ENZYMES

1
Introduction to
Enzymes
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.

What Are General Characteristics of
Enzymes?

Catalytic Efficiency
 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.
What Are General Characteristics of
Enzymes?

Specificity
 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)
What Are General Characteristics of
Enzymes?

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.
What Are General Characteristics of
Enzymes?

Regulation
 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.
2- Chemistry
 Structure of Enzymes
 Enzyme molecule contain a special pocket or cleft called as the active site .
 The active site of an enzyme is the region that binds substrates, co-factors and
prosthetic groups and contains residue that helps to hold the substrate.

 Active sites generally occupy less than 5% of the total surface area of enzyme.

 Active site has a specific shape due to tertiary structure of protein.

 A change in the shape of protein affects the shape of active site and function of
the enzyme.
 Active site
o Active site can be further divided into:

Active Site

Binding Site Catalytic Site

It chooses the substrate It performs the

and binds it to active site. Catalytic action

 Co-factors
o Co-factor is the non protein molecule which carries out chemical
reactions that can not be performed by standard 20 amino acids.

o Co-factors are of two types:

 Organic co-factors
 Inorganic cofactors
 Inorganic co-factors
o These are the inorganic molecules required for the proper activity of
enzymes.
Examples:
++
 Enzyme carbonic anhydrase requires Zn for it’s activity.
 Hexokinase has co-factor Mg++

ORGANIC CO-FACTORS
o These are the organic molecules required for the proper activity of
enzymes.
Example:
 Glycogen phosphorylase requires the small organic molecule
pyridoxal phosphate.
 Types of Organic co-factors
o A prosthetic group is a tightly bound o A coenzyme is loosely bound organic
organic co-factor e.g. Flavins, heme co-factor. E.g. NAD
groups and biotin.
+
+

Prosthetic Group Coenzyme

 Types of co-factors Continued…

 An enzyme with it’s co-factor removed is designated as apoenzyme.

 The complete complex of a protein with all necessary small organic

molecules, metal ions and other components is termed as holoenzyme of
holoprotein/ active enzyme
 Cofactor
(Coenzym
Apoenzy Active
me e or Enzyme
Inorganic
ions)
 substrate

 The reactant in biochemical reaction is termed as substrate.

 When a substrate binds to an enzyme it forms an enzyme-substrate complex.

Substrate Joins Enzyme

 3
Enzyme Nomenclature
and Classification
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).
How Are Enzymes Named?
 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.
How Are Enzymes Named?
 The hydrolysis of urea provides a typical example:

 EC Name: urea amidohydrolase

 Substrate: urea
 Functional Group: amide
 Type of Reaction: hydrolysis
How Are Enzymes Named?
 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
How Are Enzymes Named?
 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 Oxidoreductases Oxidation–reduction reactions

2 Transferases Transfer of functional groups
3 Hydrolases Hydrolysis reactions
4 Lyases Addition of double bonds or the reverse of that rxn
5 Isomerases Isomerization reactions
6 Ligases Formation of bonds with ATP cleavage
 4
The Mechanism of
Enzyme Action
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.
What Are the Mechanisms of Enzyme
Action?
 Once this complex is formed, the conversion of substrate (S)
to product (P) may take place:
General reaction:
What Are the Mechanisms of Enzyme
Action?
 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
What Are the Mechanisms of Enzyme
Action?
 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.

Lock-and-Key Model
What Are the Mechanisms of Enzyme
Action?

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

Lock-and-Key Model
What Are the Mechanisms of Enzyme
Action?

 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.

Induced-Fit Model
What Are the Mechanisms of Enzyme
Action?

 Fig: In the induced-fit model, the flexible enzyme changes

shape to match the substrate.

Induced-Fit Model
 5
Enzyme Kinetics
 Activation Energy (Ea):
 “The least amount of energy needed for a chemical reaction to
take place.”
 Enzyme (as a catalyst) acts on substrate in such a way that they
lower the activation energy by changing the route of the reaction.
 The reduction of activation energy (Ea) increases the amount of
reactant molecules that achieve a sufficient level of energy, so
that they reach the activation energy and form the product.
 Example:
 Carbonic anhydrase catalyses the hydration of 10⁶ CO₂ molecules
per second which is 10⁷x faster than spontaneous hydration.
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.

5. Effect of activators and inhibiitors.

What Factors Influence Enzyme
Activity?

 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:

Enzyme Concentration
What Factors Influence Enzyme
Activity?
 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.

Enzyme Concentration
What Factors Influence Enzyme
Activity?
 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.

Substrate Concentration
What Factors Influence Enzyme
Activity?

 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.

Substrate Concentration
What Factors Influence Enzyme
Activity?
 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.

Temperature
What Factors Influence Enzyme
Activity?
 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.

Temperature
What Factors Influence Enzyme
Activity?
 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.
The Effect of pH
What Factors Influence Enzyme
Activity?
 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 Effect of pH
Effect of Activators and inhibitors
 The activity of certain enzymes is greatly dependent of
metal ion activators and coenzymes.
 Whenever the active site is not available for the binding of

the substrate the enzyme activity may be reduced. The

substances which stop or modify the enzymatic reaction are
called inhibitors or modulators. Presence of these
substances in reaction medium can adversely affect the
rate of enzymatic reaction.
 Metals function as activators of enzyme velocity through

various mechanisms
Effect of light and radiation
 Exposure of enzymes to ultraviolet, beta, gamma and X-
rays inactivates certain enzymes due to the formation of
peroxides. e.g. UV rays inhibit salivary amylase activity.
 6
Enzyme
Inhibition
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 three categories on the basis
of how they behave at the molecular level.
1. Reversible Inhibition
2. Irreversible Inhibition
3. Allosteric inhibition
What is Enzyme Inhibition?
 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.

Irreversible Inhibitors
What is Enzyme Inhibition?
 The cyanide ion (CN2) is an example of an irreversible enzyme
inhibitor.
What is Enzyme Inhibition?

 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.

Reversible Inhibitors
What is Enzyme Inhibition?

 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.

1. Competitive Inhibitors
What is Enzyme Inhibition?
 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.
What is Enzyme Inhibition?
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:
What is Enzyme Inhibition?
 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.
What is Enzyme Inhibition?
2. Non-Competitive
Inhibitors
 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.
 Suicide Inhibition
 It is a special type of irreversible noncompetitive inhibition. In this type of
inhibition, substrate analogue is converted to a more effective inhibitor with
the help of the enzyme to be inhibited. The so formed new inhibitor binds
irreversibly with the enzyme.
 Examples :
 Allopurinol The best example of suicide inhibition. The drug is used in
treatment of gout, as it inhibits the enzyme xanthine oxidase thus decreasing
the uric acid formation. But allopurinol gets oxidized by the enzyme xanthine
oxidase itself to form “alloxanthine” a more potent effective and stronger
inhibitor of xanthine oxidase thus potentiating the action of allopurinol.
 Allosteric inhibition
 There is a mixed kind of inhibition when the inhibitor binds to the enzyme at
a site other than the active site but on a different region in the enzyme
molecule called allosteric site. Allosteric inhibition does not follow the
Michaelis-Menten hyperbolic kinetics. Instead it gives a sigmoid kinetics.
 When the inhibitor is present it fits into its site and there is a conformational
change in the enzyme molecule
 The enzyme’s molecular shape changes
 The active site of the substrate changes
 The substrate cannot bind with the substrate.
 The reaction slows down
 This is not competitive inhibition but it is reversible
 When the inhibitor concentration diminishes the enzyme’s conformation
changes back to its active form
Switching off
 These enzymes
have two
receptor sites
 One site fits the
substrate like Inhibitor
other enzymes Substrate molecule
 The other site fits cannot fit
an inhibitor into the
molecule active Inhibitor fits
site into
allosteric
site
The allosteric site the enzyme “on-off”
switch

Active
site

E Alloste
Substrate ric site Conformation E
fits into empty al change Inhibitor
the molecul
Substrate
active e is
cannot fit
site The presen
into the
inhibitor active t
Inhibitor
molecule site fits into
is absent allosteric
site
Applications of inhibitors

 Negative feedback: end point or end product inhibition

 Poisons snake bite and plant alkaloids.
 Medicine antibiotics, sulphonamides, sedatives and

stimulants