everything about enzymes

1. ENZYMES
Dr. KHUSHBU SONI
Assistant Professor
AIMS,Dewas

2. o Enzymes are biological catalysts that speed up the rate
of the biochemical reaction without undergoing
permanent change in overall process.
o Substrate is reactant on which enzyme acts and converts
it into a product.
o Zymogens (pro enzyme) are inactive form of enzymes.
E.g. proelastase, chymotrypsinogen

3. Nomenclature of enzymes
Enzymes are classified by two ways
Recommended name systematic name
o Trivial name (trypsin, pepsin)
o Convenient
o Easy to everyday use
o Suffix –ase is used after substrate
e.g. lactase, urease
o after type of reaction
e.g. oxidase

4. A systematic classification of enzymes has been developed
by International union of biochemistry.
This classification is based on the type of reactions
catalyzed by enzymes.
There are six major classes.

5. Enzyme Code (E.C.) = Four Digits
1. First (main class)= Type of Reaction
2. Second (subclass) = Type of Group involved
3. Third (sub-subclass) = denotes Substrate
4. Fourth = Individual enzyme name & serial number
E.C. 1. Oxidoreductases
E.C. 2. Transferases
E.C. 3. Hydrolases
E.C. 4. Lyases
E.C. 5. Isomeraes
E.C. 6. Ligases

6. 1. Oxidoreductases
Catalyzes a variety of oxidation-reduction reaction
With help of NADH, NADPH , FADH2 , FMN
Common examples
Dehydrogenases - Oxidases
Peroxidases - Reductases

7. 2. Transferases
Catalyzes transfer of functional group from one molecule
to another.
 Carboxyl
 Methyl
 Acyl
 Glycosyl
 amino
 Kinase transfer of Phosphate group

9. 3. Hydrolases
Cleavage of C-C, C-O, C-N & other Covalant bonds
By addition of water.
Example
Protease (Trypsin, Chymotrypsin, Pepsin ,Collagenase)
Amylase
Lipase
Phosphatase
Urease

11. 4. Lyases
Removal of group from substrates or break bonds
by mechanism other than hydrolysis or oxidation.
Example
Aldolase
Enolase
Fumarase
Arginosuccinase
Pyruvate decarboxylaes
HMG CoA lyase

13. 5. Isomerases
 produce Optical or Geometric isomer of substrate
Example
Racemases
Epimerases
Triose phosphate isomerase
Mutase

14. 6. Ligases
Link two substrate Usually with help of ATP
Example
Synthetase
Pyruvate carboxylase
DNA Ligase

15. Co-factors and Co-enzymes
Some enzymes require molecules other than proteins
for their action.
Apoenzyme = Enzyme (Protein moiety) = inactive
Holo-enzyme = Apoenzyme + Non-protein component

16. Non-protein component
Inorganic metal ion Organic molecule
Co-factor Co-enzyme
Metalloenzymes
e.g. Zn++ - carbonic anhydrase
Mg++ - hexokinase
Prosthetic groupCo-substrate

17. Co-substrate Prosthetic group
o When co-enzyme is loosely
bound with enzyme
o After reaction, dissociates
in altered state
o Need to be recycled by different
reaction
o e.g. NAD+
o When co-enzyme is tightly
bound with enzyme
o After reaction, returns to
original form
o E.g. FAD

19. The term Apoenzyme is applicable to:
a) Simple enzyme
b) Protein part of conjugate enzyme
c) Organic co-factor of a conjugate enzyme
d) Inorganic co-factor of a conjugate enzyme

20. Zymogen is:
a) Enzyme modulator
b) Enzyme inhibitor
c) Enzyme precursor
d) Enzyme poison

21. Pyruvate carboxylase is:
a) Kinase
b) Lyase
c) Transferase
d) ligase

23. Pyruvate decarboxylase is:
a) Oxido-reductase
b) Hydrolase
c) Ligase
d) Lyase

24. Characteristics of enzymes
o Most enzymes are three dimensional globular proteins
(tertiary and quaternary structure).
o Some special RNA species also act as enzymes and are
called Ribozymes
o Water soluble
o Not consumed during reaction

25. o Their presence does not effect the nature and properties of
end product.
o Enzymes are sensitive to change in pH, temperature and
substrate concentration
o Active site: special pocket or cleft that binds
substrates, co-factors and prosthetic groups and
contains residue that helps to hold the substrate.
o generally occupy less than 5% of the total surface
area of enzyme.

26. o has a specific shape due to tertiary structure of protein.
o Contains substrate binding site and catalytic site.
o Contains amino acid side chains involved in substrate
binding and catalysis called as “catalytic residues”
o Binding occurs by non covalent forces.
o A change in the shape of protein affects the shape of active
site and function of the enzyme.

28. Catalytic efficiency
enzyme catalyzed reactions are 1000 times faster than
uncatlayzed one.
Turnover number
defined as the number of substrate molecules transformed
per second by one enzyme molecule.
Action of enzyme can be regulated depending on the
production need of cell.
Cellular location: some localized in specific organelles, some
are secreted out of the cell.

29. Specificity: ability of enzyme to discriminate between two
competing substrates.
types
reactionsubstrate stereo
broadrelativeabsolute
bondgroup
e.g. urease e.g. hexokinase
e.g. alpha-amylasee.g. trypsin
L-lactate dehydrogenase

30. Lock and key model
o Proposed by EMIL FISCHER.
o Lock and key hypothesis assumes the active site of an
enzymes are rigid in its shape.
o There is no change in the active site before and after a
chemical reaction.

32. Koshland’s Induced Fit Theory
According to this theory, exposure of an enzyme to substrate
cause a change in enzyme, which causes the active site to
change it’s shape to allow enzyme and substrate to bind.

33. Reactions have an energy barrier
That energy barrier separate substrates and products.
It is difference between energy of the reactants and a high-
energy intermediate that occurs during the formation of
product.
Energy barrier = free energy of activation
Mode of action of enzymes

35. Rate of Reaction
 To reach transition state
 Substrate must contain sufficient energy.
 Enzyme
 Rate of reaction is determined by the number of such
energized molecules.
In general, enzymes…
1. Lower the free energy of activation
2. More molecules have sufficient energy to pass
3. Easily reach to transition state
4. Faster the rate of the reaction.

37. Enzyme enhances rate of a biochemical reaction, as it:
a) Increases activation energy
b) Decreases activation energy
c) Increases substrate concentration
d) None of above

38. Mechanism of enzyme catalysis
Catalysis by
proximity
Metal ion
catalysis
Covalent
catalysis
Acid base
catalysis

39. Catalysis can occur through proximity
and orientation effects
o Enzymes are usually much bigger than their substrates
o By oriented binding and immobilization of the substrate,
enzymes facilitate catalysis by:
1. bring substrates close to catalytic residues
2. Binding of substrate in proper orientation
3. Stabilization of transition state by electrostatic interactions

40. Substrate stabilization in Transition state
The active site acts as a flexible molecular template.
Binds the substrate in a geometrically favorable manner.
And activate transition state of the molecule
By stabilizing the substrate in its transition state, the
enzyme increases the concentration of the reactive
intermediate.
That can be converted to product.

41. Visualization of Transition state
Conversion of substrate to product can be visualized as
being similar to removing a sweater from an uncooperative
infant.
We can en-vision a parent acting as an enzyme.
Parent comes in contact with the baby (forming ES)
Guide baby's arms to remove sweater. (ES transition
state)
Guidance (conformation) = facilitate the process.
Removal of Sweater + Disrobed baby (Product)

43. o Enzymes contain catalytic residues at their active site
o Side chains of amino acids offer a variety of
nucleophilic centers for catalysis
o Can form temporary covalent bond with substrate
molecule
o Enzyme-substrate intermediate
o At the end of reaction, the covalent bond must be
broken to regenerate enzyme.
Covalent catalysis

46. Acid-base catalysis
o Active site may contains residue like histidine
o Participate in hydrogen ion transfer,
o by transferring hydrogen ion, the active site may:
• Activate nucleophiles required in catalysis
• Stabilize charged groups
• Facilitate electrostatic interactions that may
stabilize transition state

50. Metal ion catalysis
o Metal ions like Zn, Mg, Fe etc.. are used as co-factor
by various enzymes.
o Metal atoms lose electron easily and exist as cations
o The positive charges on metal ions allow them to:
• Stabilize transient and intermediate structures in
the reaction
• Assist in forming strong nucleophilic group
• Hold the substrate inside the active site

51. Carbonic anhydrase

53. Factors Affecting Enzyme Reaction

54. 1. Substrate concentration
Rate of reaction increases with substrate concentration
Until Vmax is reached.
At high conc. of substrate = enzyme full saturated with
substrate.

56. 2. Temperature
 Maximum reaction velocity at Optimum temperature.
 Optimum temperature for most human enzymes is 35° -
40°C.
 Human enzymes start to denature above 40°C temperature.

57. Effect of Temperature on Enzyme activity

58. 3. pH
o Concentration of H+ affects active site
o So Velocity reaction affected
o Change in pH can denature enzyme
o Optimum pH is different for different enzyme.

59. What change can occur at active site, because of
change in pH?

60. Effect of pH
 If the pH changes much from the optimum
 Chemical nature of the amino acids can change.
 Change in Ionization of amino acid at active site.
 Result in a change in the bonds.
 Active site will be disrupted.
 Enzyme will be denatured.

61. Different enzyme with it’s optimum pH

62. 4. Enzyme concentration
 Rate of the reaction is directly proportional to the
enzyme concentration at all substrate
concentrations.

64. 5. Product concentration
 As product concentration increases, enzymatic
reaction slow down.
 Higher Product concentration Inhibits reaction.

66. 6. Enzyme activation
 Activation by co-factors.
 In presence of certain metallic ions, some enzyme
shows higher activity.
 Salivary amylase = chloride
 Lipase = calcium
 Conversion of an enzyme precursor.
 Specific proteolysis is a common method of activating
enzymes and other proteins in biological system.

67. Zymogen activation by proteolytic cleavage

70. Velocity &Vmax of reaction
 Rate or Velocity of a reaction (V) is the number of
substrate molecules converted to product per unit time.
Vmax is the maximum velocity of the reaction.
Expressed as µmol of product formed per minute.

73. Michaelis-Menten Equation
Reaction model
 Leonor Michaelis and Maude Menten
 In this model,
 Enzyme reversibly combines with its substrate
 Form an ES complex
 Subsequently yields product
 Regenerating the free enzyme.

74. where:
 S is the substrate
 E is the enzyme
 ES-is the enzyme substrate complex
 P is the product
 K1,K-1 and K2 are rate constants

75. Michaelis-Menten Equation

76. Km (Michaelis constant)
It is the [S] for achieving half of the Vmax.
Km = Substrate concentration at ½Vmax.
Reflects the affinity of the enzyme for
substrate.

77. Small Km
 High affinity of the enzyme for substrate.
 Because a low concentration of substrate is needed to
reach ½Vmax of velocity.
Large Km
 Low affinity of enzyme for substrate
 Because a high concentration of substrate is needed to
reach ½Vmax of velocity.

80. Assumptions in the Michaelis-Menten equation
Relative concentrations of E and S
 [S] is much greater than [E], so that the percentage of
total substrate bound by the enzyme at any one time is
small.
Steady-state assumption
 [ES] does not change with time (the steady-state
assumption).
 The rate of formation of ES is equal to that of the
breakdown of ES (to E + S & to E + P).

81. Initial velocity
Initial reaction velocities (Vo) are used in the analysis of
enzyme reactions.
This means that the rate of the reaction is measured as soon
as enzyme and substrate are mixed.
At that time, the concentration of product is very small and,
therefore, the rate of the back reaction from P to S can be
ignored.

85. ENZYME REGULATION

86. Allosteric
regulation
Reversible
covalent
modification
Proteolytic
cleavage
Induction and
repression

87. o It permits changing needs of the cell to meet its energy
and resource demands.
o If a product is available in excess, enzyme regulation
could then divert the resources to other needy reactions.
Why?

88. o Regulatory enzymes : in a multi-step enzymatic process,
there will be one enzyme which will be responsible for
overall rate of that process.
o Key enzyme or rate limiting enzyme
o Can be affected by signal molecules

89. Allosteric regulation
o Allosteric enzymes are a class of regulatory enzymes.
o Large and composed of many subunits.
o Contains allosteric site different from active site.
o Regulatory molecules bind at allosteric site.
o Can be affected by regulatory molecules = allosteric
effectors (modulator)

90. o Binding can enhance or reduce enzyme activity.
o Modulator may have positive effect or negative effect.
o Two types of allosteric enzyme based on nature of
modulator:
Homotropic allosteric enzymes
Heterotopic allosteric enzymes

93. o Typically, allosteric regulation occurs via FEEDBACK
mechanisms.
o Negative feedback positive feedback
o Allosteric enzymes show variation in kinetics.
o They do not follow michaelis menten kinetics.
o They show sigmoidal curve instead of hyperbolic curve
when velocity [v] is plotted against [s].

95. Positive feedback

97. Covalent modification
o Enzyme activity may be regulated by reversible covalent
modification.
o Separate enzymes are used to add or to remove modifying
groups.
o Phosphorylation is the most common type.
o Addition of phosphate group to Ser,Tyr,Thr.
o ATP and GTP donates phosphate.

101. Zymogen activation by proteolytic cleavage

102. Induction and Repression
Regulate the amount of enzyme.
Act at Gene level.
Altering rate of enzyme synthesis.
Increase enzyme synthesis = Induction
Decrease enzyme synthesis = Repression
Induction / Repression = Slow (hours to days)
Allosteric regulation = Fast (seconds to minutes)

103. Feedback regulation

105. ISOENZYMES
Catalyze the same reaction
Two or more polypeptide chains
Different polypeptide chains are products of different genes
Differ in AA sequence and physical properties
Separable on the basis of charge
Are tissue specific
“They are physical distinct forms of the same enzyme ”
Different allosteric effectors and different kinetics

107. Type
of
LDH
Composition Location
LDH 1 HHHH Myocardium
LDH 2 HHHM RBC
LDH 3 HHMM Lung
LDH 4 HMMM Kidney &
Pancreas
LDH 5 MMMM Skeletal muscle
& Liver

109. Creatine Kinase - Dimer
Type of CK Composition Location
CK- 1 (CK-BB) BB Brain
CK- 2 (CK-MB) MB Myocardium
CK- 3 (CK-MM) MM Skeletal
Muscle

111. Identification of Isoenzymes
1. Electrophoresis
2. Heat stability : BALP
3. Inhibitors
4. Substrate specificity (Km value)
o e.g. Hexokinase & Glucokinase
5. Cofactor requirement
o e.g. Mitochondrial ICD – NAD+ dependent
Cytoplasmic ICD – NADP+ dependent
6. Tissue location
7. Specific antibody

112. Isoenzymes of Alkaline Phosphatase
Depending on number of sialic acid residue
1. Alpha – 1 ALP (10%) Biliary Canaliculi
2. Alpha – 2 heat labile ALP (25%) Hepatic cells
3. Alpha – 2 heat stable ALP (1%) Regan Isoenzyme
Placental cell
4. Pre – beta ALP (50%) Bone disease
5. Gamma – ALP (10%) Intestinal cells
6. Leucocyte ALP Leucocyte

114. Organ Specific Enzyme
Heart CK-MB , AST (GOT) , LDH
Liver ALT , AST , LDH , Alkaline Phosphatase
Gamma Glutamyl Transferase
Pancreas Lipase ,Amylase
Muscle Aldolase , CK-MM , CK-Total , AST
Bone Alkaline Phosphatase
Prostate Acid Phosphatase
(Prostate isoform – inhibited by Tartrate)
RBC LDH
Acid Phosphatase (Erythrocyte isoform –
inhibited by formaldehyde & cupric ion)

116. Principal SourcesDiagnostically Important Enzyme
LiverAlanine aminotransferase(ALT)
Liver, Gall Bladder, Erythrocytes
Skeletal muscle, Heart, Kidney,
Aspartate aminotransferase(AST)
I (cytosol) & II (mitochondria)
Hepatobilliary tract, KidneyGamma Glutamyl Transferase
Hepatobilliary tract5’ Nucleosidase
Bone, Gall Bladder ,Liver,
Intestinal mucosa, Placenta,
Kidney
Alkaline Phosphatase (ALP)
Prostate, ErythrocytesAcid Phosphatase
Pancreas ,Salivary glands, OvariesAmylase
PancreasLipase

117. Enzyme as Therapeutic Agents
1. Streptokinase & Urokinase
• Lysis of intravascular clot
• Use in myocardial infarction
2. Asparaginase
• Used as anticancer drugs.

118. 1. Glucose oxidase & Peroxidase (GOD-POD)
2. Urease
3. ELISA test
4. Restricted Endonuclease
Enzyme as Diagnostic Agents