Enzymes

ENZYMES
DR.Usman Saleem
Pharm.D R.Ph

What Are Enzymes?
In 1878, Kuhne coined the term enzyme from the greek enzumos , which refers
to the leavening of bread by yeast.
However, the modern term refers to:
The biological catalysts in the form of globular proteins that
facilitate chemical reactions in the cell of living organisms.
The vast majority of all known enzymes are globular proteins.
Ribozymes are enzymes made of ribonucleic acids.

What Are General Characteristics of Enzymes?
Enzymes are well suited to their essential roles in living organisms in three
major ways:
1. They have enormous catalytic power,
2. They are highly specific in the reactions they catalyze, and
3. Their activity as catalysts can be regulated.

Enzymes are true catalysts that speed
chemical reactions by lowering activation
energies and allowing reactions to achieve
equilibrium more rapidly.
They increase reaction rates by anywhere
from 109 to 1020 times.

Enzymes, unlike other catalysts, are often quite specific in the type of reaction
they catalyze and even the particular substance that will be involved in the
reaction.
For example, strong acids catalyze the hydrolysis of any amide, the dehydration
of any alcohol, and a variety of other processes.
However, the enzyme urease catalyzes only the hydrolysis of a single amide,
urea. (Absolute specificity)

Other enzymes display relative specificity by catalyzing the reaction of
structurally related substances.
For example, the lipases catalyze the hydrolysis of any triglycerides.
The specificity of enzymes also extends to stereochemical specificity.
For example, the enzyme arginase hydrolyzes the amino acid L-arginine but has
no effect on its enantiomer, D-arginine.

A third significant property of enzymes is that their catalytic behavior can be
regulated.
Even though each living cell contains thousands of different molecules that
could react with each other in an almost unlimited number of ways, only a
relatively small number of these possible reactions take place because of the
enzymes present.
The cell controls the rates of these reactions and the amount of any given
product formed by regulating the action of the enzymes.

How Are Enzymes Named?
Some of the earliest discovered enzymes were given names ending with -in to indicate
their protein composition.
For example, three of the digestive enzymes that catalyze protein hydrolysis are named
pepsin, trypsin, and chymotrypsin.
However, these names provide no information regarding enzyme function or the
substrate on which enzyme is acting.
For this purpose, the International Union of Biochemistry (IUB) adopted a systematic
nomenclature of enzymes that was prepared by its Enzyme Commission (EC).

In the EC system, each enzyme has an unambiguous (and often long) systematic
name that Specifies:
 The substrate (substance acted on),
 The functional group acted on, and
 the type of reaction catalyzed.
All EC names end in -ase.

The hydrolysis of urea provides a typical example:
EC Name: urea amidohydrolase
Substrate: urea
Functional Group: amide
Type of Reaction: hydrolysis

Enzymes are also assigned common names, which are usually shorter and more
convenient.
Common names are derived by adding -ase to the name of the substrate or to a
combination of the substrate name and type of reaction.
For example, urea amidohydrolase is assigned the common name urease:
Substrate: Urea
Common Name: Urea + ase = Urease

The enzyme name alcohol dehydrogenase is an example of a common name
derived from both the name of the substrate and the type of reaction:
Substrate: alcohol (ethyl alcohol)
Reaction type: dehydrogenation (removal of hydrogen)
Common name: alcohol dehydrogenation + ase = alcohol dehydrogenase

How Are Enzymes Classified?
 According to IUB system of enzyme classification, enzymes are grouped into six major
classes on the basis of the reaction catalyzed.
NO. GROUP NAME TYPE OF REACTION CATALYZED
1
2
3
4
5
6
Oxidoreductases
Transferases
Hydrolases
Lyases
Isomerases
Ligases
Oxidation–reduction reactions
Transfer of functional groups
Hydrolysis reactions
Addition to double bonds or the reverse of that reaction
Isomerization reactions
Formation of bonds with ATP cleavage

Typical Example
Aspartate aminotransferase or Aspartate
transaminase
Reaction Catalyzed
Typical Example
Lactate dehydrogenase
Reaction Catalyzed

Typical Example
Acetylcholinesterase
Reaction Catalyzed
Typical Example
Aconitase
Reaction Catalyzed

Typical Example
Phosphohexose isomerase
Reaction Catalyzed
Typical Example
Tyrosine-tRNA synthetase
Reaction Catalyzed

What Is the Terminology Used with Enzymes?
Many enzymes are simple proteins, whereas many others function only in the
presence of specific nonprotein molecules or metal ions.
If these nonprotein components are tightly bound to and form an integral part
of the enzyme structure, they are true prosthetic groups.
However, often a nonprotein component is only weakly bound to the enzyme
and is easily separated from the protein structure. This type of nonprotein
component is referred to as a cofactor.
The cofactors may be metallic ions, such as Zn2+ or Mg2+ , or organic compounds.
When the cofactor is an organic substance, it is called a coenzyme.

The protein portion of enzymes requiring a cofactor is called the apoenzyme.
Thus, the combination of an apoenzyme and a cofactor produces an active
enzyme:
Apoenzyme
Cofactor
(Coenzyme
or Inorganic
ions)
Active
Enzyme

An interesting feature of coenzymes is that many of them are formed in the
body from vitamins.
For example, the coenzyme nicotinamide adenine dinucleotide (NAD+), which is
a necessary part of some enzyme-catalyzed oxidation–reduction reactions, is
formed from the vitamin precursor nicotinamide.

What Are the Mechanisms of Enzyme Action?
About 100 years ago, Arrhenius suggested that catalysts speed up reactions by
combining with the substrate to form some kind of intermediate compound.
In an enzyme-catalyzed reaction, this intermediate is called the enzyme–
substrate (ES) complex.
The ES complex is formed when a substrate molecule binds to the active site of
an enzyme.
This binding occurs through hydrophobic interactions, hydrogen binding, and/or
ionic binding.

Once this complex is formed, the conversion of substrate (S) to product
(P) may take place:
General reaction:

The chemical transformation of the substrate occurs at the active site, usually
aided by enzyme functional groups that participate directly in the making and
breaking of chemical bonds.
After chemical conversion has occurred, the product is released from the active
site, and the enzyme is free for another round of catalysis.
To account for the high substrate specificity of most enzyme-catalyzed
reactions, a number of models have been proposed.
A. Lock-and-Key Model
B. Induced-Fit Model

According to the lock-and-key theory, enzyme surfaces will accommodate only
those substrates having specific shapes and sizes.
Thus, only specific substrates “fit” a given enzyme and can form complexes with
it, just as only the proper key can fit exactly into a lock and turn it open.
A limitation of the lock-and-key theory is the implication that enzyme
conformations are fixed or rigid.

Fig: In the lock-and-key model, the rigid enzyme and substrate have matching shapes.

Induced-fit model was introduced by an American biochemist, Daniel Koshland.
Induced-fit model proposes that enzymes have somewhat flexible
conformations to accommodate incoming substrates.
The active site has a shape that becomes complementary to that of the
substrate only after the substrate is bound.

Fig: In the induced-fit model, the flexible enzyme changes shape to match the substrate.

What is Enzyme Activity?
Enzyme activity refers in general to the catalytic ability of an enzyme to
increase the rate of a reaction.
Enzyme activity is measured by turnover number, which is defined as:
“The number of substrate molecules converted into product per unit time,
when the enzyme is fully saturated with substrate.”
For most enzymes, the turnover numbers fall between 1 to 104 per
second.
The turnover number of 600,000/sec for carbonic anhydrase is one of the
largest known.

What Factors Influence Enzyme Activity?
Several factors affect the rate of enzyme-catalyzed reactions.
The most important factors are:
1. Enzyme concentration,
2. Substrate concentration,
3. Temperature, and
4. pH.

In an enzyme-catalyzed reaction, the concentration of enzyme is normally very
low compared with the concentration of substrate.
When the enzyme concentration is increased, the concentration of ES also
increases in compliance with reaction rate theory:

Thus, If we keep the concentration of
substrate constant and increase the
concentration of enzyme, the rate
increases linearly.
That is, if the enzyme concentration
doubles, the rate of conversion of
substrate to product doubles as well.

Conversely, if we keep the
concentration of enzyme constant and
increase the concentration of
substrate, we get a saturation curve.
In this case, the rate does not increase
continuously.
Instead, a point is reached after which
the rate stays the same even if we
increase the substrate concentration
further.

This happens because at the saturation point, substrate molecules are bound to
all available active sites of the enzymes and the reaction is proceeding at its
maximum rate (symbolized by Vmax).
Increasing the substrate concentration can no longer increase the rate because
the excess substrate cannot find any active sites to which to bind.

Enzyme-catalyzed reactions, like all
chemical reactions, have rates that
increase with temperature.
However, because enzymes are
proteins, there is a temperature limit
beyond which the enzyme becomes
vulnerable to denaturation.

Thus, every enzyme catalyzed reaction
has an optimum temperature, usually
in the range 25°C–40°C.
Above or below that value, the
reaction rate will be lower.

As the pH of its environment changes
the conformation of a protein, we
would expect pH-related effects to
resemble those observed when the
temperature changes.
Each enzyme operates best at a
certain optimum pH.
Many enzymes have an optimum pH
near 7, the pH of most biological fluids.

Once again, within a narrow pH range,
changes in enzyme activity are
reversible.
However, at extreme pH values (either
acidic or basic), enzymes are
denatured irreversibly and enzyme
activity cannot be restored by
changing back to the optimal pH.

The Michaelis Menten Approach to Enzyme
Kinetics
A particularly useful model for the kinetics of enzyme-catalyzed reactions was
devised in 1913 by Leonor Michaelis and Maud Menten.
It helps to describe many enzymatic reactions under the following assumptions:
 The reaction has only one substrate
 The substrate concentration is much higher than that of the enzyme in the system
 Only the initial rate of enzyme activity is measured.
A typical reaction might be the conversion of some substrate, S, to a product, P.
The stoichiometric equation for the reaction is:
E S

Kinetics
The overall reaction can be written as:
In this reaction, an enzyme (E) combines with the substrate (S) to form ES
complex with a rate constant k1.
The ES complex formed can dissociate back to E and S with a rate constant k-1, or
it can give rise to a product (P) and regenerated enzyme with a rate constant k2 .
1

Kinetics
In the Michaelis–Menten model, the initial rate, V, of the formation of product
depends only on the rate of the breakdown of the ES complex,
V = k2 [ES]
The rate of formation of enzyme-substrate complex, ES is:
Rate of formation of ES = k1 [E][S]
The ES complex breaks down in two reactions, by returning to enzyme and
substrate or by giving rise to product and releasing enzyme.
2
3

Kinetics
The rate of disappearance of complex is the sum of the rates of the two
reactions.
Rate of breakdown of ES = k-1 [ES] + k2 [ES]
OR
Rate of breakdown of ES = (k-1 + k2 ) [ES]
Enzymes are capable of processing the substrate very efficiently, and a steady
state is soon reached in which the rate of formation of the enzyme–substrate
complex equals the rate of its breakdown.
4
5

Kinetics
According to the steady-state theory, then, the rate of formation of the enzyme–
substrate complex equals the rate of its breakdown,
k1 [E][S] = (k-1 + k2 ) [ES]
By rearranging the above equation, we obtain
[E][S] / [ES] = (k-1 + k2 ) / k1
Collecting all the rate constants or the individual reactions,
KM = (k-1 + k2 ) / k1
Where KM is called the Michaelis constant.
6
7
8

Kinetics
Inserting equation 8 into equation 7, we get
[E][S] / [ES] = KM
and solving for [ES] yields
[ES] = [E][S] / KM
To solve for the concentration of the complex, [ES], it is necessary to know the
concentration of the other species involved in the reaction.
The initial concentration of substrate is a known experimental condition and
does not change significantly during the initial stages of the reaction.
9
10

Kinetics
The substrate concentration is much greater than the enzyme concentration.
The total concentration of the enzyme, [E]T, is also known, but a large
proportion of it may be involved in the complex.
The concentration of free enzyme, [E], is the difference between [E]T, the total
concentration, and [ES], which can be written as an equation:
[E] = [E] T – [ES]
Substituting for the concentration of free enzyme, [E], in Equation 10, we get
[ES] = ([E] T – [ES]) [S] / KM
11
12

Kinetics
[ES] = [E]T [S] – [ES] [S] / KM
[E]T [S] – [ES] [S] = [ES] KM
[E]T [S] = [ES] (KM + [S])
[ES] = [E]T [S] / KM + [S]
By substituting this expression for [ES] into equation 2, we obtain
V = k2 [E]T [S] / KM + [S]
13
14
15
16
17

Kinetics
The maximal rate, V max, is attained when the catalytic sites on the enzyme are
saturated with substrate that is, when
[ES] = [E]T
Thus,
Vmax = k2 [E]T
Substituting equation 18 into equation 17 yields the Michaelis-Menten
equation:
V = Vmax [S] / KM + [S]
18
19

Kinetics
Figure shows the effect of increasing
substrate concentration on the observed
rate.
In such an experiment, the reaction is run at
several substrate concentrations, and the
rate is determined by way of any convenient
method.
At low-substrate concentrations, first-order
kinetics are observed.

Kinetics
At higher substrate concentrations, when
the enzyme is saturated, the constant
reaction rate characteristic of zero-order
kinetics is observed.
This constant rate, when the enzyme is
saturated with substrate, is the Vmax for
the enzyme, a value that can be roughly
estimated from the graph.
The value of KM can also be estimated
from the graph.

Kinetics
When experimental conditions are adjusted so that
[S] = KM
Then,
V = Vmax / 2
Thus, KM is equal to the substrate concentration at which the reaction rate is
half its maximal value.
KM is an important characteristic of an enzyme-catalyzed reaction and is
significant for its biological function.

What is Enzyme Inhibition?
An enzyme inhibitor is any substance that can decrease the rate of an enzyme-
catalyzed reaction.
Such a process is known as enzyme inhibition.
Enzyme inhibitors are classified into two categories on the basis of how they
behave at the molecular level.
1. Reversible Inhibitors
2. Irreversible Inhibitors

An irreversible inhibitor forms a covalent bond with a specific functional group
of the enzyme and as a result renders the enzyme inactive.
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.
A number of very deadly poisons act as irreversible inhibitors.

The cyanide ion (CN2) is an example of an irreversible enzyme inhibitor.
It is extremely toxic and acts very rapidly. The cyanide ion interferes with the
operation of an iron-containing enzyme called cytochrome oxidase.
The ability of cells to use oxygen depends on the action of cytochrome oxidase.
When the cyanide ion reacts with the iron of this enzyme, it forms a very stable
complex and the enzyme can no longer function properly.
As a result, cell respiration stops, causing death in a matter of minutes.

A reversible inhibitor (in contrast to one that is irreversible) reversibly binds to
an enzyme.
A reversible inhibitor dissociates very rapidly from its target enzyme because it
becomes very loosely bound with the enzyme.
There are two types of reversible inhibitors:
a. Competitive and
b. Non-competitive.

A competitive inhibitor binds to the active site of an enzyme and thus
“competes” with substrate molecules for the active site.
Competitive inhibitors often have molecular structures that are similar to the
normal substrate of the enzyme.

The nature of competitive inhibition is
represented in Figure.
There is competition between the
substrate and the inhibitor for the
active site.
Once the inhibitor combines with the
enzyme, the active site is blocked,
preventing further catalytic action.

Example 1
The competitive inhibition of succinate dehydrogenase by malonate is a classic
example.
Succinate dehydrogenase catalyzes the oxidation of the substrate succinate to
form fumarate by transferring two hydrogens to the coenzyme FAD:

Malonate, having a structure similar to succinate, competes for the active site of
succinate dehydrogenase and thus inhibits the enzyme.
The enzyme can be also inhibited by oxalate and glutarate because of the
similarity of this substance with succinate.

Example 2
The action of sulfa drugs on bacteria is another example of competitive enzyme
inhibition.
Folic acid, a substance needed for growth by some disease-causing bacteria, is
normally synthesized within the bacteria by a chemical process that requires p-
aminobenzoic acid (PABA).
Sulfanilamide and other sulfa drugs are structural analogues of PABA.

Because sulfanilamide, the first sulfa
drug, resembles p-aminobenzoic acid
and competes with it for the active site
of the bacterial enzyme involved, it can
prevent bacterial growth.
This is possible because the enzyme
binds readily to either of these
molecules.

Therefore, the introduction of large quantities of sulfanilamide into a patient’s
body causes most of the active sites to be bound to the wrong (from the
bacterial viewpoint) substrate.
Thus, the synthesis of folic acid is stopped or slowed, and the bacteria are
prevented from multiplying.
Human beings also require folic acid, but they get it from their diet.
Consequently, sulfa drugs exert no toxic effect on humans.

A noncompetitive inhibitor bears no
resemblance to the normal enzyme
substrate and binds reversibly to the
surface of an enzyme at a site other than
the catalytically active site.
The interaction between the enzyme and
the noncompetitive inhibitor causes the
three-dimensional shape of the enzyme
and its active site to change.

The enzyme no longer binds the normal substrate, or the substrate is
improperly bound in a way that prevents the catalytic groups of the active site
from participating in catalyzing the reaction.
Unlike competitive inhibition, noncompetitive inhibition cannot be reversed by
the addition of more substrate because additional substrate has no effect on the
enzyme bound inhibitor.
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 :
Enz—SH + Ag+ ⇄ Enz—SH—Ag + H+

How Are Enzymes Regulated?
Enzymes work together in an organized yet complicated way to facilitate all the
biochemical reactions needed by a living organism.
For an organism to respond to changing conditions and cellular needs, very
sensitive controls over enzyme activity are required.
Three mechanisms by which this is accomplished are:
a) Activation of zymogens,
b) Allosteric regulation, and
c) Genetic control.

The Activation of Zymogens
Some enzymes are manufactured by the body in an inactive form.
To make them active, a small part of their polypeptide chain must be removed.
These inactive forms of enzymes are called proenzymes or zymogens.
After the excess polypeptide chain is removed, the enzyme becomes active.
For example, trypsin is manufactured in the pancreas as the inactive
molecule trypsinogen (a zymogen).

Allosteric Regulation
A second method of enzyme regulation involves the combination of the enzyme
with some other compound such that the three-dimensional conformation of
the enzyme is altered and its catalytic activity is changed.
Compounds that alter enzymes this way are called modulators of the activity.
Modulator may affect the enzyme in either of two ways:
a. It may inhibit enzyme action; inhibitors (negative modulation) or
b. It may stimulate enzyme action; activators (positive modulation).

Enzymes that have a quaternary
protein structure with distinctive
binding sites for modulators are
referred to as allosteric enzymes.
The site to which a modulator
attaches is called a regulatory site.
Specific modulators can bind
reversibly to the regulatory sites.
For example, the enzyme depicted in
Figure is an allosteric enzyme.

Example:
An excellent example of allosteric regulation—the control of an allosteric
enzyme—is the five-step synthesis of the amino acid isoleucine.
Threonine deaminase, the enzyme that catalyzes the first step in the conversion
of threonine to isoleucine, is subject to inhibition by the final product,
isoleucine.
Isoleucine exerts an inhibiting effect on the enzyme activity.

This type of allosteric regulation in which the enzyme that catalyzes the first
step of a series of reactions is inhibited by the final product is called feedback
inhibition.

Genetic Control
One way to increase production from an enzyme-catalyzed reaction, given a
sufficient supply of substrate, is for a cell to increase the number of enzyme
molecules present.
The synthesis of all proteins, including enzymes, is under genetic control by
nucleic acids.
An example of the genetic control of enzyme activity involves enzyme
induction, the synthesis of enzymes in response to a temporary need of the cell.

Enzymes

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