This document discusses enzymes and their properties. It defines enzymes as biocatalysts that are proteins synthesized by living cells. Enzymes lower the activation energy of chemical reactions and catalyze the formation of products. Enzymes can exist as single peptides or complexes of multiple subunits. They are classified based on the type of reaction catalyzed and have specific active sites that bind substrates. Many factors influence enzyme activity such as temperature, pH, substrate and inhibitor concentrations. The mechanisms of enzyme action and inhibition are also described.

1. DR.S.Sethupathy,M.D.,Ph.D
Rajah Muthiah Medical College,
Annamalai University

2. Enzymes – Biocatalysts, synthesized by
living cells
Proteins, colloidal, thermo labile, specific
in action
Naming : -ase was added to substrate
(suffix) -Lipid- lipase
Protein- protease, carbohydrate-
amylase (starch- amylose)

3.  Chemical reactions need an initial
input of energy to occur.
 Known as activation energy.
 Enzyme increases the rate of
chemical reaction by lowering the
activation energy
 ie: Free energy barrier that separate
reactants from the products.

4. Activation energy

5. More than one peptide - multimeric
enzymes or oligomeric enzymes
Eg : Lactate dehydrogenase( LDH)
(tetramer) e.g. creatine kinase (Dimer)
Multienzyme complexes
e proteins having more than one catalytic
domains (enzyme activities)
distinct parts of a polypeptide or subunits or
both.
Eg. Pyruvate dehydrogenase, fatty acid
synthase

6. Proenzymes
 Proenzymes are inactive enzymes
which are mostly proteolytic in
nature.
 Activated by certain agents on
structural modification
 e.g pepsinogen becomes pepsin by
HCl., trypsinogen is cleaved by
enteropeptidase to form trypsin.

7. Location
 Intracellular enzymes
 The enzymes are localized in specific
organelles e.g. enzymes of Kreb’s cycle are
in mitochondria.
 Extra cellular enzymes:
 These enzymes are synthesized by cells but
are active in the extracellular
environment .Eg: Digestive enzymes such
as trypsin, amylase , lipase , coagulation
factors etc.

8. Classification
IUBMB – basic principles for
nomenclature & classification named
by 4 digits(1:1:1:1)
Six classes
O T H L I L

9.  1. Oxidoreductases are involved
in oxidation, reduction reactions.
 e.g. alcohol dehydrogenase,
catalase etc.
 2. Transferases catalyze the
transfer of a functional group
from one substrate to another.
 e.g. Aspartate amino transferase,
Alanine amino transferase.

10.  3. Hydrolases catalyze the cleavage
of a substrate by the addition of water
.Eg: pepsin, amylase, glucose-6-
phosphatase.
 4. Lyases catalyze the removal of a
small portion of such as NH3, CO2 from
a large substrate molecule without
addition of water .e.g. pyruvate
decarboxylase , glutamate
decarboxylase

11.  5. Isomerases catalyze the intramolecular
rearrangement and interconversion of
optical, geometric or positional isomers.
 e.g. epimerases, mutases, isomerases
Phosphohexoisomerase catalyzes the inter
conversion of glucose-6-phosphate and
fructose -6-phosphate.
 6. Ligases or synthetases are involved in the
synthesis of a molecule by joining two
compounds together at the expense of ATP.
Eg: Acetyl CoA carboxylase combines acetyl
CoA and CO2 to form malonyl CoA for which
ATP is required.

12. Enzyme commission
(EC)number
 Four digits.
 specify enzyme-catalyzed reactions.
 First digit represents the class. (One to Six)
 Second digit represents the subclass.
 Third digit denotes sub-subclass or
subgroup.
 Fourth digit gives the particular enzyme.

13. Enzyme specificity
 Absolute substrate specificity:
 Enzyme will act on only one substrate
type and catalyze one reaction . Eg:
Urease acts on urea only
 Glucokinase acts on glucose only.

14. Relative substrate
specificity
 Group specificity
 Enzymes which act on specific group are called
group specific enzymes.
 Eg: Phosphatases are involved in the
hydrolytic removal of phosphate
 Chymotrypsin acts hydrolyzes only the
peptide bonds attached to aromatic amino
acid residues.


15. Bond specificity
Enzyme act on more than one
substrate containing a particular
kind of bond .
Eg: Salivary amylase acts only on α-
1,4-glycosidic bonds in any
carbohydrate, lipase acts on ester
bond in lipids.

16. Broad substrate specificity
 Enzyme acts on more than one
structurally related substrates.
 Eg: Hexokinase catalyzes the
phosphorylation of glucose,
fructose, mannose.

17. Stereo specificity
Enzyme acts only on
particular stereoisomer.
Eg: Human enzymes act on L-
amino acids and D-
carbohydrates.

18. Active site
 Active site of an enzyme is the region
of the enzyme where substrate binds
for catalysis
 Three dimensional, native
conformation of the enzymes is
required for catalysis
 Enzyme-substrate complex formation
is the prerequisite for catalysis.

19. Mechanism of enzyme action
 Formation of enzyme substrate complex is
the first step
 Lock and key model
 Fisher’s template theory
 Three dimensional structure of the active
site is complementary to the substrate
 similar to lock and key.
 This explains enzyme specificity but does
not explain the mechanism of regulation.

20. Induced fit model- Hand in
Glove model)
 Enzyme conformational changes are
induced by the initial binding of
substrate
 Active site can be modified by binding
of the substrate.
 This can explain the specificity and
regulation of enzyme activity by
allosteric modulators.

21. Allosteric site
 The sites on the enzyme other
than (catalytic site) active site are
called allosteric sites.
 The enzyme activity modulators
acting on allosteric sites are called
as allosteric modulators.

22. Multisubstrate reactions
 Simple enzymes will involve one substrate
binding to the enzyme and producing a
product plus the enzyme.
 But majority of enzymes are more
complex and catalyze reactions involving
multiple substrates.
 Binding of two substrates can occur
through two mechanisms:
 sequential mechanism and non-
sequential mechanism.

23. sequential mechanism
 single displacement reactions- both
substrates bind the enzyme to form a
ternary complex
 Reaction proceeds to form products
and are released from the enzyme.
 The reaction is bimolecular where the
second substrates such as NAD or
FAD are used.

26. Non-sequential mechanism
 also known as the "ping-pong" mechanism
 The enzyme acts like a ping-pong
ball, bouncing from one state to another.
 The characteristic of the ping-pong
mechanism is that one product is formed
and released before the second substrate
binds.
 Eg: Chymotrypsin, aminotransferases.

28. Co-factors
 Helper molecules for enzyme activity
 Can be either organic or inorganic
 If they are loosely bound to enzyme
then called as Coenzymes .
 Coenzymes mostly non-protein
organic , vitamins
 If tightly bound, then called as
prosthetic group

29.  Metal ion as the prosthetic group are
called as metalloenzymes .eg: Copper
in tyrosinase enzyme.
 enzymes require metal ions in the
medium for their activity are called as
metal activated enzymes. Eg: Calcium
ions activate pancreatic lipase.
 without the cofactor – inactive
protein part - called Apoenzyme
 Complete enzyme- apoprotein +
cofactor is the Holoenzyme.

30. Co-enzymes
 A non-protein organic substance
which is dialyzable, thermo-stable
and loosely attached to the protein
part.
 Most of the vitamins act as
coenzymes. Coenzymes act as
recyclable shuttles or group transfer
agents.

31. First group of coenzymes
 Involved in the transfer of hydrogen or
electrons in the reactions catalyzed by
oxidoreductases.
 Act as co-substrate or secondary
substrates
 Eg: Nicotinamide adenine dinucleotide
(NAD), flavin adenine dinucleotide (FAD)
and flavin mono nucleotide (FMN).

32. Second group of enzymes
 They are involved in the transfer of
groups other than hydrogen.
 Eg: Thiamine pyrophosphate
transfers hydroxyethyl group.
 Pyridoxal phosphate transfers amino
group.
 Biotin transfers carbon dioxide.

33. Pyruvate dehydrogenase
enzyme complex
 It requires five organic cofactors and one metal
ion.
 They are loosely bound thiamine
pyrophosphate (TPP)
 Covalently bound lipoamide and flavin adenine
dinucleotide (FAD)
 Co-substrates nicotinamide adenine
dinucleotide (NAD+) and coenzyme A (CoA)
 metal ion (Mg2+).

35. Mechanism of catalysis
 Catalysis by bond strain
 a strain is created in a particular
bond in substrate.
 This weakens the bond and
breaks it to form products. Most
of the lyases act in this way.

36. Bond strain

37. Proximity
More reactants
More collision
Faster reaction rate
=
Less reactants
Less collision
Slower reaction rate
=

38. Acid base catalysis

40. Acid base catalysis
 Proton donors and acceptors, i.e. acids and
bases, may donate and accept protons in
order to stabilize developing charges in the
transition state.
 Nucleophile (Negatively charged) and
electrophile (positively charged) groups are
involved
 Histidine can accept and donate protons.
Example Human Immunodeficiency
Virus(HIV) protease.

41. Covalent catalysis

42.  Covalent catalysis involves the
formation of a covalent bond
between the enzyme and the
substrates.
 It involves the substrate forming a
transient covalent bond with
residues in the active site or with a
cofactor
 Eg: chymotrypsin, trypsin,
 Schiff’s base formation

43. Schiff’s base

45. Factors influencing enzyme
activity
 Enzyme concentration
 Substrate concentration
 Product concentration
 Temperature
 pH
 Conezymes
 Activators or inhibitors

46. Factors affecting enzyme
activity
 Temperature
 Increasing temperature increases the
kinetic energy of the molecules.
 more random collisions and the
reaction velocity also increases.
 App. doubles for each 10 o
C which is
called temperature coefficient (Q10).

47.  Maximum activity is observed at optimum
temperature
 Declines there after due to denaturation of
proteins . So it gives a bell shaped curve.

48. pH
 Hydrogen ions interfere with
hydrogen and ionic bonds in active
site
 Influences enzyme action.
 Optimal pH is the pH at which the
charges in the active site are
appropriate for optimal activity.
 e.g. Opt.pH - 2 for pepsin , 6.8 for
salivary amylase, 9.4 for arginase

49. Optimum pH

50. Enzyme concentration
 Enzyme concentration
 The velocity of a reaction is directly
proportional to the amount of
enzyme
 substrate concentration is unlimited.
 This property is used for
determination of enzyme levels in
plasma, serum and tissues.

51. Enzyme concentration
Unlimited
Substrate
Velocity
Enzyme concentration

52. Substrate concentration
 Initial phase, velocity is directly
proportional to substrate concentration
(first order kinetics).
 Middle phase, velocity increases but not
proportionally to substrate concentration
and this is called mixed order reaction.
 Final phase, the active sites of enzyme are
saturated, there is no increase in rate
 Reaches a plateau (zero order kinetics).

53. Hyperbolic curve
 If the velocity of enzyme activity is
plotted against the substrate
concentration, initially the plot is
linear but later it is hyperbolic in
nature.
 Finally it reaches plateau and that is
velocity maximum (Vmax).

54. Substrate concentration

55. Initial velocity Vo
-Formula
 Vo = Vmax [S] / Km+[S]
 Vo- initial velocity
 [S] – substrate concentration
 Vmax – maximal velocity

56. Equilibrium
 Enzyme does not alter the
equilibrium
 Only increase the rate of
reaction and achieves
equilibrium quicker than un-
catalyzed reaction.

57. Enzyme molecules are saturated at higher
(s).
Further increase (s) have no effect on
velocity - Maximal velocity – Vmax
Michaelis-Menten theory
Enz-substrate complex
E + S ES E + P
↑ [S] - ↑ K1 & K3
k1
k2
k3

58. Michaelis – Menten constant (km)
V 0 = Vmax [s]
Km + [s]
If V = ½ Vmax then
½ Vmax = Vmax [s]
Km + [s]
Km + [s] = 2 Vmax [s] / Vmax
Km + [s] = 2 [s] So Km = [s]

59. Km or Michaelis –Menten
constant
 It is the substrate concentration (expressed
as moles/L) at half maximal velocity (1/2
Vmax).
 Every enzyme has the characteristic Vmax
and Km which are sensitive to changes in pH,
temperature and ionic strength.
 Km is characteristic feature of a particular
enzyme for a specific substrate.
 High km means low affinity for substate

60. Glucokinase and hexokinase
 Glucokinase- high km - 10 mmol/l – liver and
β-cells of pancreas – vmax –high – Glucose-6 p
does not inhibit. Inducible in liver insulin
controls. In diabetes , less activity. Maintains
blood glucose.
 Hexokinase- low km – 0.05mmol/l- Most
tissues- vmax – low - Glucose-6-p inhibits- not-
inducible- maintains intracellular glucose conc.-
 Deficiency causes hemolytic anemia.

61. Glucokinase / Hexokinase

62. Double reciprocal plot
 It is difficult and impractical to attain high
substrate concentrations to reach maximal
velocity.
 Km or half Vmax cannot be determined.
 Data as reciprocals at lower concentrations
can be plotted and extrapolated to get the
reciprocal of Vmax.
 It is Lineweaver – Burk plot or double
reciprocal plot.

63. Lineweaver-Burk plot

64. Michaelis Menten constant- Km

65. Enzyme Inhibition
Enzyme inhibitor - a
substance which binds with
the enzyme and decrease
the velocity by inhibiting
catalysis.

66. Enzyme inhibition
 Types of enzyme inhibition
1.Competitive enzyme inhibition
2.Uncompetitive enz. Inhibition
3.Non-competitive enz.inhibition
4.Suicide inhibition
5.Allosteric enz.inhibition

67. Competitive enzyme
inhibition
 The inhibitor resembles with the
substrate.
 It competes with substrate for active site
and inhibits product formation.
 Can be reversed by increasing substrate
concentration.
 In this type of inhibition,
 Vmax is unaltered
 Km is increased.

68. Competitive enzyme inhibition

69. Competitive enz.inhibition

70. Disulfiram inhibits aldehyde
dehydrogenase - competes for active
site

72. Enzyme Substrat
e
Inhibitor importance
Xanthine
oxidase
xanthine allopurinol Used in gout
Dihydro folate
reductase
FH2 Methotrexate Anticancer
drug
Transpeptidase D-ala-D-
ala moiety
of
peptides
Penicillin Antibiotic
Succinate
dehydrogenase
Succinate Malonate TCA cycle

74. Uncompetitive
enz.inhibition The inhibitor binds directly to the
enzyme substrate complex
 Does not bind to the free
enzyme.
 The inhibitor affects the catalytic
function but not its substrate
binding.
 Both vmax and km reduced

75. Uncompetitive
enz.inhibition

76. Uncompetitive inhibitors
 Enzyme- Liver alcohol dehydrogenase
 Methanol or ethylene glycol
poisoning
 Drugs -the inhibitors
 Tetramethylene sulfoxide
 3-butyl thiolene 1-oxide

77. Non-competitive enzyme
inhibition
 Bind to free enzyme or enzyme
substrate complex
 It affects both substrate binding and
catalysis.
 Vmax is decreased but km value is
usually not altered.

78. Non-compettive inhibition

79. Non-competitive
enz.inhibition

80. Non-competitive inhibitors
 Heavy metal ions Ag+
, Pb+
, Hg2+
react
with cysteinyl SH groups
 Iodoacetate inhibits enzymes having
sulphydryl group in the active centers.
 Cyanide inhibits cytochrome oxidase.
 Fluoride inhibits enolase.

82. Lead poisoning

83. Non-competitive
enz.inhibition Non-competitive inhibitors bind the
enzyme at allosteric sites.
 Not all inhibitors that bind at
allosteric sites are non-competitive
inhibitors.
 Vmax is reduced.
 Km valus is usually unaltered

86. Suicide inhibition
 Known as mechanism based inactivation
 It is irreversible .
 Structural analog is converted to a more
effective inhibitor with enzyme inhibited.
Eg: Allopurinol inhibits xanthine oxidase
 Allopurinol is oxidized by xanthine oxidase
and forms alloxanthine which is a strong
inhibitor of xanthine oxidase.

87. Suicide inhibition

88. Allosteric enzyme
modulation Low molecular weight substances bind to an
enzyme at allosteric site causing modulation
of enzyme activity.
 Allosteric modulation
 The substances are allosteric modifiers.
 1. Allosteric activators
 2. Allosteric inhibitors
 Eg. ADP is an allosteric activator of
hexokinase and ATP is allosteric inhibitor of
hexokinase..

89. Allosteric modifiers

91. Allosteric inhibition

92.  Sigmoid saturation kinetics due
to co-operativity
 there are K series and V series
enzymes.
 K series enzymes have altered km
but same V max.
 V series have altered V max but
same Km

93. Allosteric – Sigmoid curve

94. V series enzymes- v max altered
but km same-

95. K series enzymes- km altered but
v max same

96. Allosteric inhibitor

97. Feedback inhibition
 The enzyme is inhibited by the final
product of biosynthetic pathway .
 This is also called as end product
inhibition.
 The product acts at the allosteric site
of the first enzyme of the synthetic
pathway and causes inhibition.

98. Feed back inhibition

99. Regulation of enzyme
 By changing the enzyme quantity
 Synthesis and degradation - regulated
 Inducible enzyme synthesis is increased
by specific inducers
 They cause derepression by blocking the
repressors
 eg: Glucokinase is induced by insulin.

100. Changing the quality of enzyme
 Activity of enzymes may be increase
or decrease by covalent modification
 Zymogen activation by partial
proteolysis is an example irreversible
activation – digestive enzymes-
pepsinogen to pepsin , trypsinogen to
trypsin and coagulation factors

101. Enzyme degradation
 Enzyme degradation occurs by
 1.ATP and ubiquitin dependent
proteolysis
 2. ATP independent proteolysis.
 The susceptibility of enzyme to
proteolysis is dependent on the
presence of substrate, coenzyme and
metals.

102. Ubiquitin dependent
degradation

103. Addition or removal of a group
 Phosphorylation inactivates glycogen
synthese

104. ADP-Ribosylation

105. Cholera toxin

106. Ribozymes
RNA molecule removes introns
from primary mRNA transcript

107. Isoenzymes
 Isoenzymes are physically distinct forms
of the enzyme having same catalytic
activity.
 They differ from origin, structure, km value,
electrophoretic mobility, immunological
properties etc.
 But they catalyze the same reaction.
 Same type of subunits- homomultimer
 Different types of subunits- heteromultimer

108. Lactate dehydrogenase
 A tetramer and two types of sub units.
 ie: H (heart type), M (muscle type).
 In heart, LDH 1 - HHHH subunits.
 In muscle and liver LDH 5 - MMMM .
 In other tissues LDH 2 - HHHM.
 LDH 3 - HHMM
 LDH 4 - HMMM.
 In heart disease- serum LDH1 increased
 In liver disease, LDH 5 increased

109. Creatine phosphokinase – two
subunits – B-Brain M-Muscle – CK-MM-
muscle, Ck-MB –heart, CK-BB brain

110. Name Sources Increased In
1 Amylase Pancreas Pancreatitis
Salivary Gland Parotitis
2 Alkaline Phosphatase Bone Bone Disorders (Rickets)
Liver Obstructive Jaundice
3 Acid Phosphatase Prostate Prostatic Cancer
4 Creatine Kinase
(MB)
Heart Myocardial Infarction
5 Creatine Kinase
(MM)
Skeletal Muscle Muscular Dystrophy
6 Aspartate Transaminase
(AST)
Liver Hepatitis
Heart Myocardial Infarction
7 Alanine Transaminase
(ALT)
Liver Hepatitis
8 Lipase Pancreas Pancreatitis
9 5-nucleotidease hepatobiliary tree obstructive jaundice

111. Diagnsotic importance
Myocardial infarction
CPK-MB 6-18 hr 24-40 2-3 days
AST 12-24 hrs 48 hrs 4-5 days
LDH 24-36 hr 3-4 days 10-15 days

112. Myocardial infarction- serum markers

113. Liver diseases –
Viral hepatitis , cirrhosis of liver
Serum Alanine transaminase, Aspartate
transaminase, , LDH levels increased.
- Obstructive jaundice –
- Elevated Serum Alkaline phosphatase , 5
nuclestidase
- In Alcoholic liver disease, serum Gamma
glutamyl transpeptidase elevated.

114. Therapeutic enzymes
 Eg: streptokinase from
streptococci, is used for clearing
blood clots in myocardial
infarction.
 Tissue plasminogen activator is
also used for the same purpose

115. Thank you