ENZYMES

Learning Outcomes:
At the end of the three hours lecture –discussion on enzymes, the students will be able to:
1. identify the functions of enzymes,
2. familiarize the nomenclature of enzymes,
3. classify the different types of enzymes,
4. give the roles of coenzyme,
5. differentiate the two models of enzyme activity,
6. trace the enzymatic cycle,
7. describe the specificity of enzyme –substrate complex,
8. enumerate the environmental factors that regulate enzyme activity,
9. determine the different types of inhibition.

INTRODUCTION
Enzymes are proteins that are considered as biological catalyst. They speed up the
biological reactions in our body. In the absence of enzymatic catalysis, most biochemical
reactions are so slow that they would not occur under the mild conditions of temperature and
pressure that are compatible with life. Enzymes accelerate the rates of such reactions by well
over a million-fold, so reactions that would take years in the absence of catalysis can occur in
fractions of seconds if catalyzed by the appropriate enzyme. Cells contain thousands of
different enzymes, and their activities determine which of the many possible chemical reactions
actually take place within the cell. Catalysts are substances that increase product formation by
(1) lowering the energy barrier (activation energy) for the product to form and (2) increase the
favorable orientation of colliding reactant molecules for product formation to be successful.

Below is an illustration of how activation energy of a reaction is lowered by a catalyst;

 Enzymes lower activation energy and speed up reactions by several mechanisms:

1. Active site can hold two or more reactants in the proper position so they may react.
2. Induced fit of the enzyme's active site may distort the substrate's chemical bonds, so less
thermal energy (activation energy) is needed to break them during the reaction.
3. Active site might provide a micro-environment conducive to a particular type of reaction
(e.g., localized regions of low pH caused by acidic side chains on amino acids at the active
site). 4. Side chains of amino acids in the active site may participate directly in the reaction.

5.1 NOMENCLATURE OF ENZYMES

An enzyme's name is often derived from its substrate or the chemical reaction it
catalyzes, with the word ending in -ase. Examples are lactase, alcohol dehydrogenase and
DNA polymerase.
The International Union of Biochemistry and Molecular Biology have developed another
nomenclature for enzymes, the EC numbers; each enzyme is described by a sequence of four
numbers preceded by "EC". The first number broadly classifies the enzyme based on its
mechanism:

Number Classification Biochemical Properties

Act on many chemical groupings to add or remove hydrogen

EC1. Oxidoreductases
atoms.

Transfer functional groups between donor and acceptor

molecules. Kinases are specialized transferases that regulate
EC2. Transferases
metabolism by transferring phosphate from ATP to other
molecules.

EC3. Hydrolases Add water across a bond, hydrolyzing it.

Add water, ammonia or carbon dioxide across double bonds,

EC4. Lyases
or remove these elements to produce double bonds.

Carry out many kinds of isomerization: L to D isomerizations,

EC5. Isomerases
mutase reactions (shifts of chemical groups) and others.

Catalyze reactions in which two chemical groups are joined (or

EC6. Ligases
ligated) with the use of energy from ATP.

Examples of these six major classifications of enzymes that catalyzed a certain type of reactions
are as follows:
1. OXIDOREDUCTASES
- enzymes that catalyze oxidation-reduction reaction. These enzymes are
named as oxidoreductase.

2. TRANSFERASES

- enzymes that catalyze the transfer of functional groups from one molecule to
another. They are named as transaminase, kinase, transmethylase.

3. HYDROLASES

- enzymes that encourage the addition of water molecule to a compound resulting in

a bond breakage.

4. LYASES

- speed up the attachment of a group to a double bond or the removal of a

functional group to form a double bond.

5. ISOMERASES

- enzymes that rearrange the position of a functional group in a molecule and

converts one isomer to another.
6. LIGASES

- enzymes that break or make a C-C, C-S, C-O or C-N bond.

5.2 CLASSIFICATION OF ENZYMES BASED ON THEIR COMPOSITION

Enzymes are also classified on the basis of their composition. Enzymes composed wholly
of protein are known as simple enzymes in contrast to complex enzymes, which are composed
of protein plus a relatively small organic molecule.
Complex enzymes are also known as holoenzymes. In this terminology the protein
component is known as the apoenzyme, while the non-protein component is known as the
coenzyme or prosthetic group where prosthetic group describes a complex in which the small
organic molecule is bound to the apoenzyme by covalent bonds; when the binding between
the apoenzyme and non-protein components is non-covalent, the small organic molecule is
called a coenzyme.
Many prosthetic groups and coenzymes are water-soluble derivatives of vitamins. It
should be noted that the main clinical symptoms of dietary vitamin insufficiency generally arise
from the malfunction of enzymes, which lack sufficient cofactors derived from vitamins to
maintain homeostasis.
The non-protein component of an enzyme may be as simple as a metal ion or as
complex as a small non-protein organic molecule. Enzymes that require a metal in their
composition are known as metalloenzymes if they bind and retain their metal atom(s) under all
conditions that is with very high affinity. Those which have a lower affinity for metal ion, but still
require the metal ion for activity, are known as metal-activated enzymes

5.3 ROLE OF COENZYMES

The functional role of coenzymes is to act as transporters of chemical groups from one
reactant to another. The chemical groups carried can be as simple as the hydride ion (H + + 2e-)
carried by nicotinamide adenine dinucleutide (NAD) or the mole of hydrogen carried by flavine
adenine dinucleutide (FAD); or they can be even more complex than the amine (-NH2) carried
by pyridoxal phosphate.
Since coenzymes are chemically changed as a consequence of enzyme action, it is
often useful to consider coenzymes to be a special class of substrates, or second substrates,
which are common to many different holoenzymes. In all cases, the coenzymes donate the
carried chemical grouping to an acceptor molecule and are thus regenerated to their original
form. This regeneration of coenzyme and holoenzyme fulfills the definition of an enzyme as a
chemical catalyst, since (unlike the usual substrates, which are used up during the course of a
reaction) coenzymes are generally regenerated.

5.4 MODELS OF ENZYME ACTIVITY

1. Lock and Key Model

According to this model, each enzyme molecule may have as few as one active site on
the surface of the enzyme molecule itself. An active site is an indentation or cavity whereby a
reactant molecule (substrate) is attracted to. This is called the enzyme-substrate complex. The
polar and non-polar groups of the active site attract compatible groups on the substrate
molecule so that the substrate molecule can effectively lock into the cavity and position itself for
the necessary collisions and bond breaks and formations that must take place for successful
conversion to a product molecule. Once the product molecule has been formed the electrical
attractions that made the substrate molecule adhere to the active site no longer are present,
and the product molecule can disengage itself from the active site thus freeing the site for
another incoming substrate molecule. This process occurs in a highly efficient manner hundreds
or even thousands of times in a short time span. This model assumes that molecules that lock into
the active site must form a perfect fit. Also the assumption is that the active site conformation is
ridged.

Enzyme Substrate Enzyme – Substrate Complex

2. Induced Fit (Hand and Glove) Model

Modification of the lock and key model assumes that the active site has a certain
amount of elasticity whereby the active site can expand or contract in a limited way in order to
accommodate the substrate molecule. The analogy is like a hand fitting into a glove. The glove
adjusts in shape and size to fit various sized hands within a certain range. This tolerance would
explain why bogus molecules of slightly different size compared to the true substrate molecule
can still be accommodated by the elastic active site. Small changes in temperature would
distort the active site conformation but not so much that the active site could not still
accommodate the substrate molecular size. pH changes which would also change the active
site conformation but not so much that the active site could not flexibly accommodate the
substrate molecule. The Induced Fit Model seems to explain why there is some flexibility in the
ability of the active site to accommodate other molecules and at limited temperature and pH
ranges.

5.5 THE ENZYMATIC CYLCE

Below is an illustration of the entire enzymatic cycle which is quite rapid.

The enzymatic cycle has the following steps:

a. An enzyme, depending on its degree of specificity reacts with the substrate.
b. The enzyme-substrate complex is formed either in the lock and key method or the
induced key method. Either way, it can be seen that there now starts a reaction
between the two.
c. The continued interaction between the two (enzyme-substrate) change the position
and shape of the substrate, thus, they are energetically unstable. The enzymes and
substrate is held by a covalent bonding.
The unstable energy might be derived form:
➢ during the continued interaction, the enzyme might have added stress to the
substrate in order to break the bond
➢ An enzyme might bring the substrate to its close proximity so that the reaction
would hasten.
 ➢ An enzyme might change the pH of the environment of the substrate by
donating or accepting H+.

d. The substrate is converted to a product, but it is still attached to the enzyme. This is
otherwise known as the enzyme-product complex.
e. Finally, the newly converted product detaches itself from the enzyme. The enzyme is
now ready to take another substrate for product conversion.

5.6 FACTORS AFFECTING ENZYME ACTIVITY

Each enzyme has optimal environmental conditions that favor the most active enzyme
conformation. The following are environmental factors that will affect enzymatic activity;

A. Temperature

Every enzyme has a temperature range of optimum activity. Outside that temperature
range the enzyme is rendered inactive and is said to be totally inhibited. This occurs because as
the temperature changes these supplies enough energy to break some of the intramolecular
attractions between polar groups (Hydrogen bonding, dipole-dipole attractions). Most enzymes
(and there are hundreds within the human organism) within the human cells will shut down at a
body temperature below a certain value which varies according to each individual. This can
happen if body temperature gets too low (hypothermia) or too high (hyperthermia).

Optimal temperature allows the greatest number of molecular collisions without denaturing
the enzyme. Enzyme reaction rate increases with increasing temperature. Kinetic energy of
reactant molecules increases with rising temperature, which increases substrate collisions with
active sites.
Beyond the optimal temperature, reaction rate slows. The enzyme denatures when
increased thermal agitation of molecules disrupts weak bonds that stabilize the active
conformation

B. pH

Changes in the pH or acidity of the environment can take place that would alter or
totally inhibit the enzyme from catalyzing a reaction. This change in the pH will affect the polar
and non-polar intramolecular attractive and repulsive forces and alter the shape of the enzyme
and the active site as well to the point where the substrate molecule could no longer fit, and the
chemical change would be inhibited from taking place as efficiently or not . Optimal pH range
for most enzymes is pH 6- 8.

Some enzymes operate best at more extremes of pH. For example, the digestive enzyme,
pepsin, found in the acid environment of the stomach has an optimal pH of 2. In an acid
solution any basic groups such as the Nitrogen groups in the protein would be protonated. If the
environment was too basic the acid groups would be deprotonated. This would alter the
electrical attractions between polar groups. Every enzyme has an optimum pH range outside of
which the enzyme is inhibited. Some enzymes like many of the hydrolytic enzymes in the
stomach such as Pepsin and Chymotrypsin effective operate at a very low acidic pH. Other
enzymes like alpha amylase found in the saliva of the mouth operate most effectively at near
neutrality. Still other enzymes like the lipases will function most effectively at basic pH values.
 If the pH drops in the blood called acidosis then enzymes in the blood will be inhibited
outside their optimal pH range. If the pH climbs to an unacceptably high value called alkalosis
then enzymes ceases to function effectively. Normally, these conditions do not take place
because of the highly efficient buffers found in the blood that restrict the pH of the blood to a
very narrow range.

Buffers are a substance or mixtures of substances that resist any change in the pH.
There are many buffer systems found in the body to adjust the pH so that enzymes might
continue to catalyze their reactions.

5.7 REGULATION OF ENZYME ACTIVITY

One of the major ways in which enzymes differ from nonbiological catalysts is that the
activity of the enzyme is often regulated by the cell. The production of enzyme depends on the
availability of the substrate and the need in body’s processes. The body’s cells are the ones that
regulate the enzyme activity. They send messages to the different parts of the body which is
interpreted as a command. When the cell can sense that the body lacks an enzyme for the
substrate to be converted to products, then it sends signal such that enzymes would be
produced by the cells. On the other hand, if there are more converted products present than
what is needed for the body, the cell again sends signal to stop the production.

There are five main ways that enzyme activity is controlled in the cell:

1. Enzyme production (transcription and translation of enzyme genes) can be enhanced or

diminished by a cell in response to changes in the cell's environment. This form of gene
regulation is called enzyme induction and inhibition. For example, bacteria may become
resistant to antibiotics such as penicillin because enzymes called beta-lactamases are
induced that hydrolyse the crucial beta-lactam ring within the penicillin molecule. Another
example are the enzymes in the liver called cytochrome P450 oxidases, which are
important in drug metabolism. Induction or inhibition of these enzymes can cause drug
interactions.
2. Enzymes can be compartmentalized, with different metabolic pathways occurring in
different cellular compartments. For example, fatty acids are synthesized by one set of
enzymes in the cytosol, endoplasmic reticulum and the Golgi apparatus and used by a
different set of enzymes as a source of energy in the mitochondrion, through β-oxidation.[
3. Enzymes can be regulated by inhibitors and activators. For example, the end product(s) of
a metabolic pathway are often inhibitors for one of the first enzymes of the pathway
(usually the first irreversible step, called committed step), thus regulating the amount of
end product made by the pathways.
4. Enzymes can be regulated through post-translational modification. This can include
phosphorylation, myristoylation and glycosylation. For example, in the response to insulin,
the phosphorylation of multiple enzymes, including glycogen synthase, helps control the
synthesis or degradation of glycogen and allows the cell to respond to changes in blood
sugar. Another example of post-translational modification is the cleavage of the
polypeptide chain. Chymotrypsin, a digestive protease, is produced in inactive form as
chymotrypsinogen in the pancreas and transported in this form to the stomach where it is
activated. This stops the enzyme from digesting the pancreas or other tissues before it
enters the gut. This type of inactive precursor to an enzyme is known as a zymogen.
5. Some enzymes may become activated when localized to a different environment (eg.
from a reducing (cytoplasm) to an oxidising (periplasm) environment, high pH to low pH
 etc). For example, hemagglutinin in the influenza virus is activated by a conformational
change caused by the acidic conditions; these occur when it is taken up inside its host cell
and enters the lysosome.

5.8 REASONS FOR REGULATING THE ENZYME ACTIVITY

1. For energy consideration – conservation of cellular activity.

If the cell runs out of chemical energy, it will die. Example, It is a great waste of energy to
produce an enzyme if the substrate is not available. Similarly, if the product of an enzyme –
catalyzed reaction is present in excess, it is a waste of energy for the enzyme to continue to
catalyze the reaction, thereby producing more of the unwanted product.

2. Use of allosteric enzymes

Allosteric enzymes are enzymes that have two binding sites. The activity of this enzyme is
regulated by a small molecule. Allosterism is divided into two:

1. Positive allosterism - when the effector molecule converts the active site to the active
configuration. An example of this is when the body experiences a strenuous activity. More
demand of energy is needed; therefore, the effector molecule enhances the conversion of the
substrate into products.

2. Negative allosterism - when the effector molecule converts the active site in an
inactive configuration. An example to this is when the body is at rest or not needing so much
energy, then the effector molecule binds to the enzyme to deactivate its binding site.
Negative feedback mechanism can effectively adjust the rate of synthesis of
intermediate metabolites according to the demands of the cells. This helps allocate materials
and energy economically, and prevents the manufacture of excess end products. Like other
homeostatic devices, the control of enzymatic action helps to maintain a stable internal
environment in living organisms.

Allosteric regulation

Allosteric site = Specific receptor site on some part of the enzyme molecule other than the
active site.
Most enzymes with allosteric
sites have two or more
polypeptide chains, each with
its own active site. Allosteric
sites are often located where
the subunits join.
Allosteric enzymes have two
conformations, one
catalytically active and the
other inactive
Binding of an activator to an
allosteric site stabilizes the
active conformation. Binding
of an inhibitor (noncompetitive
inhibitor) to an allosteric site
stabilizes the inactive
conformation.

Enzyme activity changes continually in response to changes in the relative proportions of

activators and inhibitors (e.g., ATP/ADP). ´ Subunits may interact so that a single activator or
inhibitor at one allosteric site will affect the active sites of the other subunits.

3. Feedback inhibition
Feedback inhibition = Regulation of a metabolic pathway by its end product,
which inhibits an enzyme within the pathway.
Feedback inhibition is also a process of regulating the enzyme activity by using the
product as an effector molecule in one of the series of catalytic process. Consider this example;

In the process of converting the substrate A to a final product C Substrate A binds to E 1

to be converted to a sub-product B. Sub-product B binds to E2 to be converted to a final
product C. During the cellular activity, if product C is not needed, then it would be used to stop
its own production. Product C would act as an effector molecule of E 1, such that when
substrate A attaches to E1, sub-product B would not be produced. Non-production of sub-
product B would mean that there will be no binding in E 2, thus, A is stopped from being
produced . From this process, it is the product itself that causes its own production.

4. Cooperativity The phenomenon where substrate binding to the active site of one subunit
induces a conformational change that enhances substrate binding at the active sites ofthe
other subunit

Substrate molecules themselves may enhance enzyme activity.

5. Production of Zymogens
The production of enzyme in an inactive form called zymogen or proenzyme. It is then
converted, usually by proteolysis (hydrolysis of protein), to the active form when it has reached
the site of activity. This can often be seen in enzymes containing toxic materials or enzymes
living in a pH lower than what is normal and standard. Enzymes in the stomach, having a pH
optimum of 2 – 3 can destroy or kill the cell that produces then if they are released in active
form. Protein enzymes secreted in the stomach are produced in an inactive form. They only
become active when they are in contact with an acid. Thus, to buffer the acidity of the
stomach, a zymogen is released.
ZYYMOGEN OF THE DIGESTIVE TRACT
ZYMOGEN ACTIVATOR ENZYME
Proelastase Trypsin elastase
Trypsinogen Trypsin trypsin
Chymotrypsinogen A Trypsin + chymotrypsin chymotrypsin
Pepsinogen Acid pH + pepin pepsin
Procarboxypeptidase trypsin Carboxypepetidase A or B

6. Protein Modifications
An enzyme modification that either turns off or activates it. This is done by adding or
cleaving a chemical group from the cleaving process.

5.9 TYPES OF INHIBITION OF ENZYME ACTIVITY

A. Competitive Inhibition

Competitive Inhibition occurs when a bogus molecule that is close enough to the shape
of the true substrate will fit into the active site. Once locked into position, the blocker molecule
prevents the true substrate molecule from getting into position. This effectively blocks the active
site. The bogus molecule competes for the active site with the true substrate molecule. Many
toxic substances owe their toxic properties to their ability to act as inhibitors to important
enzymes responsible for catalyzing important biochemical processes. Once the enzyme is
inhibited the process cannot take place, and a toxicological symptom occurs that often leads
to paralysis, coma or even death of the organism. For example, cyanide poisoning is due to the
cyanide ion competitively inhibiting the active site of the cytochromases enzymes responsible
for catalyzing the Oxidation and Reduction processes of the Electron Transport System which is
responsible for cellular respiration.

B. Non-Competitive Inhibition

Other inhibitors attached themselves not to the active site itself but to some portion of
the enzyme molecule close to the active site which results in the changing of the shape of the
active site. This is referred to as non-competitive inhibition. Many heavy metals like Lead,
Mercury,and Chromium will function as non-competitive inhibitors. Toxicology is the study of how
toxicological substances can interfere with life sustaining enzymes via inhibition.

The pesticide and herbicide industries make use of competitive and Non-Competitive
Inhibitors Biological warfare owes its success to enzyme inhibition but so does the life giving
chemotherapeutic treatment of cancerous tumor growths with agents that inhibit important
cancel cell enzymes. All in all the use of inhibitors can be used for the benefit of mankind or its
destruction.

5.10 TYPES OF ENZYME INHIBITORS

Enzyme inhibitors are classified into:

A. IRREVERSIBLE INHIBITOR
These are chemicals that hinders the formation of an enzyme-substrate complex
by blocking the active site of it; thus, effectively eliminating catalysis. This kind of
inhibitor affects many enzymes.
B. REVERSIBLE COMPETITIVE INHIBITOR
An inhibitor in which the substrate competes for the molecule that resemble its
structure and charge distribution. These molecules are referred to as structural
analogs. The inhibitor of product formation depends on the concentration of the
substrate and structural analogs. If there are more substrates than the structural
analogs, inhibition will fail because more product will be converted. Consequently, if
more analogs are present, then inhibition is successful. The term competitive comes
from the fact that enzymes and structural analogs will compete in binding to the
active binding site.

C. REVERSIBLE NON-COMPETITIVE INHIBITORS

A temporary state of inhibition. The inhibitor acts or binds temporarily to the
enzyme making the enzyme unproductive. But when the inhibitor detaches from the
enzyme, it makes the enzyme active and ready for a reaction with the substrate. It
should be noted that this inhibitor does not bind to the active site of the enzyme.

Illustrations: