Allosteric Enzymes

Models of Cooperativity
 Allosteric Enzymes
• Allosteric: Greek allo + steric, other shape
• Allosteric 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 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.
6 The Concerted Model
• Wyman, Monod, and Changeux - 1965
• The enzyme has two conformations
• R (relaxed): binds substrate tightly; the active form
• 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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6 C o n c e rte d M o d e l
• a h yp o th etica l p ro tein w ith tw o s u b u n its
• b o th ch a n g e fro m T to R at th e sam e tim e
F ig u re 6.4(a) M o n o d -W ym a n -C h an g eau x m o d el

© 2 0 0 3 T h o m s o n Le a rn in g , In c .
All rig h ts re s e rve d
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6 C o n c e rte d M o d e l
• a n a l lo s t e r ic a c t i v a t o r ( A ) b i n d s t o a n d s t a b ili z e s t h e R
(a c tiv e ) fo rm
• a n a l lo s t e r ic in h ib it o r ( I) b i n d s t o a n d s t a b i liz e s t h e T
( i n a c t iv 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

© 2 0 0 3 T h o m s o n L e a r n in g , In c .
A ll r ig h t s r e s e r v e d
6 -1 4
6 S e q u e n tia l M o d e l
F ig u r e 6 .7 S e q u e n tia l m o d e l fo r c o o p e r a tiv e b in d in g o f
s u b s tr a t e to a n a llo s te r ic e n z y m e

© 2 0 0 3 T h o m s o n L e a r n in g , In c .
A ll r ig h t s r e s e r v e d
6 -1 6
 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
6 ATCase
• Figure 6.3 Organization of ATCase
• catalytic unit: 6 subunits organized into 3 trimers
• regulatory unit: 6 subunits organized into 3 trimers

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 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.
6 ATCase NH2
N an allosteric
inhibitor of ATCase
O O O O N
-
O- P-O- P-O- P-O-CH2
O- O- O- O
H H
H H an allosteric
OH OH activator of ATCase
Cytidine triphosphate (CTP)
NH2
N N
O O O
- N N
O- P-O- P-O- P-O-CH 2
O- O- O- O
H H
H H
OH OH
Adenosine triphosphate (ATP)

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

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6

Biochemistry
PowerPoint by
4e, by Mary K. Campbell
William H. Brown & Shawn O. Farrell
Beloit College

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