This presentation provides a comprehensive account on allosteric enzymes and the mechanism of allosteric inhibition.

2.  Regulatory enzymes
 Allosteric enzymes
 Allosteric inhibition
 ATCase as an allosteric enzyme
 Phosphofructokinase as an allosteric enzyme

3. Most metabolic reactions are multi-step cascade processes.
In each enzyme system there is at least one enzyme that sets
the rate of the overall sequence because it catalyzes the rate-
limiting reaction.
These regulatory enzymes exhibit increased or decreased
catalytic activity in response to certain signals.

4. In most multi-enzyme systems the first enzyme that is specific
for that sequence is a regulatory enzyme.
Catalyzing even the first few reactions of a pathway that leads
to an unneeded product, diverts energy and metabolites from
more important processes.
An excellent place to regulate a metabolic pathway, therefore,
is at the point of commitment to the pathway.

5. The activity of regulatory enzymes is modulated through
various types of signal molecules, which are generally small
metabolites.
There are two major classes of enzyme regulation in
metabolic pathways.
These are reversible covalent modification and reversible non-
covalent modification.

6. Allosteric enzymes function through reversible, non-covalent
binding of a regulatory metabolite called a modulator.
The second class includes enzymes regulated by reversible
covalent modification.
Both classes of regulatory enzymes tend to have multiple
subunits.
In some cases the regulatory site(s) and the active site are on
separate subunits.

7. Regulatory Enzymes
Reversible non-covalent
modification:
AllostericRegulation
Allosteric
activation
Allosteric
Inhibition
Reversible covalent
modification
Adenylation
Uridylation ADP-
Ribosylation
Phosphorylation
Methylation

8. Allosteric enzymes are those having "other
shapes" or conformations induced by the
binding of modulators.
These enzymes have tworeceptor sites.
One site fits the substrate like other
enzymes.
The other site fits an inhibitor or activator
molecule.
Allosteric enzymes are very important in
feedback regulation.

10. In multi-enzyme systems the regulatory
enzyme is inhibited by the end product of the
pathway.
When the regulatory enzyme reaction is
slowed, all subsequent enzymes operate at
reduced rates.
The rate of production of the pathway's end
product is thereby brought into balance with
the cell's needs.
This type of regulation is called feedback
inhibition.
Buildup of the pathway's end product
ultimatelyslowsthe entire pathway.

12. Eg: bacterial enzyme system that catalyzes the
conversion of L-threonine into L-isoleucine.
In this system, the first enzyme, threonine
deaminase, is inhibited by isoleucine - the product.
Isoleucine is quite specific as an inhibitor.
Isoleucine binds not to the active site, but to another
specific site on the enzyme molecule, the regulatory
site: allosteric site.
This binding is non-covalent and thus readily
reversible.
Thus threonine dehydratase activity responds
rapidly and reversibly to fluctuations in the
concentration of isoleucine in the cell.

13. Regulatory enzymes for which substrate and modulator are
identical are called homotropic.
When the modulator is a molecule other than the substrate
the enzyme is heterotropic.
The properties of allosteric enzymes are significantly different
from those of simple non-regulatory enzymes.
Some of the differences are structural.

14. • In addition to active or catalytic sites, allosteric enzymes generally
have one or more regulatory or allosteric sites for binding the
modulator .
• Just as an enzyme's active site is specific for its substrate, the
allosteric site is specific for its modulator.
• Enzymes with several modulators generally have different specific
binding sitesfor each.
• In homotropic enzymes the active site and regulatory site are the
same.

15. Allosteric enzymes are also generally larger and more complex
than simple enzymes.
Most of them have two
or more polypeptide chains
or subunits.
Aspartate transcarbamoylase, has 12 polypeptide chains
organized into catalytic and regulatory subunits.

18. Allosteric enzymes show relationships
between V0 and [S] that differ from
normal Michaelis-Menten behavior.
They exhibit saturation with the
substrate when [S] is sufficiently high.
When V0 is plotted against [S] a
sigmoid saturation curveresults.
The symbol [S]0.5 or K0.5 is used to
represent the substrate concentration
giving half maximal velocity.

19. Substrate-activity curves for representative allosteric enzymes. Three examples of complex
responses given by allosteric enzymes to their modulators.
(a) The sigmoidcurve given by an allosteric enzyme, in which the substrate also serves as a
positive (stimulatory) modulator.
(b) The effects of a positive modulator, a negative modulator, and no modulator (K-type)
(c) Vmax is modulated with K0.5 nearly constant (Vtype)

20.  Sigmoid kinetic behavior generally reflects cooperative interactions
between multiple protein subunits.
 The principles are similar to those for cooperativity in oxygen binding to
the non-enzyme protein hemoglobin.
 The substrate can function as a positive modulator (an activator)
because the subunits act cooperatively.
 The binding of one molecule of the substrate to one binding site greatly
enhances the binding of subsequent substrate molecules.

21. The sigmoidal dependence of V0 on [S] reflects
subunit cooperativity, and has inspired two models
to explain these cooperative interactions.
In the concerted (symmetry model), proposed by
Jacques Monod and colleagues in 1965, an allosteric
enzyme can exist in only two conformations, active
and inactive
All subunits are in the active form or all are inactive.
In the second model (the sequential model),
proposed by Koshland in 1966, there are still two
conformations, but subunits can undergo the
conformational change individually.
Binding of substrate increases the probability of the
conformational change.

22. Allosteric
Enzymes
Regulatory
Enzymes
Have active
and
modulator
sites
Activated by
substrates and
other positive
modulators
Inhibited by
end product
Do not obey
Michaelis
Menten
kinetics.
Catalyze
irreversible
reactions
Normally composed
of multiple subunits
(identical/different)

23.  Aspartate transcarbamoylase (ATCase) is an allosteric enzyme which has 12 polypeptide
chains organized into catalytic and regulatory subunits.
 The enzyme catalyzes the first step in the synthesis of pyrimidines.
 The enzyme functions to catalyze the condensation of aspartate and carbamoyl
phosphate to form N-carbamoylaspartate and orthophosphate.
 The enzyme ultimately catalyzes the reaction that will yield cytidine triphosphate
(CTP).
 This allosteric enzyme is unique in that for high concentration of the final product
CTP, the enzyme activity is low.
 However, for low concentrations of the final product CTP, the enzymatic activity is
high.

28. • Phosphofructokinase (PFK) catalyzes the rate-limiting step in glycolysis and is the most important control
point.
• It catalyzes the first irreversible stepthat is unique to the glycolytic pathway;
• PFK is allostericallyinhibitedby ATP,PEP and allosterically activatedby ADP
• ATP binds to a site on PFK distinct from the active site, causing a conformational change resulting in rotation
of the positionsof Arg162 and Glu161.
• Movement of the side chain of this arginine from the active site lowers the affinity for fructose 6-phosphate.
• In the high affinity state, the positive charge on Arg 162 stabilizes the negative charge on phosphate of F6P
and Km is low.
• In the low affinity state, the negative charge on Glu 161 repels F6P and Km is high.

29. BlueandViolet:Subunits ADP(activator):Red;
Fructose -1,6-biphosphate: Green ADP-Product:Yellow

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Biochemistry , Fourth edition by Donald Voet and Judith G. Voet. Pages 467-479.
Ma k’ basic medical biochemistry: A clinical approach Second Edition by Colleen Smith and
Allan D. Marks Pages 138-154.
Biochemistry Fourth Edition by Reginald H. Garret and Charles M Grisham. Pages 452-480.
Principles of Biochemistry, Fifth Edition by Morgan, Horton, Scrimgeour and Perry. Pages
134-161.