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