5 enzyme kinetics-inhibition

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Enzyme Kinetics - Inhibition

Types of Inhibition Competitive Inhibition Noncompetitive Inhibition Uncompetitive Inhibition Irreversible Inhibition

Competitive InhibitionEnzyme

S

IIn competitive inhibition, the inhibitor competes with the substrate for the same binding site

Competitive Inhibition - Reaction Mechanism

E+S + I EI

ES

E+P

In competitive inhibition, the inhibitor binds only to the free enzyme, not to the ES complex

General Michaelis-Menten Equation

Vmax,app [S] v= Km,app + [S]This form of the Michaelis-Menten equation can be used to understand how each type of inhibitor affects the reaction rate curve

In competitive inhibition, only the apparent Km is affected (Km,app> Km), The Vmax remains unchanged by the presence of the inhibitor.

.

Competitive inhibitors alter the apparent Km, not the Vmx aVmax Reaction Rate - Inhibitor + Inhibitor Vmax 2

Vmx,app a Km pp ,aKm Km,app [Substrate]

= Vmx a > Km

The Lineweaver-Burk plot is diagnostic for competitive inhibition1 = Km,app 1 + 1 v Vmax [S] VmaxIncreasing [I]

1 v1 Vmax -1 Km,app

Km,app Slope = Vmax

1 [S]

Relating the Michaelis-Menten equation, the v vs. [S] plot, and the physical picture of competitive inhibitionInhibitor competes with substrate, decreasing its apparent affinity: Km,app > Km.

S

IReaction Rate

.

Vmax

- Inhibitor + Inhibitor

E+S + I EI

ES

E+P

Vmax 2

Formation of EI Formation of EI complex shifts reaction complex shifts reaction to the left: Km,appp > Kmm to the left: Km,ap > K

Km,app > Km Vmax,app = VmaxKm Km,app [Substrate]

Example - Competitive InhibitionNH2

folic acid

p-aminobenzoic acidNH2

COOH

SO2 NH2

Sulfanilamide is a competitive inhibitor of p-aminobenzoic acid. Sulfanilamides (also known as sulfa drugs, discovered in the 1930s) were the first effective systemic antibacterial agents. Because we do not make folic acid, sulfanilamides do not affect human cells.

sulfanilamide

Practical case: Methanol poisoningA wealthy visitor is taken to the emergency room, where he is diagnosed with methanol poisoning. You are contacted by a 3rd year medical student and asked what to do? How would you suggest treating this patient?

Methanol (CH3OH) is metabolized to formaldehyde and formic acid by alcohol dehydrogenase. You advisethe third year student to get the patient very drunk. Since ethanol (CH3CH2OH) competes with methanol for the same binding site on alcohol dehydrogenase, it slows the metabolism of methanol, allowing the toxic metabolites to be disposed of before they build up to dangerous levels. By the way, the patient was very grateful and decided to leave all their worldly possessions to the hospital. Unfortunately, after being released from the hospital, he went to the casinos and lost everything he had.

.

Noncompetitive InhibitionI IEnzyme

Enzyme

S

S

IEnzyme

S

IEnzyme

S

the inhibitor does not interfere with substrate binding (and vice versa)

Noncompetitive Inhibition Reaction Mechanism

E+S + I EI + S

ES + I ESI

E+PIn noncompetitive inhibition, the inhibitor binds enzyme irregardless of whether the substrate is bound

.

Noncompetitive inhibitors decrease the Vmx,app , but dont affect the Km aVmax

- Inhibitor

Reaction Rate

Vmax,app1 V 2 max 1 V 2 max,app

+ Inhibitor

Vmx,app aKm [Substrate] Km,app

< Vmx a

Km pp = Km ,a

Why does Km pp = Km for ,a noncompetitive inhibition?

E+S + I EI + S

ES + I ESI

E+PThe inhibitor binds equally well to free enzyme and the ES complex, so it doesnt alter apparent affinity of the enzyme for the substrate

The Lineweaver-Burk plot is diagnostic for noncompetitive inhibition1 1 = Km 1 + v Vmax,app [S] Vmax,appIncreasing [I]

1 v1

Km Slope = Vmax,app

Vmax,app -1 Km

1 [S]

Relating the Michaelis-Menten equation, the v vs. [S] plot, and the physical picture of noncompetitive inhibition.

I SEnzyme

I

Enzyme

S

Inhibitor doesnt interfere with substrate binding, Km pp = Km ,a

IEnzyme

S

IEnzyme

.

SReaction Rate

Vmax

- Inhibitor

E+S ES E+P + + Even at high substrate levels, I Formation inhibitor still binds, I of EI EI + S[E]t < [ES] complex shifts reaction m ax to the left: Vmax,appp VKm Km,ap

Vmax,app1 V 2 max 1 V 2 max,app

+ Inhibitor

V Km max Vmax,app m,app ==Vmax K KmKm Km,app [Substrate]

ESI

Noncompetitive inhibitors decrease the apparent Vmax , but do not alter the Km of the reaction

Example of noncompetitive inhibition: fructose 1,6-bisphosphatase inhibition by AMP-

fructose 1,6diphosphate

O O P OO H2 C H H

-

O HO

O O P OO CH2 OH

fructose 6phosphate

OH

H

fructose 1,6diphosphate

Pi

fructose 1,6bisphosphatase

fructose 1,6bisphosphatase

fructose 1,6bisphosphatase

EAMP AMP AMPfructose 1,6diphosphate-O

E.SAMP AMP AMPO P OO H2 C H H-O

E+P

O HO

O P OO CH2 OH

fructose 1,6diphosphate

OH

H

fructose 1,6bisphosphatase

fructose 1,6bisphosphatase

E.I

E.S.I

Fructose 1,6-bisphosphatase is a key regulatory enzyme in the gluconeogenesis pathway. High amounts of AMP signal that ATP levels are low and gluconeogenesis should be shut down while glycolysis is turned on. High AMP levels inhibit fructose 1,6-bisphosphatase (shutting down gluconeogenesis) and activate phosphofructokinase (turning on glycolysis). Regulation of fructose 1,6-bisphosphatase and phosphofructokinase by AMP prevents a futile cycle in which glucose is simultaneously synthesized and broken down.

Uncompetitive InhibitionEnzyme.

Enzyme

S

S

EnzymeI I

Enzyme

In uncompetitive inhibition, the inhibitor binds only to the ES complex

I

S

Uncompetitive Inhibition Reaction Mechanism

E+S

ES + I ESI

E+PIn uncompetitive inhibition, the inhibitor binds only to the ES complex, it does not bind to the free enzyme

Uncompetitive inhibitors decrease both the Vmx,app and the Km pp a ,a.

Vmax,app < VmaxVmax

- Inhibitor

Km,app < KmNotice that at low substrate concentrations, uncompetitive inhibitors have little effect on the reaction rate because the lower Km,app of the enzyme offsets the decreased Vmax,app

Reaction Rate

Vmax,app1 V 2 max 1 V 2 max,app

+ Inhibitor

Km,app

Km

[Substrate]

Uncompetitive inhibitors decrease both the Vmx,app and the Km pp of the enzyme a ,a

E+S

ES + I ESI

E+PNotice that uncompetitive inhibitors dont bind to the free enzyme, so there is no EI complex in the reaction mechanism

The Lineweaver-Burk plot is diagnostic for uncompetitive inhibition1 = Km,app 1 1 + v Vmax,app [S] Vmax,app = Km 1 1 + Vmax [S] Vmax,app

1 v

Increasing [I]

Slope =

Km Vmax

Vmax,app Km,app -1

1

1 [S]

Relating the Michaelis-Menten equation, the v vs. [S] plot, and the physical picture of uncompetitive inhibitionEnzyme.

Enzyme

VmaxS

- Inhibitor

S

EnzymeI I

Reaction Rate

Vmax,app1 V 2 max 1 V 2 max,app

+ Inhibitor

Inhibitor increases the amount of enzyme bound to substrate Km pp < Km ,a

Enzyme

Vmax,app < Vmax Km,app < Km

I

S

Km,app

Km.

[Substrate]

E+S

ES E+P + Even at high Formation of EI levels, I substrate binds, inhibitorcomplex shifts reaction [E]t < [ES] to the left: Km,app > V m V < K

ESI

m ,ap ax p

m ax

Uncompetitive inhibitors decrease the apparent Km of the enzyme and decrease the Vmax of the reaction

Example of uncompetitive inhibition: alkaline phosphatase inhibition by phenylalanine.

Alkaline phosphatase

Alkaline phosphatase

Alkaline phosphatase

Phe

O

-

Phe

Alakaline Phosphatase

-

O

Phe

O O P O-

-

O

-

O

O O P O-

-

O OP O-

O

O O-

P

At alkaline pH, alkaline phosphatase catalyzes the release of inorganic phosphate from phosphate esters. It is found in a number of tissues, including liver, bile ducts, intestine, bone, kidney, placenta, and leukocytes. Alkaline phosphatase plays a role in the deposition of hydroxyapetite in osteoid cells during bone formation. The function of alkaline phosphatase in other tissues is not known. Serum alkaline phosphatase levels are important diagnostic markers for bone and liver disease.

Irreversible InhibitionEnzymeO

I

S

In irreversible inhibition, the inhibitor binds to the enzyme irreversibly through formation of a covalent bond with the enzyme , permanently inactivating the enzyme

Irreversible Inhibition - Reaction Mechanism

E+S + I EI

ES

E+P

In irreversible inhibition, the inhibitor permanently inactivates the enzyme. The net effect is to remove enzyme from the reaction.

Vmx decreases a No effect on Km

.

The Michaelis-Menten plot for an irreversible inhibitor looks like noncompetitive inhibitionVmax

- Inhibitor

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