ii

1. ENZYMES
BY
PROFESSOR DR. M. SHAFIQUE
MBBS,M.Phil.,Ph.D

2. Enzymes
 Definition and Explanations
 Mechanism of action
 Classification
 Properties of enzymes
 Factors affecting enzyme activity
 Enzyme kinetics
 Regulatory enzymes
 Allosteric enzymes
 Enzymes of clinical interest

3. 3
What Are Enzymes?
 Most enzymes are
Proteins (tertiary
and quaternary
structures)
 Act as Catalyst to
accelerates a
reaction
 Not permanently
changed in the
process

4. 4
Enzymes
 Are specific
for what they
will catalyze
 Are Reusable
 End in –ase
-Sucrase
-Lactase
-Maltase

5. Definitions
 Enzyme: is a biological catalyst, synthesized by living
cells, increases the velocity or rate of chemical reaction
and is not consumed during that reaction. It is
functional unit of cell metabolism. It is colloidal,
thermo labile and mostly protein in nature. Holoenzyme =
apoenzyme + coenzyme
 Coenzyme: is an organic cofactor which is thermo
stable, non-proteinous, low molecular weight, required
for the action of certain enzymes, it can be dialyzed out
and separated from the enzyme.
Many coenzymes are active forms of vitamin B-complexes
Thiamine : TPP , TTP Riboflavin : FMN , FAD
Niacin : NAD , NADP Pyridoxine: Pyr Phosphate
Pentothenic acid : CoASH Vitamin B12 : Cobamide
Folic Acid : FH4

7. 7
How do enzymes Work?
Enzymes work
by weakening
bonds which
lowers
activation
energy

8. 8
Enzymes
Free
Energy
Progress of the reaction
Reactants
Products
Free energy of activation
Without Enzyme
With Enzyme

9. 9
Enzyme-Substrate Complex
The substance
(reactant) an
enzyme acts on is
the substrate
Enzyme
Substrate
Joins

10. 10
Active Site
 A restricted region of an enzyme molecule which
binds to the substrate.
Enzyme
Substrate
Active
Site

11. 11
Induced Fit
 A change in the
shape of an
enzyme’s active site
 Induced by the
substrate

12. 12
Induced Fit
 A change in the configuration of an
enzyme’s active site (H+ and ionic
bonds are involved).
 Induced by the substrate.
Enzyme
Active Site
substrate
induced fit

13. Mechanism of Action
 ENZYME SUBSTRATE
COMPLEX
 Template or lock and key model
 Induced fit model
 CONVERSION OF SUBSTRATE
TO PRODUCT
 ES-------------EP
 RLEASE OF PRODUCT FROM
ENZYME
 EP-------------E + P
The sequence is E + S----ES----EP----E
+ P

14. ENZYME ACTION

15. Enzymes
Lower a
Reaction’s
Activation
Energy

16. Lowering of the activation energy of Hydrogen peroxide
Reaction
condition
Activation free
energy
(K.cal/mol)
Relative rate
No catalyst 18.0 1
Platinum
surface
11.7 2.77×104
catalase 5.5 6.51×108

17. Classification of Enzymes
I. Oxidoreductases
II. Transferases
III. Hydrolases
IV. Lyases
V. Isomerases
VI. Ligases

18. I.Oxidoreductases :catalyze oxidation-reduction
reactions
Further divided into four subgroups
 Oxidases
 Dehydrogenases
 Hydroperoxidases
 Oxygenases
1. Oxidases
 Oxygen is added and H atoms are removed from the substrate forming H2O
or H2O2
 xanthine oxidase
 A.A oxidase

19. 2. Dehydrogenase
 They remove hydrogen from substrate, but are not
able to use oxygen as hydrogen acceptor
 Special hydrogen acceptors such as NAD+, NADP+,
FAD are used.
 Examples
1. Lactate dehydrogenase (LDH)
2. Succninate dehydrogenase
3. Glucose 6-Phosphate dehydrogenase
3. Hydroperoxidases
 Catalase and Peroxidase
2H2O2→2H2O+ O2

20. II. Transferases : Catalyze transfer of amino,
phosphoryl, methyl, glycosyl groups
1.Transaminases – The catalyze the exchange of
NH2 group between amino acid and keto acids. The
keto acid become amino acid and amino acid become
keto acid. e.g
i. G.O.T (AST)
ii. G.P.T (ALT)
2. Phaspho transferases: kinases
3. Transmethylases transfer
of methyl group from methionine
to form creatine & adrenaline etc.

21. 21
Transaminations
Glutamate a-Ketoglutarate
+ +
Pyruvate Alanine
Glutamate a-Ketoglutarate
+ +
Oxaloacetate Aspartate
Glutamate-Pyruvate
Aminotransferase(GPT)
(Alanine Transferase ALT)
Glutamate-Oxaloacetate
Aminotransferase(GOT)
(Aspartate Transferase AST)
Blood levels of these aminotransferases, also called transaminases,
are important indicators of liver disease

22. III. Hydrolases (catalyze hydrolysis )
1. Protein hydrolyzing enzymes
A. Exopeptidase: carboxypeptidase
B. Endopeptidases:
pepsin,trypsin,chymotrypsin,& elastase
2. Carbohydrases-: amylases, sucrase, lactase
3. Lipid hydrolyzing enzymes: lipases
4. Deaminases: adenase, guanase

23.  LYASES
Cause addition of NH3 ,H2O or CO2 to double bonds
or removal of these from double bonds.
FUMARIC ACID------------MALIC ACID
 ISOMERASES
Cause isomerization of substrate, may be isomerases
or mutases.
GLUCOSE 6-P------------FRUCTOSE 6-P
GLUCOSE 6-P------------GLUCOSE 1-P
 LIGASES
Involve in joining together two substrates with help
of ATP,GTP etc
ACETYL CoA------------MALONYL CoA
SUCCINIC ACID-------SUCCINYL CoA

24. IUB-Classification of Enzymes

25. ENZYME PROPERTIES
 SPECIFICITY
 Absolute Specificity
 Relative Specificity
 Reaction Specificity
 Substrate Specificity
 Stereo Specificity
 Bond Specificity
 PROTEIN NATURE
 ENZYME INHIBITION
 DIRECTION OF ENZYME REACTION
 PROENZYMES OR ZYMOGENS
 INDUCTION OF ENZYME SYNTHESIS
 REPRESSION OF ENZYMES
 ISOENZYMES

26. ISOENZYMES (ISOZYMES)
These are multiple forms of an enzyme that differ in structure
and properties but catalyze the same reaction.
i. LACTATE DEHYDROGENASE:LDH
ii. CREATININE KINASE:CK OR CPK
iii. ALKALINE PHOSPHATASE:ALP
LDH has five isoenzymes LDH1-LDH5.Each type has 4
polypeptide chains which are of two types M (muscle) and
H (heart)
M4 Chains: M4,M3H,M2H2,MH3
H4 Chains: H4,H3M,H2M2,HM3

27. ISOENZYMES
LDH SUBUNITS
LDH1-I1 HHHH
LDH2-I2 HHHM
LDH3-I3 HHMM
LDH4-I4 HMMM
LDH5-I5 MMMM
CREATINE KINASE has 3 isozymes with 2 polypeptides B & M
CK1-BB (Brain), CK2- MB (Myocardium),CK3-MM(Muscle)
Ckmb is raised in myocardial infarction
Alkaline phosphatase-ALP
α1-ALP, α 2 ALP, pre-βALP & γ-ALP etc

28. 28
What Affects Enzyme
Activity?
 Three factors:
1. Environmental Conditions
2. Cofactors and Coenzymes
3. Enzyme Inhibitors

29. 29
1. Environmental Conditions
I. Extreme Temperature are the most
dangerous
- high temps may denature (unfold) the
enzyme.
II. pH (most like 6 - 8 pH near neutral)
III. Ionic concentration (salt ions)

30. 30
2. Cofactors and Coenzymes
 Inorganic substances (zinc, iron) and
vitamins (respectively) are sometimes need
for proper enzymatic activity.
 Example:
Iron must be present in the quaternary
structure - hemoglobin in order for it to pick
up oxygen.

31. 31
3. Examples of Enzyme
Inhibitors
a. Competitive inhibitors: are chemicals
that resemble an enzyme’s normal
substrate and compete with it for the
active site.
Enzyme
Competitive inhibitor
Substrate

32. 32
Inhibitors
b. Noncompetitive inhibitors:
Inhibitors that do not enter the active site, but
bind to another part of the enzyme causing the
enzyme to change its shape, which in turn
alters the active site.
Enzyme
active site
altered
Noncompetitive
Inhibitor
Substrate

33.  Enzyme and substrate concentrations

34. Effect of substrate concentration
a) At low substrate concentration, the velocity of
reaction is directly proportional to the substrate
level (part A in graph).
a) In the second phase (part B), the substrate conc. is
not directly proportional to enzyme activity.
c) In the third & final phase (part C), the reaction is
independent of the substrate concentration.

35.  Temperature and pH effect enzyme action

36.  Temperature and pH effect enzyme action

38. Michaelis-Menten Equation
1. Reaction Model
2. Michaelis - Menten equation
 Illustrates in mathematical term the relationship b/w
initial reaction velocity (Vi) & substrates
concentration [S].

39. Michaelis-Menten Kinetics
 Shows saturation at high substrate concentrations
 Vmax – rate at saturation for a given enzyme
concentration in moles per unit time
 Km – Michaelis constant – substrate concentration
that gives ½ maximal velocity
 
 
 
S
K
S
V
V
m 
 max

40. The equation of Lineweaver-Burk plot
(Double reciprocal plot)
The equation
The plot

41. What is km ?
 The michaelis-Menten constant (Km) is the substrate conc. at
which Vi is half maximal velocity (Vmax/2) attainable at
particular concentration of enzymes.
 It indicates that half of the enzyme molecules(50%) are bound
to substrates molecules when the substrates con.
equals the Km value.

49. Allosteric enzyme (allo-other)
 Definition: Some of the enzymes possess addition sites,
known as allosteric sites, besides the active sites.
 Allosteric effector or modulator
.Positive allosteric effector
.Negative allosteric effector

50. Drugs clinically used as inhibitors
Naturally
occurring
substances
Competitive
inhibitors (anti -
metabolites)
Enzyme inhibits Chief action &
use
1. p-Amino
Benzoic Acid
(PABA)
Sulfonamides e.g
Septran
(sulfamethoxazole)
Dihydro-pteroate
synthetase
Inhibit the formation
of folic acid by the
bacterial cell which
is needed by them.
The bacteria
therefore die out.
2. Folic Acid
Pteroylglutamate
(PGA)
Methotrexate
(MTX)
Metotrexate
Dihydro-folate
reductase
(FH2 reductase)
Inhibit the formation
tetrahydro-folic acid,
which is needed for
DNA synthesis. Used
as anti-cancer drug.
3. Hypoxanthine
and xanthine
Allopurinol
(zyloric)
Xanthine Oxidase
(purine catabolism)
Inhibit formation of
uric acid; used in
treatment of gout.

51. Uses of Enzymes
1. For diagnosis & prognosis of diseases by its
estimation in serum.
i.e Cardiac enzymes: CK, AST, LDH, Troponin T & Troponin I (cTnI)
Liver enzymes: Transaminases, Alk. Phosphatase, GGT
2. Used as Therapeutic agents.
i.e Streptokinase( M.I), Serrato peptidase( anti-inflammatory)
3. Used as catalytic agents in laboratory.
i.e Glucose Oxidase, Urease, Uricase (for respective blood tests)

52. Principal serum enzymes used in clinical diagnosis
Serum Enzyme Major Diagnostic Use
Aminotransferses
Aspartate aminotransferase (AST or SGOT)
Alanin aminotransferase (ALT, or SGPT)
Myocardial infarction
Viral Hepatitis
Amylase Acute pancreatitis
Ceruloplasmin Hepatolenticular degeneration (Wilson’s
disease)
Creatine kinase Muscle disorder & myocardial infarction
γ – Glutamyl transpeptidase Various liver diseases (alcoholism)
Lactate dehydrogenase (isozyme) Myocardial infarction
Lipase (urinary) Acute pancreatitis
Phosphatase, acid Metastatic carcinoma of the prostate
Phosphatase, alkaline Various bone disorders, obstructive liver
diseases

53. Important Question
 Short notes: Coenzymes, cofactors, isoenzymes, Km,
allosteric enzymes, enzyme specificity, enzyme inhibition,
active site, turnover rate.
 SEQs:
1. Define enzyme & classify them with example.
2. Discuss the mechanism of enzyme action.
3. Describe the various factors affecting enzyme activity.
4. Enlist the important serum enzymes of clinical interest.
5. Give at least three important uses/applications of enzymes.
6. Enumerate four important properties of enzyme.
7. Write down the MM equation & lineweaver-Burk equation.
8. Enlist the cardiac & liver disease diagnostic enzymes.

54. MCQs
 Enzymes markedly elevated in obstructive jaundice :
i. GGT ii. 5-Nucleotidase iii. Alk.phosphatase iv. All of them
 Enzyme used as medicine in bleeding disorders :
i. Streptokinase ii Thrombine iii.Kallekarein iv.Pepsin v. Hyaluronidase
 Enzymes increase the rates of reaction by :
a) Increasing free energy of activation
b) Decreasing free energy change of reaction
c) Decreasing the energy of activation.
d) Changing equilibrium constant of reaction
 An allosteric enzyme influences the enzyme activity by :
i. Changing enzyme conformation by binding to site other than active site.
ii. Competing for the catalytic site with the substrate
iii. Changing the specificity of enzyme for the substrate
iv. Changing the nature of the products formed
 A competitive inhibitor of enzyme has which of the following property ?
i. It is frequently a feedback inhibitor
ii. It becomes covalently attached to enzyme
iii. It causes irreversible inactivation of enzyme
iv. It interferes with substrate binding to enzyme.

55.  Y-Glutamyl transpeptidase activity in serum is elevated in
(a) Pancreatitis (b) Muscular dystrophy (c) Myocardial infarction (d) Alcoholism.
 In recent years, a non-protein compound has been identified to bring about catalysis in
biological system. The name of the compound is
(a) DNA (b) RNA (c) Lipids (d) Carbohydrates.
 Methotraxate is a anticancer drug. It competitively inhibit folic acid. Which of the
following enzyme inhibited by this drug?
a) Dihdropteroate synthesis b) Dihydro folate reductase c) Xanthine oxidase
d) Lactate dehydrogenase e) Succinate dehydrogenase
 In obstructive liver disease, which of the following enzyme is elevated ?
a) Acid phosphatase b) Alkaline phosphatase c) Amylase d) AST e) ALT
 Which of the following isoenzyme of lactate dehydrogenase is elevated in
Myocardial infarction?
a) LDH1 b) LDH2 c) LDH3 d) LDH4 e) LDH5
 Allopurinol is drug used against gout which of following enzyme inhibited by this drug?
a) Succinate dehydrogenase b) Xanthine oxidase c) Creatinine Kinase d) Kinas
 Thiamine pyrophosphate (T.P.P) is the coenzyme. It is derived from which of the
following Vitamins?
a) Vit. B1 b) Vit. B2 c) Vit. B6 d) Vit. B12 e) Biotin

56.  Elevation of which of the following serum enzyme markers would be most useful in
diagnosing a myocardial infarction in a patient who comes to your office 3 days after
an episode of severe and prolonged substernal chest pain?
a. LDH isoenzymes b. CKMB c. Troponin I d. Myoglobin
e. Enzyme markers are no longer useful 3 days after an MI
 When [S] is equal to Km, which of the following condition exists ?
a) Half of the enzyme molecules are bound to substrate.
b) The velocity of the reaction is equal to Vmax
c) Enzyme is completely saturated with substrate
d) The reaction has reached the equilibrium
 "Lock& Key" model enzyme action proposed by Fischer implies that:
a) The active site is flexible& adjusts to substrate
b) The active site requires removal of PO4
c) The active site is complementary in shape to substrate.
d) All of them v. None of them
 In competitive inhibition of enzyme action :
i. Apparent Km is decreased ii. Apparent Km is increased.
iii. Vmax is increased iv. Vmax is decreased
 A person aged 48 years presented in CCU with history of retrosternal chest pain
radiating to left arm, breathlessness & sweating since 18 hours.
a) What is your possible diagnosis?
b) What enzymes would be raised in this patient?
c) Why are enzymes raised in certain functional diseases?
d) What other investigations will you suggest?
e) Along with treatment what will you advise this patient for future care?

57. Thank You