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Enzyme Kinetics - Inhibition

Types of Inhibition

• Competitive Inhibition

• Noncompetitive Inhibition

• Uncompetitive Inhibition

• Irreversible Inhibition

S

I

Enzyme

Competitive Inhibition

In competitive inhibition,

the inhibitor competes

with the substrate for the

same binding site

Competitive Inhibition

- Reaction Mechanism

In competitive inhibition, the

inhibitor binds only to the

free enzyme, not to the ES

complex

E + S ES E + P

EI

+I

General Michaelis-Menten Equation

This form of the Michaelis-Menten equation

can be used to understand how each type of

inhibitor affects the reaction rate curve

v =[S]

Km,app + [S]

Vmax,app

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 Vmax

Vmax

Vmax

2

Km Km,app

[Substrate]

Reaction Rate

- Inhibitor

+ Inhibitor

Vmax,app = Vmax

Km,app > Km

The Lineweaver-Burk plot is

diagnostic for competitive inhibition

Slope =Km,app

Vmax

1Vmax

-1Km,app

1[S]

Increasing [I]

1v

v=

1

Vmax

Km,app

Vmax

1+

[S]

1

Vmax

Vmax

2

Km Km,app

[Substrate]

Reaction Rate

- Inhibitor

+ Inhibitor

.

S

I

Inhibitor

competes with

substrate,

decreasing its

apparent affinity:

Km,app > Km

Formation of EI

complex shifts reaction

to the left: Km,app > Km

Km,app > Km

Vmax,app = Vmax

E + S ES E + P

EI

+I

Formation of EI



Relating the Michaelis-Menten equation, the v vs. [S]

plot, and the physical picture of competitive inhibition

Example - Competitive Inhibition

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.

COOH

NH2

p-aminobenzoic acid

folic acid

SO2NH2

NH2

sulfanilamide

Practical case: Methanol poisoning

A 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.

S

I

IS

S

I

I

S

Enzyme

Enzyme

Enzyme

Enzyme

Noncompetitive Inhibition

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

E + S ES E + P

EI

+I

ESI

+I

S+

Noncompetitive Inhibition -

Reaction Mechanism

In noncompetitive

inhibition, the

inhibitor binds

enzyme

irregardless of

whether the

substrate is bound

Noncompetitive inhibitors decrease

the Vmax,app, but don’t affect the Km

Vmax,app < Vmax

Km,app = Km

Vmax

Vmax2

1

2

1Vmax,app

Km

Km,app

[Substrate]

Reaction Rate

- Inhibitor

+ Inhibitor

Vmax,app

The inhibitor binds

equally well to free

enzyme and the ES

complex, so it doesn’t

alter apparent affinity

of the enzyme for the

substrate

Why does Km,app = Km for

noncompetitive inhibition?

E + S ES E + P

EI

+I

ESI

+I

S+

The Lineweaver-Burk plot is diagnostic

for noncompetitive inhibition

v=

1

Vmax,app

Km

Vmax,app

1+

[S]

1

Slope =Vmax,app

Km

1Vmax,app

-1Km

1[S]

Increasing [I]

1v

Formation of EI



Km,app > Km

Vmax,app = Vmax

.

S

I

IS

S

I

I

S

Enzyme

Enzyme

Enzyme

Enzyme

.

Vmax

Vmax2

1

21Vmax,app

Km Km,app

[Substrate]

Reaction Rate

- Inhibitor

+ Inhibitor

Vmax,app

Inhibitor doesn’t interfere with substrate binding,

Km,app = Km

E + S ES E + P

EI

+I

ESI

+I

S+

Even at high

substrate levels,

inhibitor still binds,

[E]t < [ES]

Vmax,app < Vmax

Vmax,app < Vmax

Km,app = Km

Relating the Michaelis-Menten equation, the v vs. [S] plot,

and the physical picture of noncompetitive inhibition

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 1,6-bisphosphatase

AMP

OH2C

H

HO

H

CH2

OH

O O

H

OH

P O-

O-OP O-

O-O


AMP

fructose 1,6-diphosphate


AMPAMPAMPAMP



OH2C

H

HO

H

CH2

OH

O O

H

OH

P O-

O-OP O-

O-O




fructose 6-phosphate

Pi

E E.S E + P

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 Inhibition

In uncompetitive

inhibition, the

inhibitor binds

only to the ES

complex

.

S

I

S

Enzyme Enzyme

I

Enzyme

S

Enzyme

I

Uncompetitive Inhibition -

Reaction Mechanism

In uncompetitive

inhibition, the

inhibitor binds only

to the ES complex,

it does not bind to

the free enzyme

E + S ES E + P

ESI

+I

Uncompetitive inhibitors decrease

both the Vmax,app and the Km,app

Vmax,app < Vmax

Km,app < Km

Notice 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

Vmax

Vmax2

1

2

1Vmax,app

KmKm,app[Substrate]

Reaction Rate

- Inhibitor

+ Inhibitor

Vmax,app

Uncompetitive inhibitors decrease both the

Vmax,app and the Km,app of the enzyme

E + S ES E + P

ESI

+I

Notice that

uncompetitive inhibitors

don’t bind to the free

enzyme, so there is no

EI complex in the

reaction mechanism

The Lineweaver-Burk plot is

diagnostic for uncompetitive inhibition

v=

1

Vmax,app

Km,app

Vmax,app

1+

[S]

1

=Vmax

Km

Vmax,app

1+

[S]

1

1Vmax,app

-1Km,app

1[S]

Increasing [I]1v

Slope =Vmax

Km

.

Vmax

Vmax2

1

2

1Vmax,app

KmKm,app[Substrate]

Reaction Rate

- Inhibitor

+ InhibitorVmax,app

Formation of EI



.

S

I

S

Enzyme Enzyme

I

Enzyme

S

Enzyme

I

E + S ES E + P

ESI

+I

Even at high

substrate levels,

inhibitor binds,

[E]t < [ES]

Vmax,app < Vmax

Inhibitor increases

the amount of enzyme bound

to substrate

Km,app < Km

Vmax,app < Vmax

Km,app< Km

Relating the Michaelis-Menten equation, the v vs. [S]

plot, and the physical picture of uncompetitive inhibition

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

Alkalinephosphatase

O P

O-

O-O

Alkalinephosphatase

Phe

AlakalinePhosphatase

Phe

PheOPO -

O-O

O P

O-

O-O

P

O-

O-O-O

Alkalinephosphatase

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 Inhibition

In irreversible

inhibition, the

inhibitor binds to the

enzyme irreversibly

through formation of

a covalent bond with

the enzyme ,

permanently

inactivating the

enzyme

Enzyme

SO I

Irreversible Inhibition - Reaction

Mechanism

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

Vmax decreases

No effect on Km

E + S ES E + P

EI

+I

The Michaelis-Menten plot for an irreversible

inhibitor looks like noncompetitive inhibition

Vmax,app < Vmax

Km,app = Km

Vmax

Vmax2

1

2

1Vmax,app

Km

Km,app

[Substrate]

Reaction Rate

- Inhibitor

+ Inhibitor

Vmax,app

Irreversible inhibition is distinguished from

noncompetitive inhibition by plotting Vmax vs [E]t

Enzyme is

inactivated

until all of the

irreversible

inhibitor is

used up

[E]t

Vmax

+ Revers

ible

Noncom

petitive Inhib

itor

- Inhibitor

+ Irreversible Inhibitor[E]t > [I][E]t < [I]

[E]t = [I]

Irreversible inhibitors decrease

Vmax,app, but leave the apparent

Km unchanged. Irreversible

inhibitors differ from other types

of inhibitors because they

covalently modify the enzyme.

This results in the permanent

inhibition of the enzyme activity.

Examples of Irreversible Inhibitors

• diisopropylphosphofluoridate

– prototype for the nerve gas sarin

– permanently inactivates serine proteases by

forming a covalent bond with the active site

serine

Penicillin is a suicide inhibitor

Glycopeptide transpeptidase catalyzes the formation of cross-links between D-amino acids in the cell walls of bacteria. This enzyme also catalyzes the reverse reaction, the hydrolysis of peptide bonds. During the course of hydrolyzing the strained peptide bond in penicillin, the enzyme activates the inhibitor (penicillin), which then covalently modifies an active site serine in the enzyme. In effect, the enzyme “commits suicide” by hydrolyzing the strained peptide bond in penicillin.

glycopeptidetranspeptidase

OHSer

O

glycopeptidetranspeptidase

Ser

N

S CH3

CH3

COO-

H

H

C

C

O

H

N

CO

H

H

R

N

S

CH3

COO-

H

H

C

C

O

H

N

CO

H

Strainedpeptide bond

Penicillin

CH3

R

Suicide inhibitors work by

“tricking” the enzyme into

activating the inhibitor, which

then forms a covalent bond with

the enzyme, leading to its

permanent inactivation.

Summary-Enzyme Inhibition

• Competitive Inhibitor

– Binds to substrate binding site

– Competes with substrate

– The affinity of the substrate appears to be decreased

when inhibitor is present (Km,app >Km)

• Noncompetitive inhibitor

– Binds to allosteric site

– Does not compete with the substrate for binding to

the enzyme

– The maximum velocity appears to be decreased in

the presence of the inhibitor (Vmax,app <Vmax)

• Uncompetitive Inhibitor

– Binds to the enzyme only after the substrate has

bound

– The affinity of the substrate appears to be increased

and the maximum velocity appears to be decreased

when inhibitor is present (Km,app <Km,

Vmax,app <Vmax),

• Irreversible Inhibitor

– Covalently modifies and permanently inactivates the

enzyme

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