Allosteric Enzyme Models

Allosteric Enzymes

Models of Cooperativity

Allosteric Enzymes

• AllostericAllosteric:: Greek allo + steric, other shape• Allosteric enzymeAllosteric enzyme:: an oligomer whose biological

activity is affected by other substances binding to it– these substances change the enzyme’s activity by

altering the conformation(s) of its 4° structure

• Allosteric effectorAllosteric effector:: a substance that modifies the behavior of an allosteric enzyme; may be an– allosteric inhibitor– allosteric activator

Common Properties

• An allosteric protein/enzyme is one in which the binding of a molecule to one site affects the binding properties of another site on the same protein

• Allosteric proteins are those having other conformations induced by the binding of substrates or modulators.

• Most have multiple chains (quaternary structure)

Modulators or Effectors

• positive effectors increase catalytic activity

• negative effectors reduce or inhibit catalytic activity

• homotropic - the normal substrate and modulator/effector are identical

• heterotropic - modulator is a molecule other than the normal substrate

Rate vs concentration of substrate

• Allosteric enzymes are co-operative systems,in which a small change in one parameter, e.g. substrate, inhibitor, activator concentration, brings about a large change in velocity. A consequence of a cooperative system is that the v vs. S plot is sigmoidal not hyperbolic.

2 Models for Cooperativity

• Sequential Model – subunits change conformation one at a time

• Concerted Model – all subunits change conformation together

• Both postulate that enzyme subunits exist in one of two conformations, tensed (T) or relaxed (R), and that relaxed subunits bind substrate more readily than those in the tense state. The two models differ most in their assumptions about subunit interaction.

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The Concerted ModelThe Concerted Model• Wyman, Monod, and Changeux - 1965

• The enzyme has two conformations•• R (relaxed)R (relaxed):: binds substrate tightly; the active form

•• T (tight or taut)T (tight or taut):: binds substrate less tightly; the inactive form

• in the absence of substrate, most enzyme molecules are in the T (inactive) form

• the presence of substrate shifts the equilibrium from the T (inactive) form to the R (active) form

• in changing from T to R and vice versa, all subunits change conformation simultaneously; all changes are concerted

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C o n certed M o d e lC o n certed M o d e l• a h yp o th etica l p ro te in w ith tw o sub un its

• b o th ch an g e fro m T to R a t th e sam e tim eF ig u re 6 .4 (a) M o n o d -W ym an -C h an g eau x m o d el

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6 - 1 4© 2 0 0 3 T h o m s o n L e a rn in g , In c .A ll r ig h ts re s e rve d

C o n c e r t e d M o d e lC o n c e r t e d M o d e l• a n a l l o s t e r i c a c t i v a t o r ( A ) b i n d s t o a n d s t a b i l i z e s t h e R

( a c t i v e ) f o r m

• a n a l l o s t e r i c i n h ib i t o r ( I ) b i n d s t o a n d s t a b i l i z e s t h e T ( i n a c t i v e ) f o r m

F i g u r e 6 . 6 E f f e c t o f b in d in g a c t i v a t o r s a n d in h ib i t o r s

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6 - 1 6© 2 0 0 3 T h o m s o n L e a r n i n g , I n c .A l l r i g h t s r e s e r v e d

S e q u e n t i a l M o d e lS e q u e n t i a l M o d e lF i g u r e 6 . 7 S e q u e n t i a l m o d e l f o r c o o p e r a t i v e b i n d i n g o f s u b s t r a t e t o a n a l l o s t e r i c e n z y m e

Sequential Model

• Developed by Daniel Koshland• 3 Assumptions

– Only 2 conformational states possible; T and R– Binding of substrate induced conformational

change in that one subunit– This conformational change can increase or

decrease the substrate binding affinity of the other subunits. Subunits interact even if they are in different conformational states.

Schematic for Sequential Model

• T = tense, low-affinity state

• R = relaxed, high affinity state

• Rs = relaxed state with substrate bound

• TT k1 RsT k2 RsRs

– k2 > k1

Allosteric Regulation

• Allosteric enzymes are often regulatory enzymes – they control the rate of pathways

Example - ATCase

• Aspartate transcarbamoylase (ATCase) catalyzes the condensation of aspartate and carbamoyl phosphate, the committed step in the pathway for the synthesis of pyrimidine nucleotides such as cytidine triphosphate (CTP)

• ATCase is inhibited by CTP, the final product of the pathway – feedback or end-product inhibition

• CTP is structurally very different from substrates and products of ATCase

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ATCaseATCase• Figure 6.3 Organization of ATCase

• catalytic unit: 6 subunits organized into 3 trimers

• regulatory unit: 6 subunits organized into 3 trimers

Interaction of subunits

• Active site(s) located by crystallizing with PALA• PALA bind at sites lying at the boundaries

between pairs of C chains within the catalytic trimer. Most of the residues belong to one subunit, two key residues belong the a neighbor unit.

• Each R chain interacts with a C chain through a structural domain stabilized by a zinc ion bound to four cysteine residues.

Which model fits?

• There is a remarkable change in quaternary structure upon binding of PALA. The whole enzyme expands on binding.

• Concerted mechanism

• The position of the equilibrium between T and R states depends on the number of active sites that are occupied by substrate.

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ATCaseATCase

-O-P-O-P-O-P-O-CH2O

OHOH

HHHH

N

N

NH2

OO

O- O-

O

O-

O

Cytidine triphosphate (CTP)

-O-P-O-P-O-P-O-CH2O

OHOH

HHHH

O

O- O-

O

O-

O

Adenosine triphosphate (ATP)

N

NN

N

NH2

an allosteric inhibitor of ATCase

an allosteric activator of ATCase

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ATCaseATCaseFigure 6.2(b) ATCase catalysis in presence of CTP; ATP

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BiochemistryBiochemistryPowerPoint byPowerPoint byWilliam H. BrownWilliam H. BrownBeloit CollegeBeloit College

4e, by Mary K. Campbell4e, by Mary K. Campbell& Shawn O. Farrell& Shawn O. Farrell