Allosteric enzymes

Allosteric enzymes tend to be

multi-sub unit proteins

The reversible binding of an

allosteric modulator (here a

positive modulator M) affects

the substrate binding site

T

T

R

T

[S]

vo

Mechanism and Example of Allosteric Effect

S S

R

R

SS

RS

A

I

T[S]

vo

[S]

vo

(+)

(-) X X

X

R = Relax(active)

T = Tense(inactive)

Allosteric siteHomotropic(+)Concerted

Heterotropic(+)Sequential

Heterotropic(-)Concerted

Allosteric site

Kinetics Cooperation Models

(-)

(+)

(+)

Enzyme Inhibitors

• Specific enzyme inhibitors regulate enzyme activity and help us

understand mechanism of enzyme action. (Denaturing agents are not

inhibitors)

• Irreversible inhibitors form covalent or very tight permanent bonds with

aa at the active site of the enzyme and render it inactive. 3 classes:

groupspecific reagents, substrate analogs, suicide inhibitors

• Reversible inhibitors form an EI complex that can be dissociated back

to enzyme and free inhibitor. 3 groups based on their mechanism of

action: competitive, non-competitive and uncompetitive.

Enzyme Inhibition

Competitive inhibitors

• Compete with substrate for binding to enzyme

• E + S = ES or E + I = EI . Both S and I cannot bind enzyme at the same time

• In presence of I, the equilibrium of E + S = ES is shifted to the left causing dissociation of ES.

• This can be reversed / corrected by increasing [S]

• Vmax is not changed, KM is increased by (1 + I/Ki)

• Eg: AZT, antibacterial sulfonamides, the anticancer agent methotrexate etc

Competitive Inhibition

Kinetics of competitive inhibitor

Increase [S] toovercomeinhibition

Vmax attainable,

Km is increased

Ki =dissociationconstant forinhibitor

V max unaltered, Km increased

Non-competitive Inhibitors

• Inhibitor binding site is distinct from substrate binding site. Can bind to free enzyme E and to ES

• E + I = EI, ES + I = ESI or EI + S = ESI

• Both EI and ESI are enzymatically inactive

• The effective functional [E] (and [S]) is reduced

• Reaction of unaffected ES proceeds normally

• Inhibition cannot be reversed by increasing [S]

• KM is not changed, Vmax is decreased by (1 + I/Ki)

Mixed (Noncompetitive) Inhibition

Kinetics of non-competitive inhibitor

Increasing [S] cannotovercome inhibition

Less E available,V max is lower,Km remains the samefor available E

Km unaltered, V max decreased

Uncompetitive Inhibitors

• The inhibitor cannot bind to the enzyme directly, but can only bind to the enzyme-substrate complex.

• ES + I = ESI

• Both Vmax and KM are decreased by (1+I/Ki).

Uncompetitive Inhibition

Substrate Inhibition

Caused by high substrate concentrations

E + S ES E + PKm

’ k2

KS1

+

S

ES21

2'

'

2

][][

][

][

]][[,

][

]][[

Sm

m

mSi

KS

SK

SVv

ES

ESK

ES

ESSK

Substrate Inhibition

At low substrate concentrations [S]2/Ks1<<1 and inhibition is not observed

Plot of 1/v vs. 1/[S] gives a line Slope = K’

m/Vm

Intercept = 1/Vm

][

111

][1

'

'

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations, K’m/[S]<<1, and

inhibition is dominant

Plot of 1/v vs. [S] gives a straight line Slope = 1/KS1 · Vm

Intercept = 1/Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1'

max][

0][/

SmKKS

Sddv

1/V I>0

I=0

1/Vm

-1/Km -1/Km,app 1/[S]

1/VI>0

I=0

1/Vm

-1/Km-1/Km,app 1/[S]

1/Vm,app

1/VI>0

I=0

1/Vm

-1/Km 1/[S]

1/Vm,app

1/V

1/Vm

-1/Km 1/[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] only,and competes with [S];increasing [S] overcomesInhibition by [II].

[II] binds to free [E] or [ES] complex; Increasing [S] cannot overcome [II] inhibition.

[II] binds to [ES] complex only, increasing [S] favorsthe inhibition by [II].

E + S → ES → E + P + II↓EII

←

↑

E + S → ES → E + P + + II II↓ ↓EII + S →EIIS

←

↑ ↑

E + S → ES → E + P + II ↓ EIIS

←

↑

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Km’ [S], mM

vo

[S], mM

vo

II II

Km [S], mM

Vmax

II

Km’

Vmax’Vmax’

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax & Km decreased

II

1/[S]1/Km

1/vo

1/ Vmax

II

Two parallellines

II

Intersect at X axis

1/vo

1/ Vmax

1/[S]1/Km 1/[S]1/Km

1/ Vmax

1/vo

Intersect at Y axis

= Km’

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites.

- Variation of pH changes the ionic form of the active sites.

- pH changes the three-Dimensional structure of enzymes.

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme.

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature, activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0/

/

0

/2

2

][][

][Where Ea is the activation energy (kcal/mol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=A*exp(-Ea/R*T)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate:

kd=Ad*exp(-Ea/R*T)

kd: enzyme denaturation rate constant;

Ea: deactivation energy

REFERENCES

Michael L. Shuler and Fikret Kargı, Bioprocess Engineering: Basic Concepts (2 nd Edition),Prentice Hall, New York, 2002.

1. James E. Bailey and David F. Ollis, Biochemical Engineering Fundementals (2 nd Edition), McGraw-Hill, New York, 1986.

www-nmr.cabm.rutgers.edu/academics/

biochem694/2005BioChem412/

Biochem.412_2005_Lect.18.ppt –

juang.bst.ntu.edu.tw/BCbasics/Animation.htm - 37k –

www.saburchill.com/IBbiology/chapters03/images/

ENZYME%20INHIBITION.ppt –

http://www.wiley.com/college/pratt/0471393878/student/animations/enzyme_inhibition/index.html