Alosteric enzymes by arooj arshad

OBJECTIVE
The objective of this assignment is
• To have a detailed overview of Allosteric Enzymes
• To understand the mechanism and kinetics of Allosteric Enzyme
• To understand the importance of allosteric enzyme

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Allosteric Enzymes
an overview



Enzymology
Arooj Arshad
University of Management and Technology
Enzymology

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CONTENT
Introduction
Objectives
Allosterice Enzymes
Types of Effector
Dependenc of allosteric Enzymes
Control of Allosteric Enzyme behavior and feed back inhibition
Models to determine the behavior of allosteric enzymes
Concerted Model
Sequantial Model
Examples
Referneces

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ALLOSTERIC ENZYME AN OVERVIEW

INTRODUCTION
When one talk about protein, the most flexible and the most resilient macromolecule of
the living cell: chiefly represent the composite and multiplex structure encoded by the
genes in the form of folded chain of amino acids. The conception of word protein is
from Greek word “Prota” which means “cardinal significance” forged by Jacob
Berzilus. The beacon of protein chemistry was built by Fredrick Sanger and his
companions at Cambridge University when after the research of ten years finally they
got succeeded in sorting out the primary structure of Insulin. Since that time
tremendous advancement and discoveries has been observed in the field of proteomics
and with the increasing knowledge and discoveries the importance of proteins has rose
to the peak , most scientists focused their attention to determine “How do proteins
work, how do they fold , and what are the mechanisms and incidents hidden behind the
wonderful working of proteins. Of course evolution in protein study has left behind, the
folklores and philosophies such as the philosophy of Aristotle: connecting rennet to the
fire coming from the heat of animal body. In the modern era evidences are based upon
chemical, biological and systematic facts. Scientists like Perutz, John cowdry, etc.
worked and by 1970s and 1980s work in the field made scientist wise sensible enough to

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admit that the proteins covers the most area of cell biology and that organisms has been
surviving through various chemical reactions involving proteins directly or indirectly.
Since protein has been playing various roles, one of the most formidable and
astonishing role it plays is the role which it plays as a catalyst. And when we talk about
catalyst the instead of using word protein we use the specific term “Enzyme” referring
the catalytic role of proteins. Enzymes greatly increase the speed of the chemical
reactions. This catalytic capability of enzyme proteins aid the processes of life chiefly all
kind of life forms either viruses or men. The study of enzymes since has become
another field of study called as “Enzymology”
If we has to define enzyme in simple words then ,” Enzyme is the protein catalyst that
aid chemical reactions to get completed with increased speed in less time, without
itself getting mandated during the reaction being catalyzed by them”.
In other words, enzymes don’t start any chemical reaction rather it aids the chemical
reaction to precede to finish line with more efficiency and with more speed. It’s not
being produced or utilized during the chemical reactions.
All enzymes are very large and complex and spatial structure of proteins that can be
globular and they have great molecular weighing from 14000 to 400,000 Da. Since the
structure of an enzyme is of great importance, it’s critical because it determines the
specificity and activity of enzyme according to the reaction and substrate. We are very
well familiar with terms like Active site (the pockets of an enzyme that containing
specific sequence of amino acids making it complementary to the substrate surface,
cofactors (the non-protein part of an enzyme), Holoenzyme, Apoenzyme prosthetic
group, etc. But in this assignment I will be writing about another most important
structural property of enzyme which makes it unique and more specific. And settle it on
a different place in the room full of enzymes.
And that structural property is the “Allosteric Site” which sends such enzyme to
another category of enzymes that is “The Allosteric enzymes”. So this chapter will
chiefly focus on “Allosteric Enzymes”.
OBJECTIVE
The objective of this assignment is

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 To have a detailed overview of Allosteric Enzymes
 To understand the mechanism and kinetics of Allosteric Enzyme
 To understand the importance of allosteric enzyme

ALLOSTERIC ENZYMES
Allosteric Enzyme in simple words is the enzymes that are being controlled and
regulated by the action of Modulators or modifiers or Effectors.
The term allosteric has been derived from two words “ allo means other “ and “ steric
means shape”, raising the fact that conformation affects the behavior of such proteins.

Effectors are the modifiers which bind to the enzyme on the site other than active site
and this attachment is non-covalent. Allosteric enzymes showcase adaptable and
legislate catalytic activity after binding to its modulator (Effector). This modulation can
either inhibit or stimulate the activity of the allosteric enzyme.

The mechanism of allosteric activity and modulation chiefly depends upon the flexible
design of the enzyme which permits the enzyme to flutter the functions of its active site.
Anyway the effectors or ligands for allosteric enzymes are of two types
1- Positive Effectors
2- Negative Effectors
These molecules (effectors) are very much different in structure and design of their
shape is very much different when we compare it with the design of substrate.

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Since the effectors binds on the site other than the active site, so the sites upon which
allosteric effectors bind are called “Allosteric sites or Regulatory site”.


DEPENDENCE OF ALLOSTERIC CONTROL
The allosteric control chiefly depends upon the binding sites for modulators or effectors
and on at least two conformational cases of enzyme, First one in which the binding site
has high compatibility for the substrate and leads to the strong binding of the substrate
to binding site and the second is the state during which there is very much low affinity
for substrate and hence substrate binds weakly to the binding site.

When positive effector binds to the allosteric site of an enzyme, it stabilizes the most
possible shape of the enzyme and hence activates the enzyme.
While upon binding to the negative effector the enzyme is being stabilized in an
inactive state

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BEHAVIOR OF ALLOSTERIC ENZYMES
When we talk about the behavior of enzymes then through literature study we can use
michalis-menten model (which assumes the rapid state of equilibrium between
reactants (Enzymes and substrate) and enzyme-substrate complex, but when it comes to
allosteric enzyme behavior then michilus-menten model cannot explain it.
For example: Both ATCase and Hemoglobin displays common aftermath produced
through precise change in the quaternary structure of the proteins.
On other hands allosteric enzymes show a different response in the presence of
inhibitors.

CONTROL OF ALLOSTERIC ENZYMES AND FEED BACK INHIBITION
Aspartate transcarbamoylase the classic case
The first step of the reaction for production of Cytidine triphosphate (an important
nucleoside needed for the production of DNA and RNA) is catalyzed by the ATCase,
though this reaction is consisted of many series.
The pathway for the production of RNA and DNA nucleoside is very much extravagant
and involves many steps which require high amount of energy. Which means such
reaction not only needed to proceed but overproduction is also needed to be controlled?
So the CTP is the good example for the control of the over production of the products.

CTP regulate ATCase in negative manner which means in the presence of CTP there is
no need to make more, since the product of the reaction is controlling the activity or
inhibiting the activity of the enzyme which catalyzed the first step for the production of
that product, this mechanism is called as feedback Inhibition also known as end
product inhibition.

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However feedback inhibition isn’t only confined to the behavior of allosteric enzyme
but too many metabolic processes yet the ATCase is a classic example.


The condensation of aspartate and carbonyl-phosphate is catalyzed by ATCase to form
carbomyle aspartate.

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ATCase is regulated positively by the ATP, which means more ATP leads to the
production of more pyrimidine for the DNA and RNA also the CTP Is less in amount.
ATCase exhibits sigmoidal kinetics with respect to its substrate that is aspartate
The kinetics of allosteric kinetics is being described in the term of low and high affinity.
Low affinity is the T-state and High affinity is the R-state.
In the low amount of asp, the enzyme has low affinity but when the Asp increases the
enzyme switches toward the high affinity of R-state. The monitoring of enzyme is done
through the fraction of maximum rate of catalysis that is measure of the occupancy of
catalytic site.

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 When there is only substrate present then the reaction rates follow sigmoidal
curve C.
 When CTP starts binding then Curve D favors the Low affinity state that is the T
state which shifted the curve to right side.
 In contrast when ATP binds then Curve B favors the high affinity which means
the curve is shifted to the left.
 If ATP is in high amount ( curve A) then there is no T-state hyperbolic curve is
being followed by ATCase and this curve if of pure R-state

For allosteric enzymes, the concentration of substrate giving vo/Vmax = 0.5 is
designated as K' or K0.5, and it displays the visible substrate affinity, just as P50
represents O2 affinity for hemoglobin. Although KM is derived in a similar
manner, the term KM is not used for allosteric enzymes because they don't
follow the Michaelis-Menten equation.

The existence of multiple quaternary structure is the key to allosteric behavior
and it includes both cooperatively and modifications.

There are two most important terms that is used while learning about Allosteric
enzymes

1- Homotropic effects:
literal meaning is the
change which is being
induced by same
individuals; these effects
are the allosteric
interaction which occurs
when many identical
molecules get bound to a
protein. The switching of T
to R state describes the
homotropic effect.

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2- Heterotropic effects: such interactions which occurs between different
substances.




T to R state switch by binding of ATP with ATCase shows the positive heterotropic
effect.



R to T state switched through
the binding of CTP with ATCase
also called as negative heterotropic effect

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Composition of ATCase

 The catalytic site and substrate binding site is contained within the c catalytic
subunit and it can be in T or R state.

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 ATP/CTP subunits are contained within the r regulatory subunit and this subunit
actually control the c subunit by determining its T or state and hence the further
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MODELS TO UNDERSTAND THE BEHAVIOR OF ALLOSTERIC ENZYMES
There are two models so far through which we can describe and understand the
behavior of the allosteric enzymes. The concerted and the sequential model, proposed
in 1965 and 1966; both models are significant and useful for the assumption of the
results that one can obtain during the allosteric enzyme experiments.
The concerted model is simpler when we compare it with sequential model thought
both of the models are very much realistic and describe the enzyme system very well.

CONCERTED MODEL
The concerted model was proposed by Jacquas Monord, Wyman and Pierre for the
behavior of allosteric enzyme and now has become classic of biochemical knowledge
and literature.

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Conformation of Protein
There are two conformations of protein, The R (relaxed) conformation or the active
conformation that bind tightly to the substrate. The other is T (tight or taut)
conformation it’s inactive and binds very loosely to the substrate.
The property of this model is that all of the substrates changed their conformations
together. The figure below describes that the hypothetical protein with two subunits,
both of the subunits changes conformation from tight to active conformation at the
same time. Means a concerted change occurs in the conformation of protein. The
equilibrium ration for T/R is L and is supposed to be very high.


Initially the amount of R form enzyme is small, but after the binding of substrate the
free R form is removed and causes the high production of more R form. Hence makes
up more R in order to establish the equilibrium state. The effects of inhibitors and
activators can also be assumed in terms of the exchanging the equilibrium state between
T and R forms of the enzyme. And it became possible through the concerted model or
Monod-Wyman –Changeus Model.

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When there is an Activator present (A) the binding of A shifts the forms between T and
R , and R form is most favored, as a result there is less need for the substrate. To shift
the equilibrium.

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SEQUANTIAL MODEL


The sequential model was put forward by Daniel Koshland, according to this model the
binding of the substrate shifts the T form to R form, the postulate similar to induced fit
model. It shows that it makes it easy a subunit to change their conformation through the
effect of another subunit. As a result co-operative mechanism is shown in this model.
The induced fit model also explains the activator and inhibitor binding in this model.
In short the binding of the activator or inhibitor to a subunit induces the change in the
conformation of another subunit.

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SIGNIFICANT ROLES THAT ALOSTERIC ENZYMES PLAY
 Allosteric Enzymes as biosensors for biological diagnosis
Biosensors are the meld gadgets which covert the chemical information to an
analytically beneficial source through the biochemical mechanisms.
It basically consist of the receptor system, in which a component interact with the given
analytic. And as a result get coupled with the physiochemical transducer that increases
the signals which result by such interactions up to macroscopically level.
Allosteric enzymatic regulation is very critical as the base of speedy and reliable
diagnosis through targeted molecular signal and sensing. Bothe allosteric and non-
allosteric enzymes can be manipulated and engineered for specific activities.

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Enzymes Engineered as allosteric biosensors
Beta-Galactosidase
Alkaline Phosphatase
Beta-Lactamase



 Human phenylalanine hydroxylase, a highly regulated allosteric enzyme
Phenylalanine hydroxylase (PAH) is a tetrahydrobiopterin (BH4)-dependent enzyme
through which the degradation od phenylalanine rate limiting step is catalyzed.
Excessive amounts of L-Phe is toxic to the brain and in patients with the disease
phenylketonuria (PKU), if the person donot follow the restricted protein diet then
dysfunctional PAH thus leads to the damage which is irreversible and leads to the
serious consequances.

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REFERENCES
 Campbell and farre,Introduction to Biochemistry,India Edition.

 Robert A. ,Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis.
2000 by Wiley-VCH, Inc. ISBNs: 0-471-35929-7 (Hardback); 0-471-
22063-9 (Electronic).


 Antonio Villaverde, Allosteric enzymes as biosensors for molecular diagnosis, Received 4
August 2003; revised 15 September 2003; accepted 3 October 2003,First published online 16
October 2003,Edited by Judit.

 The ¤venot, D.R., Toth, K., Durst, R.A. and Wilson, G.S. (2001) Biosens. Bioelectron. 16,
121^131.


 Yeo, G.F. and Madsen, B.W. (1998) Biochim. Biophys. Acta 1372, 37^44.
 Nakamura, H. and Karube, I. (2003) Anal Bioanal. Chem., in press.

 ROSSEN DONEV , ADVANCES IN PROTEIN CHEMISTRY AND STRUCTURAL
BIOLOGY ,Protein Structure and Diseases,Institute of Life Science College of Medicine,
Swansea University Swansea United Kingdom.


 Marte I. Flydal1,2, Martín Alcorlo3, Lars Skjærven1, Ines Muñoz4, Knut Teigen1, Juan A.
Hermoso3, Aurora Martinez1, Structural and dynamics studies of human phenylalanine
hydroxylase, a highly regulated allosteric enzyme, Poster in Acta CrystallographicaSection
A:Foundations and Advances August 2016.

 Berg, Tymoczko & Stryer, 6th ed., Chapter 10, pp. 275-283.


 ENZYMOLOGY ,Introduction,P.C. Misra Professor Department of Biochemistry Lucknow
University Lucknow-226 0078,12-May-2006 (Revised 17-Aug- 2006)
 BIOCHEMISTRY AND MOLECULAR BIOLOGY,Dr peter M.D Hardwicks,problem unit
two 1999/2000