Competitive inhibition

(1)

MODULATION OF ENZYME ACTIVITY

Effectors

Inhibitor: Activator:

decreases increases

reaction rate reaction rate

v_i v_a

Degree of inhibition: Degree of activation:

0 0

i i

v v

ε = v⁻ ⁰

0 a a

v v ε = v⁻

INHIBITION

REVERSIBLE IRREVERSIBILE

distinction:

V_max

E_O KONTROLL

+REVERZIBILIS INHIBITOR

+IRREVERZIBILIS INHIBITOR

E S ES E P EI

+ ⇋ → +

⇃↾

S 2

K k

ES

E S E P

EI

+ ←→ → +

↓

2

Competitive inhibition

Competition between S and I for the active sites of the enzyme, or mutual exclusion

I may be an:

substrate analogue alternative substrate product

MODEL 1.: Classical competitive inhibition:

S I

(2)

COMPETITIVE INHIBITION

4

MODEL 2.: steric hindranceA

I S

Binding of I to another site sterically hinders S in binding to the active site of enzyme.

COMPETITIVE INHIBITION

5

MODEL 3.: steric hindrance B

An analog part of S and I compete for a common binding site.

S I

E

MODEL 4.: overlaping

Sites 1 and 3 can bind I, 2 and 4 can bind S, but both exclude each other.

I S

1 2 3 4

COMPETITIVE INHIBITION

6

(3)

COMPETITIVE INHIBITION

MODEL 5.:

Binding of I changes the conformation of the enzyme which prevents binding of S to the active centre.

End product inhibition (feed back inhibition) is typical example of this case.

S S I

S I

I

7

Kinetics of competitive inhibition

Basic equations for competitive inhibition:

if k_app>0 than I is an alternative substrate if k_app=0 than I is a „dead end” competitive inhibitor

S 2

K ES k

app i

k

E S E P

I K

EI E P

+ ←→ → + +

→ + ′ վ

( )

s

K E S ES

= ⋅

( )

i

K E I EI

= ⋅

8

Kinetics of competitive inhibition

Alternative substrate: the enzyme is able to transform the structural analogous molecule, too.→an alternative product is formed.

Enzymes with group and region specifity have numerous alternative substrates

Example: the enzymes of liver: alcohol dehydrogenase, alde- hyde dehydrogenase:

E + S ' ←   → E + P'

(4)

Kinetics of competitive inhibition

Repeat the deduction:

product formation rate:

Mass balance of enzyme:

S 2

K ES k

app i

k

E S E P

I K

EI E P

+ →← → + +

→ + ′ վ

( )

s

K E S ES

= ⋅

( )

i

K E I EI

= ⋅

V dP dt k (ES)₂

= =

E0 = +E (ES) + (EI)

10

Kinetics of competitive inhibition

Divide the two equation:

Substitute:

2 o

V k (ES) E =E (ES) + (EI)

+ ( )

s

K E S ES

= ⋅ ^Kⁱ⁼( )^{E I}^EI^⋅

2 s o

s I

k E S

V K

S I

E E E E

K K

=

+ + ² 1

s o

s I

S

V K

S I k E

K K

= + +

V

_max

= k E

₂ _o

11

Kinetics of competitive inhibition

Simplified forms of reaction rate:

or:

max 1

s i

V S

V I

K S

K

=  

+ +

 

 

max s 1

i

V V S

K I S

K

=  

+ +

 

 

max i

I S

I

v ( S ) v

K ( I )

K ( S )

K

=  + +

 

12

(5)

Kinetics of competitive inhibition

13

S 1 K 1 I V

K V

1 V 1

I max

S max







+ +

=

HC C NH2H H

H

C HO O

L-alanin

O C H H

NH2 HC C

O NH cikloszerin

Competitive inhibition

Alternative substrates: for hexokinase: glucose, fructose

S-analogons: drugs:

14

Effect of sulfami- des (antimicrobial drugs): substrate analogon act as competitive inhibitor.

Competitive inhibition

(6)

K S 1 I K V S V

i s max

+







 +

=

K S 1 P K V S V

P s max

+







 +

=

1

1 1max

2

S1 1

S2

V V S

K 1 S S

K

=  

+ +

 

 

2

2 2max

1

S2 2

S1

V V S

K 1 S S

K

=  

+ +

 

 

Analogous inhibitions

competitive inhibition: product inhibition:

alternative or competing substrates

16

Noncompetitive inhibition

17

Noncompetitive inhibition

Inhibitor binds to an other active site of the enzyme and does not affect the binding of the substrate – does not change the affinity of the enzyme to the substrate.

It exists only when rapid equilibrium can be supposed, K_s=K_m.

Equations of noncompetitive inhibition:

K_s

kp

E + S ES E + P

+ +

I I

K_i K_s

EI + S ESI

K_i

K E.S

ES EI.S

ESI és K E.I EI

ES.I

s= = i= =ESI

V V

ES E ES EI ESI

max

= + + +

V = kp(ES)

18

(7)

V V

ES E ES EI ESI

max

= + + +

V V

S K

1 S

K I K

S.I K K vagy

V V

S

K 1 I

K S 1 I

K illetve

V = V 1

1 I

K S

max

s

s i s i

max s

i i

max

i s

=

+ + +

=

 +

 

+  +

 



 +

 

 +

V V S

K S ahol V V 1

1 I

K

maxi s

maxi max

i

= + =

+ where

or

Noncompetitive inhibition

Inhibitor changes the value of the apparent V_max, but does not change the values of K_s( or K_m).

19

V 1/V

0 1/S } ¹

Vmax 1

Vmaxi 1 I

Ki Vmax

=

+ K

V 1 I K

m

max i

+

 



K V

m max

I V_max

V_maxi

I

K_m S

Noncompetitive inhibition

The inhibitor affects the apparent V_maxvalue but does not change K_s(or K_m).

20

Noncompetitive inhibition

Examples:

H⁺ions’ effect on chymotripsine. Here a proton acceptor site exists in the active centre, which can be inhibited by increasing H⁺-ion concentration. (L-B plot shows clear noncompetitive inhibition, (but do not forget the complex effect of the pH on enzymes).

Heavy metal molecules(-SH reagensek), or cyanides.

Often these effects are irreversible.

(8)

Noncompetitive inhibition

Surface of slices apple gets brown in air: o-diphenol oxidase enzyme catalyses the catechol→ o-quinone reaction

this and other reaction products give the brown color

competitive inhibitor of o-diphenol oxidase is para-hydroxybenzoic acid (PHBA), a structural analog.

22

Noncompetitive inhibition

competitive inhibitor of o-diphenol oxidase is para-hydroxybenzoic acid (PHBA), a structural analogon

noncompetitive inhibitor is: phenyl- thiourea, bound to copper ion what is necessary to enzyme activity.

23

Uncompetitive inhibition

Fixed order: the inhibitor must join second, after the substrate

24

(9)

1 V

K V

1 S

1

V 1 I

K

m

max max i

= +  +

 



V V 1

1 I K

S K 1 I

K S

max

i m

i

=

+ ⋅

 +

 

 +

Vmax/2 Vmaxi/2

V_max Vmaxi

I

V V

1 I K

maxi max

i

= +

K K

1 I K

mi m

i

= +

K_m V

S

I

nem inhibeált

1/S 1/V

I

I=0 K_m/V_max

1/Vmax

1 1

V I K i V

i

max max

= +

− + 1 I

K K

i m

1/K_m

Uncompetitive inhibition

25

Linear mixed type inhibition

Mechanism of linear mixed type inhibition resembles to noncompetitive inhibition but presence of I modifies the enzyme affinity to substrate.

26

Linear mixed type inhibition

Expressing the change of two kinetic parameters:

(10)

competitive noncompetitive uncompetitive

mixed

28

Summary of the inhibition types

S and I mutually exclude each other from the enzyme COMPETITIVE

S and I bind to the enzyme independently on each other NONCOMPETITIVE I binds only after S UNCOMPETITIVE

Like former but I modifies the affinity of the enzyme MIXED TYPE

29

Substrate inhibition

The substrate binds to two or more sites.

If the S concentration is high, it can occur that two S bind to one and the other binding site forming inactive complex.

(also reversible inhibition).

E

-OOC

CH₂ CH₂

-OOC

E

-OOC

CH CH

-OOC

Succinate Malonate E

-OOCCH2CH2COO^-

Normal S inhibition

30

(11)

Substrate inhibition

31 V

0.0 0.1 0.2 0.3 0.4

0 50 100 150 200 250 300

Vmax=0.9,Ks=50,Ki=10 Vmax=0.9,Ks=50,Ki=50

Vmax=0.9,Ks=50,Ki=100

Szubstrát koncentráció(mg/L)