyperparathyroidism,
osteosarcoma, bony metastases etc

 This enzyme hydrolyses organic phosphate
esters at an acidic pH
 The main source of ACP is the prostate gland
 Plasma ACP is elevated in metastatic
carcinoma of prostate

 This is a digestive enzyme, synthesised in the
pancreas and the parotid gland
 Sharp elevation of plasma amylase occurs in
acute pancreatitis
 A smaller elevation occurs in acute parotitis

 This lipolytic enzyme is released into circulation
from the pancreas
 Plasma lipase rises in acute pancreatitis

Other Fluids
Adenosine Deaminase – TB Pleural effusion
LDH in CSF – Malignant tumor

Therapeutic Uses
Streptokinase – To remove the blood clots
Asparginase – In cancer treatment
Alpha 1 Antitrypsin - Emphysema

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