Download MBBS Biochemistry PPT 9 Enzyme Kinetics Lecture Notes

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Enzyme Kinetics

Enzyme Inhibition

Drugs utilizing kinetics and inhibition and its clinical utility

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Enzyme Kinetics

the quantitative measurement of the rates of enzyme-catalyzed reactions

and the systematic study of factors that affect these rates

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Catalysts

Increase rate of reaction by factor of 106

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Highly selective and specific

Not changed as a result of catalysis

Does not change the equilibrium constant

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Enzymes Alter Only the Reaction Rate

and Not the Reaction Equilibrium
Factors affecting reaction velocity

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Substrate concentration
The rate of an enzyme-catalyzed reaction increases with substrate

concentration until a maximal velocity (Vmax) is reached

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Temperature

Bell shaped curve

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Increase of velocity with temperature

Decrease of velocity with higher temperature
pH

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Bell shaped curve

Effect of pH on the ionization of the active site

Effect of pH on enzyme denaturation

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Variable pH optimum

Kinetic Order of Reaction

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Sum of the molar ratios of the reactants defines the kinetic order of the

reaction

First order

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Second order

Pseudofirst order reaction
Michaelis Menten Equation

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Vmax

1/2Vmax

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[Vo]

Km

Relationship between initial velocity and substrate concentration

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[S]

Km and its importance

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The Michaelis constant Km is the substrate concentration

at which Vi is half the maximal velocity (Vmax/2) attainable
at a particular concentration of the enzyme.

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Unit?

Reflects the af inity of the enzyme for that substrate:

inverse relationship

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Specific for enzyme substrate combination

Order of reaction
Clinical importance of Km

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Hexokinase vs Glucokinase

Line-Weaver Burk plot

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Double Reciprocal Graph

Linear curve

1

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=

Km

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+ 1

V0

Vmax [S]

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Vmax

Linear form of Michaelis menten equation to
Determine Km and Vmax.

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Units of Enzyme activity

Amount of substrate converted to product per unit time under standard

conditions of pH and Temperature

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IUB unit: Katal (?mol/min)

SI Unit: (mol/sec)

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Relative activities of Enzymes

Specific activity (Vmax/protein concentration): Impure Enzymes

Turnover number(Vmax/moles of enzyme): Homogenous Enzymes

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Catalytic constant, Kcat [Vmax/No. of active sites(St)]: unit time-1

Catalytic efficiency: Kcat/Km (Carbonic anhydrase, ADA,

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acetylylcholinesterase)
Two substrate Reactions(Bi Bi

Reactions)

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Sequential

:

Random

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Ordered

Ping Pong:

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Double displacement reactions

Two substrate Reactions

Random

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Ordered/Sequential

Ping Pong/Double Displacement
Enzyme Inhibition

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Types of Inhibitions based on kinetics

Competitive Inhibition

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Non-Competitive Inhibition

Un-Competitive inhibition
Competitive Inhibition

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Binding at substrate binding site

Inhibitor similar to substrate

Km increased

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Vmax same

Clinical Application/Drugs

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Statin Drugs

Competitive Inhibitors of HMG CoA reductase

Sulpha Drug (Str. Analogues of PABA)

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Inhibits Folic acid synthesis in Bacteria

Methanol Poisoning
Non-Competitive Inhibition

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Substrate and inhibitor binds at dif erent sites

Not structural analogues

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Decrease Vmax

Km same

Drugs/Toxins based on Non -

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Competitive Inhibition

Ferrochelatase (Inhibition by Lead)

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Acetylcholinesterase (Insecticides)

Cytochrome oxidase(Cyanide)
Uncompetitive Inhibition

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Inhibitor binds to ES Complex

With Inhibitor

Both Km and Vmax decreases

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1/V

1/S

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Examples of Drugs showing

Uncompetitive Inhibition

Lithium (Inositol monophosphatase)

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Phenylalanine(Placental ALP)
Classification based on Reversibility

Reversible

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Irreversible: Chemical modification or Covalent modification

Irreversible inhibitors Poison Enzymes

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Diisopropylflurophosphate (nerve gas): covalently binds

acetylcholinestrase

Aspirin(Cox)

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Penicil in (bacterial transpeptidase)
Mechanism Based Inhibition

Suicide Inhibition

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Contains chemical group that is transformed by catalytic machinery

Generates highly reactive group

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Binds covalently to catalytically essential residues

Drugs based on Suicide Inhibition

Al opurinol (inhibits xanthine oxidase: Oxypurinol)

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5 fluorouracil (inhibits thymidylate synthase: FdUMP)
Transition state Analogs & Abzymes

Transition state analog: A molecule with shape

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similar to transition state

Catalytic Antibodies

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Abzymes created using transition state analog

as antigens

Clinical Scenario 1

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A 45-year-old man presents to emergency with bradycardia, blurred vision,

vomiting, increased and salivation. He is a farmer using OPC Spray for his field

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and pipe ruptured. Type of inhibition?
(A) Competitive

(B) Noncompetitive

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(C) Uncompetitive

(D) Irreversible
Clinical scenario 2

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A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,

dysphagia and slurred speech. A drug is administered with following effect.

What is true

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In presence of drug

a. Competitive: Vmax same, km increased

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b. Competitive : Vmax same, km decreased

1/V

c. Non-competitive: Vmax decreased, Km same

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d. Non-competitive: Vmax decreased, Km decreased

1/S

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Clinical Scenario 3

A patient wants to go to Manali for trekking. He took a medicine for mountain

sickness with following kinetics. What is the type of inhibition?

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With Drug

A. Competitive

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B. Noncompetitive

1/V

C. Uncompetitive

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D. Allosteric

1/S