5 enzyme kinetics-inhibition

40
Enzyme Kinetics - Inhibition

Upload: joel-smolanoff

Post on 29-Mar-2015

777 views

Category:

Documents


9 download

TRANSCRIPT

Page 1: 5 enzyme kinetics-inhibition

Enzyme Kinetics - Inhibition

Page 2: 5 enzyme kinetics-inhibition

Types of Inhibition

• Competitive Inhibition

• Noncompetitive Inhibition

• Uncompetitive Inhibition

• Irreversible Inhibition

Page 3: 5 enzyme kinetics-inhibition

.

S

I

Enzyme

Competitive Inhibition

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

Page 4: 5 enzyme kinetics-inhibition

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

Page 5: 5 enzyme kinetics-inhibition

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

Page 6: 5 enzyme kinetics-inhibition

In competitive inhibition, only the apparent Km is affected (Km,app> Km),

The Vmax remains unchanged by the presence of the inhibitor.

Page 7: 5 enzyme kinetics-inhibition

Competitive inhibitors alter the apparent Km, not the Vmax

.

Vmax

Vmax

2

Km Km,app

[Substrate]

Rea

ctio

n R

ate

- Inhibitor

+ Inhibitor

Vmax,app = Vmax

Km,app > Km

Page 8: 5 enzyme kinetics-inhibition

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

Page 9: 5 enzyme kinetics-inhibition

.

Vmax

Vmax

2

Km Km,app

[Substrate]

Rea

ctio

n R

ate

- Inhibitor

+ Inhibitor

.

S

I

Inhibitor competes with substrate, decreasing its apparent affinity: Km,app > Km

Formation of EIcomplex shifts reactionto the left: Km,app > Km

Km,app > Km

Vmax,app = Vmax

E + S ES E + P

EI

+I

Formation of EIcomplex shifts reactionto the left: Km,app > Km

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

Page 10: 5 enzyme kinetics-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

Page 11: 5 enzyme kinetics-inhibition

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?

Page 12: 5 enzyme kinetics-inhibition

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.

Page 13: 5 enzyme kinetics-inhibition

.

S

I

IS

S

I

I

S

Enzyme

Enzyme

Enzyme

Enzyme

Noncompetitive Inhibition

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

Page 14: 5 enzyme kinetics-inhibition

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

Page 15: 5 enzyme kinetics-inhibition

Noncompetitive inhibitors decrease the Vmax,app, but don’t affect the Km

Vmax,app < Vmax

Km,app = Km

.

Vmax

Vmax21

21 Vmax,app

Km

Km,app

[Substrate]

Re

ac

tio

n R

ate

- Inhibitor

+ InhibitorVmax,app

Page 16: 5 enzyme kinetics-inhibition

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+

Page 17: 5 enzyme kinetics-inhibition

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

Page 18: 5 enzyme kinetics-inhibition

Formation of EIcomplex shifts reactionto the left: Km,app > Km

Km,app > Km

Vmax,app = Vmax

.

S

I

IS

S

I

I

S

Enzyme

Enzyme

Enzyme

Enzyme

.

Vmax

Vmax21

21 Vmax,app

Km Km,app

[Substrate]

Re

ac

tio

n R

ate

- Inhibitor

+ InhibitorVmax,app

Inhibitor doesn’t interferewith substrate binding,Km,app = Km

E + S ES E + P

EI

+I

ESI

+I

S+

Even at highsubstrate 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

Page 19: 5 enzyme kinetics-inhibition

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

Page 20: 5 enzyme kinetics-inhibition

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

fructose 1,6-bisphosphatase

AMP

OH2C

HHO

H

CH2

OH

O O

H

OH

P O-

O-OP O-

O-O

fructose 1,6-bisphosphatase

AMP

fructose 1,6-diphosphate

fructose 1,6-diphosphate

AMPAMPAMPAMP

fructose 1,6-bisphosphatase

fructose 1,6-bisphosphatase

OH2C

HHO

H

CH2

OH

O O

H

OH

P O-

O-OP O-

O-O

fructose 1,6-bisphosphatase

fructose 1,6-diphosphate

fructose 1,6-diphosphate

fructose 6-phosphate

Pi

E E.S E + P

E.I E.S.I

Page 21: 5 enzyme kinetics-inhibition

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.

Page 22: 5 enzyme kinetics-inhibition

Uncompetitive Inhibition

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

.

S

I

S

Enzyme Enzyme

I

Enzyme

S

Enzyme

I

Page 23: 5 enzyme kinetics-inhibition

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

Page 24: 5 enzyme kinetics-inhibition

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

Vmax21

21 Vmax,app

KmKm,app[Substrate]

Re

ac

tio

n R

ate

- Inhibitor

+ InhibitorVmax,app

Page 25: 5 enzyme kinetics-inhibition

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

Page 26: 5 enzyme kinetics-inhibition

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

Page 27: 5 enzyme kinetics-inhibition

.

Vmax

Vmax21

21 Vmax,app

KmKm,app[Substrate]

Re

ac

tio

n R

ate

- Inhibitor

+ InhibitorVmax,app

Formation of EIcomplex shifts reactionto the left: Km,app > Km

.

S

I

S

Enzyme Enzyme

I

Enzyme

S

Enzyme

I

E + S ES E + P

ESI

+I

Even at highsubstrate levels,inhibitor binds,[E]t < [ES]Vmax,app < Vmax

Inhibitor increasesthe amount of enzyme boundto substrateKm,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

Page 28: 5 enzyme kinetics-inhibition

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

Page 29: 5 enzyme kinetics-inhibition

Example of uncompetitive inhibition: alkaline phosphatase inhibition by phenylalanine

.

Alkalinephosphatase

O P

O-

O-O

Alkalinephosphatase

Phe

AlakalinePhosphatase

Phe

Phe

OP

O -

O

-O

O P

O-

O-O

P

O-

O-O-O

Alkalinephosphatase

Page 30: 5 enzyme kinetics-inhibition

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.

Page 31: 5 enzyme kinetics-inhibition

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

Page 32: 5 enzyme kinetics-inhibition

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

Page 33: 5 enzyme kinetics-inhibition

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

Vmax,app < Vmax

Km,app = Km

.

Vmax

Vmax21

21 Vmax,app

Km

Km,app

[Substrate]

Re

ac

tio

n R

ate

- Inhibitor

+ InhibitorVmax,app

Page 34: 5 enzyme kinetics-inhibition

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

Vm

ax

+ Reversible

Noncompetitive In

hibitor

- Inh

ibito

r

+ Ir

reve

rsib

le In

hibi

tor[E]t > [I][E]t < [I]

[E]t = [I]

Page 35: 5 enzyme kinetics-inhibition

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.

Page 36: 5 enzyme kinetics-inhibition

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

Page 37: 5 enzyme kinetics-inhibition

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

SerN

S CH3

CH3

COO-

H

H

C

C

O

H

N

CO

H

H

R

N

S

CH3

COO-

H

H

C

CO

H

N

CO

H

Strainedpeptide bond

PenicillinCH3

R

Page 38: 5 enzyme kinetics-inhibition

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.

Page 39: 5 enzyme kinetics-inhibition

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)

Page 40: 5 enzyme kinetics-inhibition

• 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