Cooperativity and Allosterism

Team 2
Cristina Morales
Mariana Elizondo
Christian Boada

SEQUENTIAL MODEL (KNF)

Koshland, Nmthy & Filmer, 1966.

Explain anticooperative and cooperative
phenomena.
Based on the induced-fit theory:
INSTRUCTIVE MECHANISM.
The sequential model does not at all
take into account the symmetry
conservation and assumes the
existence of hybrid conformations.

 It is based in the following postulates:

The protein exists in one form A only in the absence of a ligand.
The ligand binding to one subunit induces a conformational change of
the subunit towards a conformation B.
The conformational change of the concerned subunit provokes
variations in interactions between subunits
The ligand binds preferentially to one of the two conformations. The
proposed mechanism confers a particular importance to interactions
between subunits, and the formalisms that describe the mechanism
depend on the geometry of the oligomeric molecule. If a tetramer is
considered, the nature of the interactions depends on the spatial
arrangement of the subunits.
All the sites are equivalent and independent in the absence of a ligand.

 Deviation from Michaelis law is expressed in terms of

the variation in the interaction between protomers as
the sites progressively become occupied.
Kt=[B]/[A]
Cooperative and anticooperative effects depend on:
Values of the constants KAB, KBB and KAA.
Geometry of the molecule.
Fraction of occupied sites:
KAA=1
KAA < KAB < KBB cooperativity
KAA > KAB > KBB anticooperativity
A , B: Conformations, LIGAND BINDS TO B.
Ks: Affinity constant.
Kt: Equilibrium between conformations.
KAB, KBB, KAA: Interaction variation related constants.

Ns: Average number of S bound to an enzyme molecule.

n: Number of subunits

TETRAHEDRAL TETRAMER MODEL

Species

Interactions

A3BS

3AB

A2B2S2

1BB, 4AB and 1 AA

AB3S3

3BB and 3AB

B4S4

6BB

SQUARE TETRAMER MODEL

 Tetrahedral model illustrates the importance of the geometry of the molecule in

enzymatic behavior.
In the square model, the proteins saturation and state functions are the same
since the conformational change of each protomer is induced by the substrate
binding during saturation.

ANTICOOPERATIVITY
The binding of the first ligand molecules
to an oligomeric protein decreases the
apparent affinity for the other ligand sites.
Example:
glyceraldehyde 3-phosphate dehydrogenase.

Anticooperativity arises when:

KAA > KAB > KBB.
Heterogeneity
differs
from
anticooperativity in that several site
categories exist with different substrate
affinities, which exist prior to ligand
binding.

 Half-site reactivity: The binding of the first ligand induces a structural modification
of the protein which confers a practically infinite dissociation constant to the residual
sites.
Flip-flop mechanism: Each protomer of the dimeric enzyme is alternately
phosphorylated by the substrate, while the other protomer is dephosphorylated -> E. coli alkaline phosphate.

THE GENERALIZED MODEL

Proposed by Weber in 1965.
Extension of the Adair model, taking
into account the conformational
mobility of the protein.

m conformations of the enzyme,

with n active sites. S: substrate.

Different equilibriums considered by Weber

Concerted model: protomers interaction > protein-ligand interaction

Sequential model: protein-ligand interaction > protomers interaction

THERMODYNAMICAL COUPLING BETWEEN LIGAND

BINDING ENERGY AND SUBUNIT INTERACTION ENERGY
Weber: Protein behavior analysis method for proteins displaying cooperative effects
in terms of variations in G, associated with both ligand binding and interactions
between protein subunits.
Monomer-dimer equilibrium:

Every difference in ligand affinity for the proteins two states, corresponds an equal difference
in the subunit interaction energy of the free and ligand associated form.

 If G(22) > G(12) : binding cooperative.

First order system: First S molecule

binding provokes the subunit
interaction energy change. Decreases
the subunit interaction if its
cooperative and increases if its
anticooperative.

Second order system: Second S

molecule binding induces de effect.
Increases the subunit interaction if its
cooperative and decreases if its
anticooperative.

Intermediate order system: Binding

of both ligand molecules contributes.

If G(0) > G(1) : dissociation favored.

KINETIC COOPERATIVITY:
RICARD MODEL
Ricards approach (1989) attempts to describe the way in which variation of
subunits interactions can change the catalytic constant.

Types of effect on the rate of the

catalyzed reaction due to the
interactions between subunits

Protomer arrangement contribution to

free activation energy: The interactions
change the rate of the conformational
transitions involved in the reaction.
Quaternary constraint contribution to
free activation energy: The interactions
between subunits can induce a distortion of
the catalytic site.

 Rate constant of a reaction:

KB: Boltzmann constant
h: Plancks constant
R: gas constant
T: absolute temperature

Global activation energy (G) of the catalyzed reaction:

Intrinsic energy contribution

(reaction without enzyme)

Rate constant of the catalyzed reaction:

Protomer arrangement
contribution

Quaternary constraint
contribution

 The theories developed on the basis of this formulation lie on three postulates:
In each catalytic transition state the quaternary constraints are relieved.
The minimum number of conformations is assumed (as in the MWC and
KNF bonding models).
The conformation of the ligand transition state complex is the same, or
energetically indistinguishable, for all transition states.
The three postulates take into account that maximum catalytic efficiency occurs
when the enzyme conformation is complementary to the transition state of the
reaction.

 Conclusions:
Weak interaction between subunits can imply a kinetic cooperative
phenomenon, although the substrate saturation curve is not a sigmoid.
Strong interaction between subunits:
The substrate binding cooperativity is positive and the kinetic
cooperativity must be positive.
The substrate binding cooperativity is negative and the kinetic
cooperativity can be negative or positive.
In a strong interaction between subunits, a strong catalytic cooperativity can
generate a substrate saturation curve with no sigmoidal shape.

COOPERATIVITY & ALLOSTERY

Allostery: A metabolite whose structure is different to that of the substrate,
on binding to a site, which is not the substrate binding site, induces an
alteration of the active center's properties, in terms substrate affinity and/or
catalytic efficiency (Monod et al., 1963).
Examples: Ribonucleotide reductases (monomeric structure), aspartate transcarbamylase from
certain bacteria and the yeast Saccharomyces cerevisiae.

Cooperativity: Indicates that the occupancy of one site in a oligomeric

enzyme influences the binding on the others. The substrate can generate this
phenomena.
Examples: Glyceraldehyde 3-phosphate dehydrogenase.

Sigmoidal curve
in cooperativity