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GROUP TWOS SEMINAR WORK

TOPIC: ENZYME REGULATION; ALLOSTERIC REGULATION

AND MODELS

OUTLINE:

1. Introduction

2. What is enzyme regulation?

3. Importance of enzyme regulation

4. Characteristics of regulatory enzymes

5. Devices employed in the regulation of enzyme.

6. Mechanisms of enzyme regulation

7. Allosteric regulation

7.1 Feedback inhibition

8. Models for the allosteric behavior of proteins

8.1 The symmetry model

8.2 The sequential model

9. K systems and v systems

10. Specific Examples

10.1 ATP and glucose-6-p are allosteric inhibitors of glycogen

phosphorylase

10.2 AMP is an allosteric activator of glycogen phosphorylase

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1. INTRODUCTION

Thousands of biochemical reactions occur simultaneously in a cell to

maintain its structure and functions, and hence keep it alive. Even

though the cells live in a constantly changing environment, the

intracellular environment has to be kept relatively constant. This

necessitates precise Control Mechanisms for regulating the rates ofmetabolic reactions. So that living cells neither synthesize nor

breakdown more materials than is required for normal metabolism and

growth at a given circumstance. Since enzymes are remarkable

components of these reactions (whose regulation sustain life), enzyme

regulation in turn regulate the reactions they catalyse.

2. WHAT IS ENZYME REGULATION?

Enzyme regulation is the control of enzyme catalyzed chemical

reactions that occur in living cells with the aid of devices such as

activators, inhibitor, inducers, repressors, etc, via mechanisms such as

genetic control, covalent modification, allosteric regulation, etc.

Enzymes can be controlled or regulated at two levels:

- Controlling the synthesis of the enzyme (genetic control) and

-Controlling the activity of the enzyme (post synthetic control).

3. IMPORTANCE OF ENZYME REGULATION

1. Conservation of Energy: Living cells constantly control energy

generating reactions so that they consume just enough nutrients

to meet their energy requirement. For example, mammalian cells

both catabolize and synthesize glucose. The rates at which these

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reactions occur must be regulated; otherwise, energy is wasted

by what is called a futile cycle carrying out opposing reactions

at high rates with no net substrate flow in either direction.

2. Responsiveness to Environmental Changes: Cells can make

relatively rapid adjustment to changes in temperature, pH, ionic

strength and nutrient concentrations because of their capacity to

increase or decrease the rate of specific reactions.

3. Maintenance of an Ordered State: Regulation of each

pathway result in the production of the substances required for

the maintenance of cell structure and function in a timely fashion

and without excesses.

4.CHARACTERISTICS OF REGULATORY ENZYMES

1. Inhibition of a regulatory enzyme by a feedback inhibitor does not

conform to any normal inhibition pattern, and the feedback inhibitor

bears little structural similarity to the substrate for the regulatory

enzyme. The allosteric inhibitor apparently acts at a binding site

distinct from the substrate-binding site. The term allosteric is apt,

because inhibitor is sterically dissimilar and, moreover, acts at a site

other than the site for substrate. Its effect is called allosteric

inhibition.

2. Regulatory enzymes frequently catalyze thermodynamically

irreversible reactions, that is, a large negative free energy change (-

G) greatly favors formation of a given metabolic product rather than

the reverse reaction. Thus, regulation of enzyme activity, usually at the

committed step of the pathway, is critical for supplying and

maintaining cellular metabolic and energy homeostasis.

3. Regulatory enzymes are usually the enzymes that are the rate-

limiting or committed step, in a pathway.

4. Frequently, regulatory enzymes are at or near the initial steps in a

pathway, or part of a branch point or cross-over point between

pathways (where a metabolite can be potentially converted into

several products in different pathways).

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5. Their kinetics do not obey the Michaelis - Menten equation. Their v

versus [S] plots yield sigmoid- or S-shaped curves rather than

rectangular hyperbolas. Such curves suggest a second-order (or

higher) relationship between vand [S]; that is, vis proportional to [S]n,

where n > 1. A qualitative description of the mechanism responsiblefor the S-shaped curves is that binding of one S to a protein molecule

makes it easier for additional substrate molecules to bind to the same

protein molecule. In allostery, substrate binding is cooperative.

Figure 1.Sigmoid vversus [S] plot.

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5. DEVICES EMPLOYED IN THE REGULATION OF ENZYME.

1. Effectors (Activators and Inhibitors)

2. Inducers and Repressors

3. Changes in the concentration of substrate and co-factors

4. Degradative mechanism

5. Intra-molecular conformational changes

6. Compartmentalization

These devices are employed by the various mechanisms by

which enzyme activities are regulated. For example:

Effectors are employed in allosteric regulation. Allosteric

effectors are small organic molecules that regulate the activity

of an enzyme for which it is neither the substrate nor the

immediate product. They are also known as allosteric

modulator. The effectors can be activators when they

enhance the activity of the enzyme and they can be

inhibitors when they impede the enzyme activity.

Inducers and repressors are employed by the genetic

control. The gene that codes for the enzyme can be regulated

with regard to the number of copies of corresponding mRNA

that are produced (i.e. transcribed), and therefore the number

of enzyme molecules that are produced.

6. MECHANISMS OF ENZYME REGULATION

Regulation by altering the rate of synthesis or degradation of

enzyme proteins

Zymogen activation

Isozyme activities

Regulation by compartmentalization of enzymes

Allosteric regulation

Covalent modification

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7. ALLOSTERIC REGULATION

The term allosteric derives from the Greek word for another shape

(or state), thereby indicating that all enzymes capable of allosteric

regulation can exist in two different states. In one of the two forms, theenzyme has a high affinity for its substrate, whereas in the other form,

it has little or no affinity for its substrate. Enzymes with this property

are called allosteric enzymes. The two different forms of an allosteric

enzyme are readily interconvertible and are, in fact, in equilibrium with

each other. Obviously, the reaction rate is high when the enzyme is in

its high-affinity form and low or even zero when the enzyme is in its

low-affinity form. Allosteric enzymes are regulated by molecules known

as effectors or modulators that non-covalently bind to the enzyme

site other than the active site. This site is called allosteric site.

Whether the active or inactive form of an allosteric enzyme is favored

depends on the cellular concentration of the appropriate regulatory

substance, called an allosteric effector. In the case of isoleucine

synthesis, the allosteric enzyme is threonine deaminase and the

allosteric effector is isoleucine

An allosteric effector influences enzyme activity by binding to one of

the two interconvertible forms of the enzyme, thereby stabilizing it inthat state. In other words, an allosteric enzyme can exist in either a

complexed or uncomplexed form, depending on whether it has an

effector molecule bound to it or not. In fact, some allosteric enzymes

have multiple allosteric sites, each capable of recognizing a different

effector. Effectors exert their effect by altering the conformation of the

enzyme in a way affecting the binding of the substrate to the enzyme,

or the catalytic efficiency of the enzyme. That means they may

increase or decrease the Km value or increase or decrease the Vmax.

Allosteric inhibitors increase the Km value and decrease the Vmaxwhereas allosteric activators decrease the Km value and increase the

Vmax.

When the effector is different from the substrate the effect is known as

heterotropic effect. An example is the allosteric activation of isocitrate

dehydrogenase by ADP. When the substrate itself serves as an effector

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the effect is called homotropic effect. Often used as the model of a

homotropic effect is the binding of O2 molecules to hemoglobin heme

groups, although hemoglobin is not an enzyme. The binding of one O2

molecule to one heme enhances the binding of O2 molecules to other

heme groups. This is also known as cooperative binding.

This means that, as the multiple catalytic sites on the enzyme bind

substrate molecules, the enzyme undergoes conformational

changes (intramolecular conformational change, which is one of the

devices employed in allosteric regulation) that affect the affinity of the

remaining sites for substrate. Some enzymes show positive

cooperativity, in which the binding of a substrate molecule to one

catalytic subunit increases the affinity of the other catalytic subunits

for substrate. Other enzymes show negative cooperativity, in which thesubstrate binding to one catalytic subunit reduces the affinity of the

other catalytic sites for substrate.

7 FEED BACK INHIBITION

This occurs when an end product of a metabolic sequence accumulates

and turns off one or more enzymes needed for its own formation. It is

often the first enzyme unique to the specificbiosynthetic pathway for

the product that is inhibited. When a cell makes two or moreisoenzymes, only one of them may be inhibited by a particular product.

Such type of inhibition is a common regulatory mechanism in

biosynthetic pathway. An example is aspartate transcarbomylase

feedback inhibition by CTP in bacterial pyrimidine biosynthesis. The

starting materials for pyrimidine biosynthesis are carbamoylphosphate

and aspartate. Aspartate transcarbamoylase converts aspartate and

carbamoylphosphate to carbamoylaspartate. CTP, the end product of

the pathway inhibits aspartate transcarbamoylase by feedbackallosteric inhibition.

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In allosteric feedback regulation the end product inhibits the first or the

second enzyme in the pathway to prevent wastage of energy and rawmaterials.

A second pattern of allosteric control may be referred to as precursor

activation or feed-forward control. A metabolite acting as an

allosteric effector turns on an enzyme that either acts directly on that

metabolite or acts on a product that lies further ahead in the

sequence. An actual example is provided by glycogen synthase, whose

inactive dependent or D form is activated allosterically by the

glycogen precursor, glucose 6-phosphate.

8. MODELS FOR THE ALLOSTERIC BEHAVIOR OF

PROTEINS

Two quite different models were proposed to explain the unusual

nature of the hemoglobin oxygen-binding curve. Even though they

were first proposed to explain the allosteric behavior of hemoglobin,

the models can also be used to explain other allosteric proteins,

because hemoglobin is the best understood regulatory protein and

provides a model system for understanding how other allostericproteins work.

8.1 THE SYMMETRY MODEL

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In 1965, Jacques Monod, Jeffries Wyman, and Jean-Pierre Changeux

proposed a theoretical model of allosteric transitions based on the

observation that allosteric proteins are oligomers. They suggested that

allosteric proteins can exist in (at least) two conformational states,

designated R, signifying relaxed, and T, or taut/tense and that, ineach protein molecule, all of the subunits have the same conformation

(either R or T). That is, molecular symmetry is conserved. Molecules of

mixed conformation (having subunits of both R and T states) are not

allowed by this model.

In the absence of substrate, the two states of the allosteric

protein are in equilibrium:

R0

T0

In the absence of substrate, nearly all the enzyme molecules are in the

T form. When substrate is added to a solution of the allosteric protein

at conformational equilibrium, although the relative [R0] concentration

is small, S will bind only to R0, forming R1. This depletes the

concentration of R0, perturbing the T0/R0 equilibrium. To restore

equilibrium, molecules in the T0 conformation undergo a transition to

R0. This shift renders more R0 available to bind substrate, yielding R1

and diminishing [R0], perturbing the T0/R0 equilibrium, and so on. Thus,these linked equilibria are such that substrate-binding by the R0 state

of the allosteric protein perturbs the T0/R0 equilibrium with the result

that S-binding drives the conformational transition, T0 R 0.

Hence, the proportions of enzymes in R form increases progressively

as more substrate is added, and so the binding of substrate is

cooperative. When all the enzymes are fully saturated, all of the

enzyme molecules are in R form.

The effect of allosteric activators and inhibitors can readily be

accounted for by this concerted model. An allosteric inhibitor binds

preferentially to the T form, whereas, an allosteric activator binds

preferentially to R form. Consequently, an allosteric inhibitor shifts the

R T conformational equilibrium towards T, whereas an allosteric

activator shifts it towards R.

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In this model, homotrophic effects are necessarily positive, whereas

heterotrophic effect can either be positive or negative. For example,

the cooperative binding of N-carbamoyl phosphate and aspartate by

ATCase is by homotropic effect; inhibition by CTP is a negative

heterotropic affect; and activation by ATP is a positive heterotropiceffect.

8.2 THE SEQUENTIAL MODEL

In 1966, Koshland, Nemethy and Filmer proposed an allosteric model in

which ligand-induced conformational changes caused transition to a

conformational state with altered affinities. Because they considered

ligand binding and conformational transitions to be distinct steps in a

sequential pathway, the Koshland, Nemethy, Filmer (or KNF) model is

named the sequential model for allosteric transitions. The modelmakes three assumptions

1. There are only two conformational states (R and T) accessible to

any subunit

2. The binding of substrate changes the shape of the subunit to

which it is bound. However, the conformations of the other

subunits in the enzyme molecule are not appreciably altered.

3. The conformational change elicited by the binding of substrate in

one subunit can increase or decrease the substrate-bindingaffinity of the other subunits in the same enzyme molecule.

This model differs from the symmetry/concerted model in the

following ways:

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1. It does not assume an equilibrium between R and T forms in the

absence of substrate. Rather, the conformational transition

between T and R is induced by the binding of substrate.

2. The conformational change from T to R in different subunits of an

enzyme molecule is sequential, not concerted.3. The concerted model supposes that symmetry is essential for the

interaction of subunits in oligomeric protein, therefore must be

conserved in allosteric transition. In contrast, the sequential

model assumes that subunits can interact even if they are in

different conformational states.

4. Homotropic interaction can be either positive or negative in this

model, unlike in the symmetry model where it only be positive.

Whether the second substrate can be bound more or less tightly

than the first depend on the nature of the distortion induced bythe binding of the first substrate molecule.

9. K Systems and V Systems

The allosteric models just presented is called a K system because the

concentration of substrate giving half-maximal velocity, defined as K0.5,

changes in response to effectors and Vmax is constant.

An allosteric situation where K0.5 is constant but the apparent

Vmax changes in response to effectors is termed a V system. In a V

system, all vversus [S] plots are hyperbolic rather than sigmoid. The

positive heterotropic effector activates by raising Vmax, whereas the

negative heterotropic effector, decreases it. Note that K0.5 is not

affected. This situation arises if R and T have the same affinity for the

substrate, but differ in their catalytic ability and their affinities for

activator and inhibitor. Activator and Inhibitor thus can shift the

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relative T/R distribution. Acetyl-coenzyme A carboxylase, the enzyme

catalyzing the committed step in the fatty acid biosynthetic pathway,

behaves as a V system in response to its allosteric activator, citrate.

10. SPECIFIC EXAMPLES

10.1 ATP and Glucose-6-P Are Allosteric Inhibitors of Glycogen

Phosphorylase

ATP can be viewed as the end product of glycogen phosphorylase

action, in that the glucose-1-P liberated by glycogen phosphorylase isdegraded in muscle via metabolic pathways whose purpose is energy

(ATP) production. Glucose-1-P is readily converted into glucose-6-P to

feed such pathways. (In the liver, glucose-1-P from glycogen is

converted to glucose and released into the bloodstream to raise blood

glucose levels.) Thus, feedback inhibition of glycogen phosphorylase

by ATP and glucose-6-P provides a very effective way to regulate

glycogen breakdown. Both ATP and glucose-6-P act by decreasing the

affinity of glycogen phosphorylase for its substrate Pi. Because the

binding of ATP or glucose-6-P has a negative effect on substrate

binding, these substances act as negative heterotropic effectors. When

concentrations of ATP or glucose-6-P accumulate to high levels,

glycogen phosphorylase is inhibited; when [ATP] and [glucose-6-P] are

low, the activity of glycogen phosphorylase is regulated by availability

of its substrate, Pi.

10.2 AMP Is an Allosteric Activator of Glycogen Phosphorylase

AMP also provides a regulatory signal to glycogen phosphorylase. Itbinds to the same site as ATP, but it stimulates glycogen

phosphorylase rather than inhibiting it. AMP acts as a positive

heterotropic effector, meaning that it enhances the binding of

substrate to glycogen phosphorylase. Significant levels of AMP indicate

that the energy status of the cell is low and that more energy (ATP)

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should be produced. Reciprocal changes in the cellular concentrations

of ATP and AMP and their competition for binding to the same site (the

allosteric site) on glycogen phosphorylase, with opposite effects, allow

these two nucleotides to exert rapid and reversible control over

glycogen phosphorylase activity. Such reciprocal regulation ensuresthat the production of energy (ATP) is commensurate with cellular

needs.

To summarize, muscle glycogen phosphorylase is allosterically

activated by AMP and inhibited by ATP and glucose-6-P; caffeine can

also act as an allosteric inhibitor. When ATP and glucose-6-P are

abundant, glycogen breakdown is inhibited. When cellular energy

reserves are low (i.e., high [AMP] and low [ATP] and [G-6-P]), glycogen

catabolism is stimulated.

Glycogen phosphorylase conforms to the Monod - Wyman -

Changeux model of allosteric transitions, with the active form of the

enzyme designated the R state and the inactive form denoted as the

T state. Thus, AMP promotes the conversion to the active R state,

whereas ATP, glucose-6-P, and caffeine favor conversion to the inactive

T state.

CONCLUSION

In conclusion, allosteric regulation is an efficient mechanism of enzyme

regulation which employs devices to regulate metabolic reactions and

hence, maintain orderliness and cellular homeostasis.

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