Regulatory enzymes

Allosteric enzymes

Allosteric inhibition

ATCase as an allosteric enzyme

Phosphofructokinase as an allosteric enzyme

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.

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.

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.

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.

Regulatory Enzymes

Reversible non-covalent

modification:

Allosteric Regulation

Allosteric

activation

Allosteric

Inhibition

Reversible covalent

modification

Adenylation

Uridylation ADP-

Ribosylation

Phosphorylation

Methylation

Allosteric enzymes are those having "other shapes" or conformations induced by the binding of modulators.

These enzymes have two receptor 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.

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 ultimately slows the entire pathway.

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.

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.

• 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 sites for each.

• In homotropic enzymes the active site and regulatory site are the same.

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.

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 curve results.

The symbol [S]0.5 or K0.5 is used to

represent the substrate concentration

giving half maximal velocity.

Substrate-activity curves for representative allosteric enzymes. Three examples of complex

responses given by allosteric enzymes to their modulators.

(a) The sigmoid curve 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)

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.

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.

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)

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.

• Phosphofructokinase (PFK) catalyzes the rate-limiting step in glycolysis and is the most important control point.

• It catalyzes the first irreversible step that is unique to the glycolytic pathway;

• PFK is allosterically inhibited by ATP, PEP and allosterically activated by ADP

• ATP binds to a site on PFK distinct from the active site, causing a conformational change resulting in rotation of the positions of 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.

Blue and Violet : Subunits ADP (activator) : Red;

Fructose -1,6- bi phosphate: Green ADP- Product: Yellow

Lehninger Principles of Biochemistry, Fourth Edition by David L. Nelson and Michael M.

Cox. Pages 225-233.

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.