Você está na página 1de 40

Enzyme Kinetics - Inhibition

Types of Inhibition
Competitive Inhibition Noncompetitive Inhibition Uncompetitive Inhibition Irreversible Inhibition

Competitive Inhibition
Enzyme

S I
In 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 Vmax


Vmax - Inhibitor

Reaction Rate

+ Inhibitor Vmax 2

Vmax,app = Vmax Km,app > Km


Km Km,app [Substrate]

The Lineweaver-Burk plot is diagnostic for competitive inhibition


1 = Km,app 1 + 1 v Vmax [S] Vmax
Increasing [I]

1 v
1 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 inhibition
Inhibitor competes with substrate, decreasing its apparent affinity: Km,app > Km
Reaction Rate

Vmax

- Inhibitor

+ Inhibitor Vmax 2

Formation Formationof ofEI EI complex complex shifts shiftsreaction reaction to > Kmm to the theleft: left:K K m,app m,app > K

Km,app > Km Vmax,app = Vmax


Km Km,app [Substrate]

Example - Competitive Inhibition


NH2

folic acid

COOH

p-aminobenzoic acid
NH2

SO2NH2

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 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.

Noncompetitive Inhibition
I I
S
Enzyme

Enzyme

I
Enzyme

I S
Enzyme

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

Noncompetitive Inhibition Reaction Mechanism

E+S + I EI + S

ES + I ESI

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

Noncompetitive inhibitors decrease the Vmax,app, but dont affect the Km

Vmax,app < Vmax Km,app = Km

Why does Km,app = Km for noncompetitive inhibition?

E+S + I EI + S

ES + I ESI

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

E+P

The Lineweaver-Burk plot is diagnostic for noncompetitive inhibition


1 1 = Km 1 + v Vmax,app [S] Vmax,app
Increasing [I]

1 v
1 Vmax,app -1 Km

Km Slope = Vmax,app

1 [S]

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

I S
S
Enzyme

Enzyme

Inhibitor doesnt interfere with substrate binding, Km,app = Km

I
Enzyme

I S
Reaction Rate
Enzyme
Vmax

- Inhibitor

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

Even at high substrate levels, Formation inhibitor of EI still binds, [E]t < reaction [ES] complex shifts Vmax,app < Vmax

+ Inhibitor

Vmax,app <m Vmax Km,app >K Vmax,app Km,app= =V Km max


Km Km,app [Substrate]

to the left: Km,app > Km

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 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
Enzyme
.

Enzyme

Enzyme
I I

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

Enzyme

Uncompetitive Inhibition Reaction Mechanism

E+S

ES + I ESI

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

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

Uncompetitive inhibitors decrease both the Vmax,app and the Km,app of the enzyme

E+S

ES + I ESI

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

E+P

The Lineweaver-Burk plot is diagnostic for uncompetitive inhibition


1 = Km,app 1 1 + v Vmax,app [S] Vmax,app = Km 1 1 + Vmax [S] Vmax,app

1 v

Increasing [I]

Slope =

Km Vmax

1 Vmax,app -1 Km,app

1 [S]

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

Enzyme

Vmax
S
Enzyme S

- Inhibitor

Reaction Rate

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

+ Inhibitor

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

Vmax,app < Vmax Km,app< Km

Enzyme

Km,app

Km
.

[Substrate]

Even at high substrate Formation of EI levels, inhibitor binds, complex shifts reaction [E]t < [ES] to the left: K > Km Vm,app max,app < Vmax

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

O O P OO-

O O P OO-

O O
-

O O
-

Phe

Phe

Alakaline Phosphatase

Phe

O O P OO-

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
Enzyme
O

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. Vmax decreases No effect on Km

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

- Inhibitor

Reaction Rate

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

+ Inhibitor

Vmax,app < Vmax Km,app = Km


Km Km,app [Substrate]

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

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


R C H N O H

Penicillin
S CH3 CH3 N COO-

HC C O H Strained peptide bond

R C

glycopeptide transpeptidase

O S CH3 CH3 N C H O H COO-

glycopeptide H transpeptidase H N
HC

Ser

OH

Ser

Glycopeptide transpeptidase catalyzes the formation of cross-links between Damino 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.

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