An Assignment on

Enzyme Inhibitors, Activation Energy, Enzyme Kinetics, Multienzyme complex

Submitted to,

Dr. R. Bhatnagar

Professor & Head

Department Of Biochemistry

BACA , AAU, Anand

Submitted by

Joshi Prathmesh Govind

M.Sc (Agri.) Biochemistry.

BACA, AAU, Anand.

WELCOME

Enzyme Inhibitors

Compounds which convert the enzymes into inactive substances and thus adversely

affect the rate of enzymatically-catalyzed reaction are called as enzyme inhibitors.

Such a process is known as enzyme inhibition.

Two broad classes of enzyme inhibitors are generally recognized : reversible and

nonreversible itself being subdivided into competitive and noncompetitive

inhibition. depending on whether the enzyme-inhibitor (EI) complex dissociates

rapidly or very slowly.

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Feedback Inhibition

Some enzymes catalyze the synthesis of small molecules (such as amino acids) in a

number of steps.

The enzyme catalyzing the first step in this biosynthesis is inhibited by the end

product of the reaction.

Such type of regulatory mechanism, which is called feedback inhibition, is

beautifully illustrated by the biosynthesis of isoleucine from threonine.

The reaction completes in 5 steps. The first step reaction is catalyzed by the enzyme

threomine aminase.

The activity of this enzyme is inhibited upon accumulation of high quantities of

isoleucine.

Isoleucine binds to a different site from threonine.

This is called allosteric interaction. However, when the level of isoleucine drops

sufficiently, the enzyme reactivates and isoleucine is resynthesized.

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Reversible Enzyme Inhibition

A reversible inhibitor dissociates very rapidly from its target enzyme because it

becomes very loosely bound with the enzyme.

Three general types of reversible inhibition are contents distinguished :

competitive,

noncompetitive and uncompetitive

Fig. Feedback inhibition of the first enzyme in a multistep reaction by reversible binding of the end product.

A. Competitive or Substrate analogue inhibition.

This type of competition occurs at the active site. Here the structure of the inhibitor

(I) closely resembles with that of the substrate (S).

It may, thus, combine with the enzyme (E), forming an enzyme-inhibitor (EI) complex

rather than an ES complex. the inhibitor, thus, competes with the substrate to

combine with the enzyme.

The degree of inhibition depends upon the relative concentrations of the substrate

and the inhibitor.

Thus, by increasing the substrate concentration and keeping the inhibitor

concentration constant, the amount of inhibition decreases and conversely a decrease

In substrate concentration results in an increased inhibition. It may, however, be

noted that in competitive inhibition, the enzyme can bind substrate (forming an ES

complex) or inhibitor (EI), but not both (ESI).

Thus, we see that a competitive inhibitor diminishes the rate of the reaction by

reducing the proportion of the enzyme molecules bound to a substrate.

Fig:- Competitive or substrate analogue inhibition

Example of competitive inhibition:

An enzyme, succinic acid dehydrogenase (= succinodehydrogenase) catalyzes the

conversion of succinic acid to fumaric acid. Many organic compounds, which are

structurally related to succinic acid, combine with the enzyme, thus inhibiting the

reaction.

B. Noncompetitive inhibition. Here no competition occurs between the substrate, S and the inhibitor.

The inhibitor has little or no structural resemblance with the substrate and it binds with

the enzyme at a place other than the active site (i.e., at the allosteric site).

Since I and S may combine at different sites, formation of both EI and ESI complexes

takes place.

Both ES and ESI may break down to produce the reaction product (P). It may, however,

be noted that in noncompetitive inhibition, the inhibitor and substrate can bind

simultaneously to an enzyme molecule since their binding sites are different and hence

do not overlap.

The enzyme is inactivated when inhibitor is bound, whether or not substrate is also

present.

Thus, it is apparent that a noncompetitive inhibitor acts by lowering the turnover

number rather than by decreasing the proportion of enzyme molecules that are bound to

the substrate. Noncompetitive inhibition, in contrast to competitive inhibition, cannot be

overcome by increasing substrate concentration.

Fig:- Non competitive inhibition

Example:-

Various heavy metals ions (Ag+, Hg2+, Pb2+) inhibit the activity of a variety of

enzymes. Urease, for example, is highly sensitive to any of these ions in traces. Heavy

metals form mercaptides with sulfhydryl (-SH) groups of enzymes :

C. Uncompetitive inhibition

An uncompetitive inhibitor also binds at an allosteric site (like the noncompetitive

inhibitors) but the binding takes place only with the enzyme-substrate (ES) complex, and

not the free enzyme molecule.

Irreversible Enzyme Inhibition

Although irreversible inhibition was once categorized and tested as noncompetitive

inhibition, it is now recognized as a distinct type of inhibition.

Irreversible inhibitors are those that combine with or destroy a functional group on the

enzyme that is essential for its activity.

In fact, an irreversible inhibitor dissociates very slowly from its target enzyme because it

becomes very tightly bound to its active site, thus inactivating the enzyme molecule.

The bonding between the inhibitor and enzyme may be covalent or noncovalent.

Example

Alkylating reagents, such as iodoacetamide, irreversibly inhibit the catalytic

activity of some enzymes by modifying cysteine and other side chains. Iodoacetamide

is a widely-used agent for the detection of sulfhydryl group.

Activation Energy

In all reactions there is an energy barrier that has to be overcome in order for the

reaction to proceed.

This is the energy needed to transform the substrate molecules into the transition

state an unstable chemical form part-way between the substrates and the products.

The transition state has the highest free energy of any component in the reaction

pathway.

The Gibbs free energy of activation (ΔG‡) is equal to the difference in free energy

between the transition state and the substrate.

An enzyme works by stabilizing the transition state of a chemical reaction and

decreasing G‡ .Δ

The enzyme does not alter the energy levels of the substrates or the products.

Thus an enzyme increases the rate at which the reaction occurs, but has no effect on

the overall change in energy of the reaction.

Free energy

The change in Gibbs free energy (ΔG) dictates whether a reaction will be

energetically favorable or not.

It should be noted that G is unrelated to G‡. The G of a reaction is independent of Δ Δ Δ

the path of the reaction, and it provides no information about the rate of a reaction

since the rate of the reaction is governed by G‡.Δ

A negative G indicates that the reaction is thermodynamically favorable in the Δ

direction indicated (i.e. that it is likely to occur without an input of energy), whereas a

positive G indicates that the reaction is not thermodynamically favorable and Δ

requires an input of energy to proceed in the direction indicated.

In biochemical systems, this input of energy is often achieved by coupling the

energetically unfavorable reaction with a more energetically favorable one (coupled

reactions).

It is often convenient to refer to G under a standard set of conditions, defined as when Δ

the substrates and products of a reaction are all present at concentrations of 1.0 M and

the reaction is taking place at a constant pH of 7.0.

Under these conditions a slightly different value for G is found, and this is called Δ ΔG0’.

An example of an energetically favorable reaction which has a large negative G and is Δ

commonly used to drive less energetically favorable reactions is the hydrolysis of

adenosine triphosphate to form adenosine diphosphate (ADP) and free inorganic

phosphate (Pi).

Fig:Free energy diagram for a simple chemical reaction, S P

Fig:Energy diagram, comparing the nonenzymatic and enzymatic reactions, S → P

Enzymes Kinetics

Enzyme velocity:

The rate of an enzyme-catalyzed reaction is often called its velocity.

Enzyme velocities are normally reported as values at time zero (initial velocity, symbol

V0; mol min1), since the rate is fastest at the point where no product is yet present.μ

This is because the substrate concentration is greatest before any substrate has been

transformed to product, because enzymes may be subject to feedback inhibition by

their own products and/or because with a reversible reaction the products will fuel \

the reverse reaction.

Experimentally V0 is measured before more than approximately 10% of the substrate

has been converted to product in order to minimize such complicating factors.

A typical plot of product formed against time for an enzyme-

catalyzed reaction shows an

initial period of rapid product formation which gives the linear portion of

the plot.

This is followed by a slowing down of the enzyme rate as substrate is used upand/or as

the enzyme loses activity.

V0 is obtained by drawing a straight line through the linear part of the curve, starting

at the zero time-point. The slope of this straight line is equal to V0.

Enzyme units Enzyme activity may be expressed in a number of ways. The commonest is by the

initial rate (V0) of the reaction being catalyzed (e.g. mol of substrate transformed μ

per minute; mol min1).μ

There are also two standard units of enzyme activity, the enzyme unit (U) and the

katal (kat). An enzyme unit is that amount of enzyme which will catalyze the

transformation of 1 mol of substrate per minute at 25C under optimal conditions μ

for that enzyme.

The katal is the accepted SI unit of enzyme activity and is defined as that catalytic

activity which will raise the rate of a reaction by one mole per second in a specified

system.

The term activity (or total activity) refers to the total units of enzyme in the sample,

whereas the specific activity is the number of enzyme units per milligram of protein

(units mg1).

The specific activity is a measure of the purity of an enzyme; during the purification

of the enzyme its specific activity increases and becomes maximal and constant when

the enzyme is pure.

substrate concentration :

The normal pattern of dependence of enzyme rate on substrate concentration

([S]) is that at low substrate concentrations a doubling of [S] will lead to a doubling of

the initial velocity (V0).

However, at higher substrate concentrations the enzyme becomes saturated, and

further increases in [S] lead to very small changes in V0.

This occurs because at saturating substrate concentrations effectively all of the

enzyme molecules have bound substrate.

The overall enzyme rate is now dependent on the rate at which the product can

dissociate from the enzyme, and adding further substrate will not affect this.

The shape of the resulting graph when V0 is plotted against [S] is called a hyperbolic

Curve.

Enzyme concentration:

In situations where the substrate concentration is saturating (i.e. all the enzyme

molecules are bound to substrate), a doubling of the enzyme concentration

will lead to a doubling of V0.

This gives a straight line graph when V0 is plotted against enzyme concentration.

Temperature:

Temperature affects the rate of enzyme-catalyzed reactions in two ways.

First, a rise in temperature increases the thermal energy of the substrate molecules.

This raises the proportion of substrate molecules with sufficient energy to overcome

the Gibbs free energy of activation, and hence increases the rate of the reaction.

However, a second effect comes into play at higher temperatures.

Increasing the thermal energy of the molecules which make up the protein structure

of the enzyme itself will increase the chances of breaking the multiple weak,

noncovalent interactions (hydrogen bonds, van der Waals forces, etc.) which hold the

three-dimensional structure of the enzyme together.

Ultimately this will lead to the denaturation (unfolding) of the enzyme, but even

small changes in the three-dimensional shape of the enzyme can alter the structure of

the active site and lead to a decrease in catalytic activity.

The overall effect of a rise in temperature on the reaction rate of the enzyme is a

balance between these two opposing effects.

A graph of temperature plotted against V0 will therefore show a curve, with a well-

defined temperature optimum.

For many mammalian enzymes this is around 37oc, but there are also organisms

which have enzymes adapted to working at considerably higher or lower

temperatures.

Cont…

Fig:Effect of temperature.

pH:

Each enzyme has an optimum pH at which the rate of the reaction that it

catalyzes is at its maximum.

Small deviations in pH from the optimum value lead to decreased activity due to

changes in the ionization of groups at the active site of the enzyme.

Larger deviations in pH lead to the denaturation of the enzyme protein itself,

due to interference with the many weak noncovalent bonds maintaining its

three-dimensional structure.

A graph of V0 plotted against pH will usually give a bell shaped curve.

Many enzymes have a pH optimum of around 6.8, but there is great diversity in the pH

optima of enzymes, due to the different environments in which they are adapted to

work.

For example, the digestive enzyme pepsin is adapted to work at the acidic pH

of the stomach (around pH 2.0).

Michaelis–Menten model

The Michaelis–Menten model uses the following concept of enzyme catalysis:

The enzyme (E), combines with its substrate (S) to form an enzyme–substrate

complex (ES).

The ES complex can dissociate again to form E+ S, or can proceed chemically to form E

and the product P.

The rate constants k1, k2 and k3 describe the rates associated with each step of the

catalytic process.

It is assumed that there is no significant rate for the backward reaction of enzyme and

product (E+P) being converted to ES complex.

[ES] remains approximately constant until nearly all the substrate is used, hence the

rate of synthesis of ES equals its rate of consumption over most of the course of the

reaction; that is, [ES] maintains a steady state.

The effect of (a) temperature and (b) pH on enzyme activity.

From the observation of the properties of many enzymes it was known

that the

initial velocity (V0) at low substrate concentrations is directly

proportional to

[S], while at high substrate concentrations the velocity tends towards a

maximum value, that is the rate becomes independent of [S].

This maximum velocity is called Vmax ( mol min1).μ

The initial velocity (V0) is the velocity measured experimentally before more than

approximately 10% of the substrate has been converted to product in order to

minimize such complicating factors as the effects of reversible reactions, inhibition of

the enzyme by product, and progressive inactivation of the enzyme.

Fig:The relationship between substrate concentration [S] and initial reaction velocity (V0 ).

The equation describes a hyperbolic curve of the type shown for the experimental

data.

In deriving the equation, Michaelis and Menten defined a new constant, Km, the

Michaelis constant [units: Molar (i.e. per mole), M]:

Km is a measure of the stability of the ES complex, being equal to the sum of the rates

of breakdown of ES over its rate of formation. For many enzymes k2 is much greater

than k3.

Under these circumstances Km becomes a measure of the affinity of an enzyme for

its substrate since its value depends on the relative values of k1 and k2 for ES

formation and dissociation, respectively.

A high Km indicates weak substrate binding (k2 predominant over k1), a low Km I

indicates strong substrate binding (k1 predominant over k2).

Lineweaver Burk Plot

Km can be determined experimentally by the fact that its value is equivalent to the

substrate concentration at which the velocity is equal to half of Vmax.

Because Vmax is achieved at infinite substrate concentration, it is impossible to

estimate Vmax (and hence Km) from a hyperbolic plot.

However, Vmax and Km can be determined experimentally by measuring V0 at

different substrate concentrations.

Then a double reciprocal or Lineweaver–Burk plot of 1/V0 against 1/[S] is made This plot is a derivation of the Michaelis–Menten equation:

which gives a straight line, with the intercept on the y-axis equal to 1/Vmax, and

the intercept on the x-axis equal to 1/Km.

The slope of the line is equal to Km/Vmax .

The Lineweaver–Burk plot is also a useful way of determining how an inhibitor binds

to an enzyme.

Although the Michaelis–Menten model provides a very good model of the

experimental data for many enzymes, a few enzymes do not confirm to Michaelis-

Menten kinetics.

These enzymes, such as aspartate transcarbamoylase (ATCase), are called allosteric

enzymes.

Fig:The relationship between substrate concentration [S] and initial reaction velocity (V0 ).(a) A direct plot, (b) a Lineweaver–Burk

double-reciprocal plot.

Multienzyme complex

Defination:-

A multienzyme complex in which a series of chemical intermediates remain bound to

the enzyme molecules as a substrate is transformed into the final product.

Five cofactors, four derived from vitamins, participate in the reaction mechanism.

The regulation of this enzyme complex also illustrates how a combination of covalent

modification and allosteric regulation results in precisely regulated flux through a

metabolic step.

Pyruvate dehydrogenase complex

From Escherichia coli is a large multienzyme cluster (MW = 48,00,000 ; Pigheart

has MW = 1,00,00,000) consisting of pyruvate dehydrogenase or pyruvate

decarboxylase (E1), dihydrolipoyl transacetylase (E2) and dihydrolipoyl

dehydrogenase (E3) and 5 coenzymes viz., thiamine pyrophosphate (TPP), lipoic acid

(LA), flavin adenine dinucleotide (FAD), coenzyme A (CoA) and nicotinamide adenine

dinucleotide (NAD+).

Four different vitamins required in human diet are vital components of this complex

enzyme.

These are thiamine (in TPP), riboflavin (in FAD), pantothenic acid (in CoA) and

nicotinamide (in NAD+).

Lipoic acid, however, is an essential vitamin or growth factor for many

microorganisms but not so for higher animals, where it can be made from readily

available precursors.

Fig:Space-filling model of the pyruvatedehydrogenase complex

α-Ketoglutarate dehydrogenase

A large multienzyme cluster consisting of 3 enzyme components, e.g: -α ketoglutarate

dehydrogenase or -α ketoglutarate decarboxylase (12 moles per mole of the complex),

transsuccinylase (24 moles) and dihydrolipoyl dehydrogenase (12 moles).

The complex also requires the same 5 coenzymes, as required by pyruvate

dehydrogenase complex, for activity, e.g: thiamine pyrophosphate (6 moles),lipoic and

(6 moles), flavine adenine dinucleotide (8 moles), coenzyme A and nicotinamide

adenine dinucleotide.

The compelx enzyme molecule is comparable in size to ribosomes.

The transsuccinylase (B′) component, like the transacetylase of PDC, forms the ‘core’ of

the multienzyme complex while the -α ketoglutarate dehydrogenase (A′) and

dihydrolipoyl dehydrogenase (C′) components are arranged on the periphery.

Again, A′ binds to B′ and B′ binds to C′ but A′ does not bind directly to C′ .

The -α ketoglutarate dehydrogenase (A′) and transsuccinylase (B′) components are

different from the corresponding components (A and B) in the pyruvate dehydrogenase

complex.

However, the dihydrolipoyl dehydrogenase components (C and C′) of the two enzyme

complexes are similar.

Lipoic acid is attached to the core transsuccinylase by forming an amide bond with

lysine side chains.

This places the reactive disulfide groups (—S—S—) at the end of a long flexible chain.

The ability of the chain to swing the disulfide group in contact with the different

proteins is an important feature of the enzyme complex.

As in the case of pyruvate oxidation, arsenite inhibits the reaction, causing the

substrate -ketoglutarate to accumulate.α

The -KDC is inhibited by both succinyl-CoA and NADH, the former being more α

effective.

Fatty Acid synthase complex

The core of the E. coli fatty acid synthase system consists of seven separate

polypeptides , and at least three others act at some stage of the process.

The proteins act together to catalyze the formation of fatty acids from acetyl-CoA and

malonyl-CoA.

Throughout the process, the intermediates remain covalently attached as thioesters

to one of two thiol groups of the synthase complex.

One point of attachment is the OSH group of a Cys residue in one of the seven

synthase proteins (β-ketoacyl-ACP synthase); the other is the OSH group of acyl

carrier protein.

Acyl carrier protein (ACP) of E. coli is a small protein (Mr 8,860) containing the

prosthetic group 4-phosphopantetheine.

Hydrolysis of thioesters is highly exergonic, and the energy released helps to make

two different steps in fatty acid synthesis (condensation) thermodynamically

favorable.

The 4-phosphopante-theine prosthetic group of ACP is believed to serve as a flexible

arm,tethering the growing fatty acyl chain to the surface of the fatty acid synthase

complex while carrying the reaction intermediates from one enzyme active site to the

next.Proteins of the Fatty Acid Synthase Complex of E.

coli

Tryptophan synthase

Tryptophan synthase or tryptophan synthetase is an enzyme that catalyzes the

final two steps in the biosynthesis of tryptophan.

It is commonly found in Eubacteria, Archaebacteria, Protista, Fungi, and Plantae.

However, it is absent from animalia. It is typically found as an 2 2 tetramer. The α β

α

subunits catalyze the reversible formation of insole and glyceraldehyde-3-

phosphate (G3P) from indole-3-glycerol phosphate (IGP).

The subunits catalyze the irreversible condensation of indole and serine to form β

tryptophan in a pyridoxal phosphate (PLP) dependent reaction.

Each active site is connected to a active site by a 25 angstrom long α β

hydrophobic

channel contained within the enzyme.

This facilitates the diffusion of indole formed at active sites directly to active α β

sites in a process known as substrate channeling.

The active sites of tryptophan synthase are allosterically coupled.

Enzyme Structure:-

Tryptophan synthase typically exists as an - - complex. α ββ α

The and subunits have molecular masses of 27 and 43 kDa respectivelyα β

The subunit has a TIM barrel conformation. α

The subunit has a fold type II conformation and a binding site adjacent to the β

active site for monovalent cations.

Their assembly into a complex leads to structural changes in both subunits

resulting in reciprocal activation.

There are two main mechanisms for intersubunit communication.

First, the COMM domain of the -subunit and the -loop2 of the -subunit β α α

interact. Additionally, there are interactions between the Gly181 and Ser178 α β

residues.

The active sites are regulated allosterically and undergo transitions between

open, inactive, and closed, active, states.

Mechanism of Tryptophan Synthase

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