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Enzymology: Factors affecting enzyme activity& Kinetics
Dr. Rohini C Sane
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
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
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
Factors affecting enzyme activity: concentration of product
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
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 )
(S ) –Substrate ConcentrationV ₒ -- Initial VelocityK m -- Michelis Menten constant Vmax –maximum velocity
Factors affecting enzyme activity: concentration of Substrate
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 )
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:
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
Rate of enhancement by Enzymes • Chemical reaction
Substrate transition state productNumber of collision α rate of reaction Transition state : reactive formActivation 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 ).
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 reactionActivation energy Number of molecules in transition state = number of substrate molecules into product
20*
50 70 √
Energy diagram for chemical reaction
Activation energy forCatalyzed & uncatalyzedreaction
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 ⁰COPTIMUM TEMPERATURE FOR UREASE =60 ⁰C
Factors affecting enzyme activity: p H
Optimum p H : Pepsin -1-2 ,Glucose 6-phosphtase -7.2 ,Alkaline Phosphatase- 11
Factors affecting enzyme activity: p H
Optimum p H :Pepsin -1-2 ,Urease -6.5 ,Trypsin-7.5
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
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
Factors affecting enzyme activity: Anti –enzymes • Serum containing Anti enzymes /Antibodies against enzymes eg
Anti -Trypsin, Anti –Pepsin decreased /loss of activity
Use of Anti enzyme in treatment of Myasthenia Gravis
Definition of Cofactors& Coenzymes
Factors affecting enzyme activity: Co-enzyme & Cofactors (Activators )
Factors affecting enzyme activity: co-enzyme & cofactors
Comparison of Cofactors & Coenzymes
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
LipoicAcid
Lipoic Acid Hydrogen Or Electron Pyruvate Dehydrogenase Complex
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
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
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
Factors contributing catalytic efficiency1. 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
Effects of proximity & orientation-enhancement of catalytic efficiency of enzymes
Effects of proximity & orientation-increased catalytic efficiency of enzymes
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
Induced fit hypothesis Of enzyme catalysis –Transition state stabilization leads to rate enhancement
Induced fit model of enzyme catalysis: change in three dimensional structure of enzyme & substrate is induced on binding of substrate to enzyme active site
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
INDUCED FIT OF ENZYME CATALYSIS
INDUCED FIT OF ENZYME CATALYSIS
INDUCED FIT OF ENZYME CATALYSIS/INHIBITION
Acid Base catalysis Proton is transferred From amino acid of enzyme to substrateThis results in lowering activation energy or stabilization in transition state.
Covalent Catalysis-Covalent linkage between amino acid from active site of enzyme & substrate .This results in lowering activation energy or stabilization in transition state.
Covalent Catalysis-Products have lower affinity for enzyme active site & are therefore released.Enzymes are set freeat end of reaction ( Completion of catalysis ) in unaffected form
Covalent intermediates of Enzymes in Covalent catalysisClass 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
Comparison between Acid- Base catalysis ,Covalent catalysis & Metal ion catalysis