1. Enzymology: Factors affecting
enzyme activity& Kinetics
Dr. Rohini C Sane

2. Factors affecting enzyme activity
1. Concentration of enzymes
2. Concentration of substrate
3. Concentration of products
4. Temperature
5. p H
6. Activators (Coenzymes & Cofactors )
7. Inhibitors
8. Time
9. UV light & Radiations
10. Oxidizing agent
11. Anti-enzymes

3. Factors affecting enzyme activity: concentration of enzymes
Rate of reaction directly
proportional to concentration of
enzymes (when p H ,temp
,substrate concentration etc are
constant )
Clinical application
1. Determination of enzyme
concentration
2. For diagnosis of inborn errors
in metabolism

4. Factors affecting enzyme activity: Concentration of Product
Factors affecting enzyme activity: Concentration of Product
1. E+ S P+ E
2. With increase in product concentration velocity of reaction decreases
3. Feed back mechanism
4. Inborn errors of metabolism: there is increase in substrate
concentration in plasma & product concentration decreases
5. eg Phenylketonuria ( Phenylalanine  Tyrosine- catalyzed by
Phenylalanine Hydroxylase. Enzyme is inhibited /absent
concentration of Serum Phenylalanine increases & concentration
Serum Tyrosine decreases ) A-- - B

5. Factors affecting enzyme activity: concentration of product

6. Factors affecting enzyme activity: Concentration of products
Increase in Concentration of products ( after saturation of enzyme
molecules ) decrease in velocity of reaction  Feed Back Inhibition

7. Factors affecting enzyme activity: concentration of Substrate
• 8 E + 2S ↔ 2 ES  2P + 8E ( 1HR )
• 8 E + 4S ↔ 4 ES  4P + 8E ( 1/2HR )
• 8 E + 8S ↔ 8 ES  8P + 8E ( 1/4HR )
SATURATION---------------------------------------------------------------
• 8 E + 10S ↔ 8 ES  8P + 8E ( 1/4HR )
• 8 E + 12S ↔ 8 ES  8P + 8E ( 1/4HR )
• *( Rate of formation of 2P )

8. (S ) –Substrate Concentration
V ₒ -- Initial Velocity
K m -- Michelis Menten constant
Vmax –maximum velocity
Factors affecting enzyme activity: concentration of Substrate

9. Line Weavers Burk Equation-Double Reciprocal Graph
1/VO =Km /Vmax (1/S ) + 1/Vmax
Y = mx + c ( slope = Km /Vmax )
When 1/S =0 S=∞
1/Vₒ = 1/V max
Advantage : (exact Vmax )

10. Michaelis Menten equation
• V = initial velocity
• Vmax = maximum velocity
• Km = Michaelis–Menten constant
• S = substrate concentration
Importance of Michaelis Menten equation :
1. relates substrate concentration with reaction velocity
2. Quantitative calculations of enzyme characterization
3. Analysis of enzyme inhibition
The plot provides a useful graphical method for analysis othe Michaelis–Menten equation:

11. Enzyme Kinetics
Increase in rate of reaction is observed when :
(1)increasing internal energy ( activation energy) by increase in
temperature  increase in molecules in transition state
(2) cell when uses of enzyme ,there is
a) Decrease in activation energy
b) Increase in number of substrate in transition state
c) Increase in rate of reaction

12. Rate of enhancement by Enzymes
• Chemical reaction
Substrate transition state product
Number of collision α rate of reaction
Transition state : reactive form
Activation energy : of the reaction is the amount energy in calories
required to bring all molecules in one molar of substrate at a given
temperature to transition state (at the top of energy barrier ).

13. Rate enhancement by enzyme in catalyzed reaction
Number of substrate molecule Internal energy ( kcal )
20 * √ 150
50 √ 60
30 10
Activation energy (Uncatalyzed reaction) 130
Transition state 130
Number of substrate molecules converted into product
Catalyzed reaction
Activation energy
Number of molecules in transition state = number of
substrate molecules into product
20*
50
70 √

14. Energy diagram for chemical reaction

15. Activation energy for
Catalyzed &
uncatalyzed
reaction

16. Factors affecting enzyme activity: Temperature
Each Enzyme has optimum temperature
Q 10 = 10 ⁰C reaction rate doubled
HIGHER TEMPERATURE CAUSES DENATURATION
OPTIMUM TEMPERATURE FOR MOST BODY ENZYMES
= 37 ⁰C
OPTIMUM TEMPERATURE FOR UREASE =60 ⁰C

17. Factors affecting enzyme activity: p H
Optimum p H : Pepsin -1-2 ,Glucose 6-phosphtase -7.2 ,Alkaline Phosphatase- 11

18. Factors affecting enzyme activity: p H
Optimum p H :Pepsin -1-2 ,Urease -6.5 ,Trypsin-7.5

19. Factors affecting enzyme activity : presence of Oxidizing agent
Oxidation by oxygen or by oxidizing agents
E SH + ½ O2 E S + H2O
SH S
E S + 2 R-SH ↔ E SH + R –S-SH
S SH
Reduced Sulph-hydryl group is contributed by Cysteine or Glutathione

20. Factors affecting enzyme activity: Radiation
X rays ,Beta or Gamma rays ---( high energy rays ) peroxides + E
↓
OXIDIZED ENZYMES
↓
LOSS OF ENZYME ACTIVITY
↑
LOSS OF GENE EXPRESSION

21. Factors affecting enzyme activity: Anti –enzymes
• Serum containing Anti enzymes /Antibodies against enzymes eg
Anti -Trypsin, Anti –Pepsin  decreased /loss of activity

22. Use of Anti enzyme in treatment of Myasthenia Gravis

23. Definition of Cofactors
& Coenzymes
Factors affecting enzyme activity: Co-enzyme & Cofactors (Activators )

24. Factors affecting enzyme activity: co-enzyme & cofactors

25. Comparison of Cofactors & Coenzymes

26. Coenzyme forms of Vitamin B & their functions
Vitamin Activated form- (coenzyme ) Type of catalysis Enzyme using co -
enzyme
Thiamine Thiamine Pyrophosphate(TPP ) Aldehyde or Keto Group Trans- Ketolase
Riboflavin Flavin Mono Nucleotide (FMN ) Hydrogen or Electron L -Amino oxidases
Riboflavin Flavin Adenine Dinucleotide
(FAD )
Hydrogen or Electron D -Amino oxidases
Niacin Nicotinamide Adenine
Dinucleotide (NAD )
Hydrogen or Electron LDH
Niacin Nicotinamide Adenine
Dinucleotide Phosphate (NADP )
Hydrogen Or Electron G-6 P-D
Lipoic
Acid
Lipoic Acid Hydrogen Or Electron Pyruvate Dehydrogenase
Complex

27. Coenzyme forms of Vitamin B & their functions
Vitamin Activated form-
(coenzyme )
Type of catalysis Enzyme using coenzyme
Pyridoxine Pyridoxal Phosphate Amino Group Transfer Alanine Transaminase
Pantothenic Acid Coenzyme A Acyl Group Transfer Thio Ketolase
Folic Acid Tetra Hydro Folate (TFH4 ) One Group Transfer-
formyl, Methyl
Formyl Transferase
Biotin Biotin CO2 Pyruvate Carboxylase
Cobalamine Methyl Cobalamine Methyl Malonyl Co A Mutase

28. VITAMINS AND COENZYMES
Vitamin Coenzyme Reaction type Coenzyme class
SOURCE: Compiled from data contained in Horton, H. R., et al. (2002). Principles of Biochemistry , 3rd edition. Upper Saddle River, NJ:
Prentice Hall.
B 1 (Thiamine) TPP Oxidative decarboxylation Prosthetic group
B 2 (Riboflavin) FAD Oxidation/Reduction Prosthetic group
B 3 (Pantothenate) CoA - Coenzyme A Acyl group transfer Cosubstrate
B 6 (Pyridoxine) PLP
Transfer of groups to and from
amino acids
Prosthetic group
B 12 (Cobalamin) 5-deoxyadenosyl cobalamin Intramolecular rearrangements Prosthetic group
Niacin NAD + Oxidation/Reduction Cosubstrate
Folic acid Tetrahydrofolate One carbon group transfer Prosthetic group
Biotin Biotin Carboxylation Prosthetic group
Read more: http://www.chemistryexplained.com/Ce-Co/Coenzyme.html#ixzz3oL0qkSi1

29. Factors affecting enzyme activity: Cofactors (activators )
Cofactor –inorganic ion Enzymes
Fe 2 ⁺ ,Fe3 ⁺ Peroxidase
Cu ⁺ ⁺ Cytochrome oxidase
Mg ⁺⁺ Hexokinase
Ni⁺⁺ Urease
Mn ⁺⁺ Arginase
K ⁺ Pyruvate Kinase
Zn ⁺ ⁺ DNA Polymerase
Mo⁺⁺ Nitrate Reductase
Se Glutathione Peroxidase
Ca ⁺ ⁺ Lipase
Cl⁻ Salivary Amylase

30. Factors contributing catalytic efficiency
1. Proximity & orientation of substrate in relation to catalytic group
2. Strain & orientation of the susceptible bond by induced fit of enzymes
3. General acid base catalysis
4. Covalent catalysis

31. Effects of proximity &
orientation-
enhancement of
catalytic efficiency of
enzymes

32. Effects of proximity &
orientation-increased
catalytic efficiency of
enzymes

33. Proximity & Orientation of substrate in relation to catalytic group
• Orientation : unfavorable
• Proximity : unfavorable no product formation
• Orientation : unfavorable
• Proximity : favorable no product formation
• Orientation :favorable
• Proximity : favorable product formation
ES has lower activation energy as ES IN TRANSITION STATE

34. Induced fit
hypothesis
Of enzyme
catalysis –
Transition state
stabilization
leads to rate
enhancement

35. Induced fit model of enzyme catalysis: change in three dimensional structure of enzyme &
substrate is induced on binding of substrate to enzyme active site

36. Induced Fit ,Strain & Distortion
Relaxed substrate molecule +Relaxed enzyme conformational
change  strain form of substrate molecule
1. Strain active site
2. Distortion of substrate
3. Conformational leverage on substrate
• NESSECIATES: ENZYME LARGE & PROTEIN MOLECULE

37. INDUCED FIT OF ENZYME CATALYSIS

38. INDUCED FIT OF ENZYME CATALYSIS

39. INDUCED FIT OF ENZYME CATALYSIS/INHIBITION

40. Acid Base catalysis
Proton is transferred
From amino acid of
enzyme to substrate
This results in
lowering activation
energy or stabilization
in transition state.

41. Covalent
Catalysis-
Covalent
linkage
between
amino acid
from active
site of enzyme
& substrate .
This results in
lowering
activation
energy or
stabilization in
transition
state.

42. Covalent Catalysis-
Products have lower
affinity for enzyme active
site & are therefore
released.
Enzymes are set free
at end of reaction
( Completion of catalysis )
in unaffected form

43. Covalent intermediates of Enzymes in Covalent catalysis
Class enzyme
Serine Class Phospho Glucomutase Phospho Enzyme
Serine Class Trypsin Acyl Enzyme
Serine Class Chymotrypsin Acyl Enzyme
Serine Class Acetyl Choline Esterase Acyl Enzyme
Cysteine Class Pepsin Acyl Enzyme
Cysteine Class Glyceraldehyde Phosphate
Dehydrogenase
Acyl Enzyme
Histidine Class Glucose 6 Phosphatase Phospho Enzyme
Histidine Class Succinyl –Coa Synthtase Phospho Enzyme
Lysine Class Trans Aldolase Schiff Base
Lysine Class Amino Acid Oxidase Schiff Base

44. Comparison between Acid- Base catalysis ,Covalent catalysis & Metal ion catalysis