ALLOSTERISM

Allosteric enzyme.
• Allosteric enzymes are enzymes that change
their conformational ensemble upon binding
of an effector, which results in an apparent
change in binding affinity at a different ligand
binding site.
• Allosteric means “other site”
• These enzymes have two Binding sites .
Whose properties are affected by
changes in the conformation
• Active site- site fits the substrate
like other enzymes
• The other site – allosteric site - fits
an inhibitor or activator molecule
noncovalently
An allosteric site is a site at which a small
regulatory molecule interacts with an enzyme
to inhibit or activate that specific enzyme;
which is different from the active site where
catalytic activity occurs. The binding of the
allosteric effector is in general noncovalent
and reversible.
• Cooperativity, in enzymology, a phenomenon
in which the shape of one subunit of an
enzyme consisting of several subunits is
altered by the substrate (the substance upon
which an enzyme acts to form a product) or
some other molecule so as to change the
shape of a neighbouring subunit
 NEGATIVE AND POSITIVE COOPERATIVITY
In the case of positive cooperativity the fraction of T states exceeds
that of the R state and the binding of ligand increases the amount of
R state, thus increases the ease of ligand binding.

In the case of negative cooperativity, the fraction of the T state is

smaller than that of the R state. Thus, the initial binding affinity is
high. However, the binding of ligand increases the amount of T state,
thus reducing the binding affinity.
 Allosteric Enzymes Do Not Obey
Michaelis-Menten Kinetics
• The Michaelis-Menten model has greatly assisted the development
of enzyme chemistry. Its virtues are simplicity andbroad
applicability. However, the Michaelis-Menten model cannot
account for the kinetic properties of many enzymes.An important
group of enzymes that do not obey Michaelis-Menten kinetics
comprises the allosteric enzymes. These enzymes consist of
multiple subunits and multiple active sites

• Allosteric enzymes often display sigmoidal plots of the reaction

velocity V 0 versus substrate concentration[S], rather than the
hyperbolic plots predicted by the Michaelis-Menten equation
(equation 23). In allosteric enzymes, the binding of substrate to
one active site can affect the properties of other active sites in the
same enzyme molecule.
•A possible outcome of this interaction between subunits is
that the binding of substrate becomes cooperative; that is,
the binding of substrate to one active site of the enzyme
facilitates substrate binding to the other active sites. Such
cooperativity results in a sigmoidal plot of V 0 versus [S]. In
addition, the activity of an allosteric enzyme may be altered
by regulatory molecules that are reversibly bound to specific
sites other than the catalytic sites. The catalytic properties of
allosteric enzymes can thus be adjusted to meet the
immediate needs of a cell. For this reason, allosteric enzymes
are key regulators of metabolic pathways in the cell
Michaelis-Menten Kinetics. Kinetics for an Allosteric Enzyme
 Properties of Allosteric Enzymes
• There are distinct properties of Allosteric Enzymes that makes
it different compared to other enzymes.

• (1) One is that allosteric enzymes do not follow the Michaelis-

Menten Kinetics. This is because allosteric enzymes have
multiple active sites. These multiple active sites exhibit the
property of cooperativity, where the binding of one active site
affects the affinity of other active sites on the enzyme. As
mentioned earlier, it is these other affected active sites that
result in a sigmoidal curve for allosteric enzymes.
• Allosteric Enzymes are influenced by substrate concentration. For
example, at high concentrations of substrate, more enzymes are
found in the R state. The T state is favorite when there is an
insufficient amount of substrate to bind to the enzyme. In other
words, the T and R state equilibrium depends on the concentration
of the substrate.
•Allosteric Enzymes are regulated by other molecules.
This is seen when the molecules 2,3-BPG, pH, and
CO2 modulates the binding affinity of hemoglobin to
oxygen. 2,3-BPG reduces binding affinity of O2 to
hemoglobin by stabilizing the T- state. Lowering the
pH from physiological pH=7.4 to 7.2 (pH in the
muscles and tissues)favors the release of O2.
Hemoglobin is more likely to release oxygen in CO2
rich areas in the body.
 Allosteric Regulation
Allosteric regulation is a mechanism of regulation that usually
involves key enzymes in metabolic pathways
•One common example of allosteric regulation is feedback
inhibition, where the final product of a metabolic pathway is
an allosteric inhibitor of the first enzyme in the pathway (and
effectively shuts down the pathway when the product begins
to accumulate)
 Models for Allosteric Behavior
The symmetry model of Monod, Wyman and Changeux:
•The allosteric enzyme has an oligomeric quaternary structure (i.e. a
dimer, trimer, tetramer, etc)
•All molecules within the oligomer adopt the same conformation (i.e.
homogenous with regard to structure of monomeric units)
•Two distinctly different conformational states are available to the
monomeric units. One is the so-called "relaxed" or R state, the other is
the "taut" or T state
•If no effector molecule is bound, then the R and T states are indicated by
a '0' subscript: R0 and T0 (and if effector molecule is bound, then this is
indicated by a subscript of '1', as in R1 and T1)
•The R0 and T0 states exist in equilibrium:
 •The equilibrium constant L is assumed to be large (most of the enzyme oligomers
will assume the homogenous T state in the absence of bound effector molecules)
•The R and the T states have different affinities for substrate. The substrate
dissociation constant for the R state is defined as KR and the dissociation constant for
the T state is defined as KT. A high-affinity for substrate is characterized by a low value
for the dissociation constant. The model assumes that the R state has much higher
affinity for substrate (i.e. lower dissociation constant) than the T state. One way to
think about this type of model is to say that substrate only binds to the R state (and
therefore, the T1 state essentially is never found).
 Concerted model
The concerted model of allostery, also referred to as the
symmetry model or MWC model postulates that enzyme
subunits are connected in such a way that a conformational
change in one subunit is necessarily conferred to all other
subunits. Thus, all subunits must exist in the same
conformation. The model further holds that, in the absence of
any ligand (substrate or otherwise), the equilibrium favors one
of the conformational states, T or R. The equilibrium can be
shifted to the R or T state through the binding of one ligand(the
allosteric effector or ligand) to a site that is different from the
active site (the allosteric site).
 SEQUENTIAL MODEL
The sequential model of allosteric regulation holds that subunits are not connected
in such a way that a conformational change in one induces a similar change in the
others. Thus, all enzyme subunits do not necessitate the same conformation.
Moreover, the sequential model dictates that molecules of a substrate bind via
an induced fit protocol. In general, when a subunit randomly collides with a
molecule of substrate the active site, in essence, forms a glove around its substrate.
While such an induced fit converts a subunit from the tensed state to relaxed state,
it does not propagate the conformational change to adjacent subunits. Instead,
substrate-binding at one subunit only slightly alters the structure of other subunits
so that their binding sites are more receptive to substrate. To summarize:
subunits need not exist in the same conformation molecules of substrate bind via
induced-fit protocol conformational changes are not propagated to all subunits
 COOPERATIVITY

The affinity of hemoglobin for oxygen is less than its structural analog myoglobin.
Interestingly enough, however, this does not affect hemoglobin's usefulness for the
body; on the contrary, it allows hemoglobin to be a more efficient oxygen carrier
than myoglobin. This is so because hemoglobin can release oxygen more easily than
can myoglobin. While it is important for oxygen to be carried to different areas of the
body, it is even more important for the oxygen to be released when needed. The
higher the affinity of a given protein for oxygen, the harder it will be for that protein
to release oxygen when the time comes.

Thus, hemoglobin's lower affinity for oxygen serves it well because it allows
hemoglobin to release oxygen more easily in the body. Myoglobin, on the other
hand, has a significantly higher affinity for oxygen and will, therefore, be much less
inclined to release it once it is bound.

Thus hemoglobin's lower affinity for oxygen relative to myoglobin allows it to have a
higher overall efficiency in binding and then releasing oxygen species. For this
reason, the body tends to use hemoglobin more often for oxygen-distributing
purposes, although myoglobin is used as well, particularly for carrying oxygen to
muscle cells. More can be read on myoglobin in the appropriate section.
.
When oxygen is bound to hemoglobin, the color changes to crimson red. When
oxygen is not bound, the color becomes a dark "rustic" shade of red [1] .
Hemoglobin's affinity to oxygen increases as more oxygen is bound to it. The
disassociation curve represents how hemoglobin is cooperative to oxygen with its
sigmoidal shape. - The left shift shows an increase in oxygen affinity. Hemoglobin
has a better chance to hold onto oxygen. This normally occurs with a change in
environmental factors such as low temperature, low metabolism rate, and high
pH.
- The right shift shows a decrease in affinity. Hemoglobin is more likely to release
Oxygen. This is due to high temperature, high metabolism, and low pH.
While Hemoglobin has 4 subunits, Myoglobin has one subunit. It is the enzyme of
oxygen storage within the cells (found in skeletal muscle cells). The reason
muscles are red is because they contain large amount of myoglobin. Organisms
such as diving mammals have very large amounts of myoglobin so that they can go
for an extended period of time without breathing.
As mentioned above, hemoglobin exists in two distinct states: the T-
state and the R-state. The T-state of hemoglobin is the more "Tense" of
the two; this is the deoxy form of hemoglobin (meaning that it lacks an
oxygen species) and is also known as "deoxyhemoglobin. The R-state of
hemoglobin is more "Relaxed" and is the fully oxygenated form; it is also
known as
 ATCase

• The enzyme aspartate transcarbamoylase

(ATCase) is an allosteric enzyme that catalyzes
the first step in the synthesis of pyrimidines.
 Structure of ATCase
• ATCase is made of six regulatory subunits and six catalytic subunits.
The 3 regulatory subunits (r) are dimers made of 2 chains of 17 kd
each. The smaller of the two, the regulatory subunit can bind to CTP
and thus shows no catalytic activity. The 2 catalytic subunits (c) are
trimers consisting of 3 chains, each 34 kd. The catalytic subunit is
unresponsive to CTP thus does not follow a sigmoidal behavior.
• The quaternary structure of ATCase is composed of the two
catalytic trimers stacked one on top of another. The inhibitory effect
of CTP, the stimulatory action of ATP, and the cooperative binding of
substates are all accompanied by large changes in the quaternary
structure of ATCase.
• Each r chain of each the regulatory subunit binds with a c chain of
the catalytic trimer. The region of contact on between the r chain
and c chain is stabilized by a domain of zinc bound to histidine
residues in the r chain. All c chains have contact with the regulatory
subunit.
The quaternary structure of ATCase is composed of the two catalytic trimers stacked one
on top of another. The inhibitory effect of CTP, the stimulatory action of ATP, and the
cooperative binding of substates are all accompanied by large changes in the quaternary
structure of ATCase.

Each r chain of each the regulatory subunit binds with a c chain of the catalytic trimer. The
region of contact on between the r chain and c chain is stabilized by a domain of zinc
bound to histidine residues in the r chain. All c chains have contact with the regulatory
subunit.
The catalytic and regulatory subunits can be separated by first adding a mercurial
compounds and then by ultracentrifugation. Mercurial compounds breaks the
connection because it displaces the Zinc ion, destabilizing the r-subunit domain. The
reaction does not follow a Michaelis-Menten behavior, instead it produces a sigmoidal
curve because of the response changes in the substrate concentration via regulation
by other molecules and of the changes in binding probability. The addition of more
substrate has two effects on increasing the probability that the enzyme will bind more
than one substrate molecule while increasing the average amount of substrates bound
to each enzyme. More substrate ultimately favors the R-state of ATCase since
equilibrium depends on the number of active sites that are occupied by the substrate
which is completely opposite of Michaelis-Menten behavior.
Heterotropic effects are allosteric interactions that occur when
different substances (such as inhibitor and activators) are bound to
the protein.
In the ATCase reaction,
inhibition by CTP and activation by ATP are both heterotropic
effects.