ENZYMES

SHANDELE GINNETHON
 INTRODUCTION TO ENZYMES

• Enzymes are biological polymers that catalyze the chemical

reactions which make life as we know possible.

• With the exception of a few catalytic RNA molecules, or

ribozymes, the vast majority of enzymes are proteins.

• Their catalytic activity depends on the integrity of their native

protein conformation.

• Deficiencies in the quantity or catalytic activity of key

enzymes can result from genetic defects, nutritional deficits, or
toxins.
• Substances on which enzymes act to convert them into
products are called substrates.

• For chemical reaction to take place, the reacting molecules are

required to gain a minimum amount of energy, called energy
of activation.

• Enzymes have immense catalytic powers and accelerate

reactions at least a million times by reducing the energy of
activation.
• Few enzymes are simple proteins while some are conjugated
proteins.

• In such enzymes the non-protein part is called prosthetic group

or coenzyme and the protein part is called apoenzyme.

• When many different enzyme catalyzing sites are located at

different sites of the same macromolecule, it is called
multienzyme complex. Examples: fatty acid synthase,
carbamoyl phospahte synthetase II, pyruvate dehydrogenase,
etc.
 COENZYMES

• Certain enzymes require non-protein organic coenzymes for

the activity.

• Prosthetic groups are distinguished by their tight, stable

incorporation into a protein’s structure by covalent or non-
covalent forces.

• Examples include Biotin, tertrahydrofolate, NAD+, NADP,

FMN, FAD, Co, Cu, Zn etc.

• Enzymes that contain tightly bound metal ions are termed

metalloenzymes.
 COFACTORS

• Cofactors serve functions similar to those of prosthetic groups

but bind in a transient, dissociable manner either to the enzyme
or to a substrate.

• Cofactors must be present in the medium surrounding the

enzyme for catalysis to occur.

• Enzymes that require a metal ion cofactor are termed metal-

activated enzymes.
 NOMENCLATURE AND CLASSIFICATION OF
ENZYMES
• The International Union of Biochemistry (IUB) adopted a
nomenclature system based on chemical reaction type and
reaction mechanism.

• The systemic names for enzymes include the substrate and the
type of reaction.

• According to this system, enzymes are grouped into six main

classes. They are:
1. Oxidoreductase: catalyze oxidations - reductions of their
substrates, e.g. alcohol dehydrogenase, lactate dehydrogenase.

2. Transferase: catalyze transfer of particular group from one

substrate to another, e.g. hexokinase, aspartate and alanine
transaminase (AST/ALT).

3. Hydrolase: bring about hydrolysis, the addition of water to a

chemical bond. e.g. glucose-6-phospatase, pepsin, trypsin.
4. Lyases: usually catalyze a carbon-carbon bond cleavage., e.g.
fumarase, arginosuccinase.

5. Isomerases: transfer of groups or bonds within molecules to

yield isomeric forms, e.g. UDP-glucose, epimerase.

6. Ligases: catalyze joining together two substrates coupled with

ATP hydrolysis, e.g. DNA ligase, glutamine synthetase.
 SPECIFICITY OF ENZYMES

1. Optical specificity:
• Substrates can have optical isomers, but only one of the
isomers acts as a substrate for the enzyme activity.
2. Reaction specificity:
• An enzyme can catalyze only a single type of reaction.
• A substrate can undergo many reaction, each reaction
catalysed by different enzymes.
3. Substrate specificity:
• This means that certain enzymes are specific for a certain
substrate.
• Group specificity - the enzyme will act only on molecules
that have specific functional groups, such as amino, phosphate
and methyl groups.
- E.g. Trypsin hydrolyses the residues of only lysine and
arginine, chymotrypsin hydrolyses residues of only aromatic
amino acids.

• Bond specificity - the enzyme will act on a particular type of

chemical bond regardless of the rest of the molecular structure.
- E.g. Proteolytic enzymes, glycosidases and lipases act on
peptide, glycosidic and ester bonds respectively.
 MECHANISM OF ENZYME ACTION
• According to most acceptable hypothesis, enzyme molecule
(E) first combines with substrate molecule (S) to form an
enzyme-substrate (ES) complex which further dissociates to
form product (P) and enzyme (E).

• Enzyme once dissociated from ES complex is free to combine

with another substrate and form product.

• The ES complex is an intermediate or transient complex held

together by weak non-covalent bonds such as H-bonds, Van
der Waals forces, hydrophobic interactions.
• The site at which the substrate can bind to the enzyme with
extreme specificity is called active site or catalytic site.
 MODELS OF ENZYME-SUBSTRATE COMPLEX
FORMATION

1) Template or Lock and Key Model

• This model states that the active site already exists in proper
conformation even in the absence of the substrate.
• Substrate fits into the active site as key fits into lock, hence called
lock and key model.
• Model cannot explain change in enzyme activity in presence of
allosteric modulators.
2) Induced Fit or Koshland Model
• Important feature of this model is the flexibility of active site region.
• According to this, the substrate during its binding induces
conformational changes in the active site to attain the final catalytic
shape and form.
• This model explains;
- enzymes become inactive on denaturation, saturation kinetic,
competitive inhibition, allosteric modulation.
 FACTORS AFFECTING ENZYMES

 Temperature

 pH

 Enzyme concentration

 Substrate concentartion

 Inhibitors
Temperature
• As the temperature rises, reacting molecules have more and
more kinetic energy. This increases the chances of a successful
collision and so the rate increases.
• Each enzyme is most active at a specific temperature, called
optimum temperature.
• This optimal temperature is usually around human body
temperature (37.5 oC) for the enzymes in human cells.
• The Q10 or temperature coefficient is a measure of the rate
of change of a biological or chemical system as a consequence
of increasing the temperature by 10 °C.
pH
• Extreme pH levels will cause denaturation.
• The active site is distorted and the substrate molecules will no
longer fit.
• Small changes in pH above or below
the optimum do not cause a permanent change to the enzyme,
since the bonds can be reformed.
Enzyme Concentration
• Rate of enzyme activity is directly proportional to
enzyme concentration as long as the substrate
concentration is in excess.

Substrate Concentration
• Increasing substrate concentration increases the rate of
reaction. This is because more substrate molecules will
be colliding with enzyme molecules, so more product will be
formed.
• After a certain concentration, any increase will have no
• effect on the rate of reaction, because enzymes will
effectively become saturated. The enzyme-substrate complex
has to dissociate before
the active sites are free to accommodate more substrate.

Inhibitors

• Enzyme inhibitors are substances which alter the catalytic

action of the enzyme and consequently slow down, or in some
cases, stop catalysis.
• Whenever the active site is not available for binding of the
substrate the enzyme activity may be reduced.
 ENZYME INHIBITION

• The chemical substances which inactivate enzymes are called

inhibitors and the process is called enzyme inhibition.

• Enzymes catalyze virtually all cellular processes, enzyme

inhibitors are among the most important pharmaceutical agents
known.
• For example, aspirin (acetylsalicylate) inhibits the enzyme that
catalyzes the first step in the synthesis of prostaglandins,
compounds involved in many processes, including some that
produce pain.

• Three major groups of inhibition:

1. Reversible inhibition
2. Irreversible inhibition
3. Allosteric inhibition
Reversible inhibition.

• When the active site or catalytic site is occupied by a

substance other than the substrate, its activity is inhibited.

• One common type of reversible inhibition is called

competitive inhibition.

• A competitive inhibitor [I] competes with the substrate for

binding the active site of a free enzyme.
• Many competitive inhibitors are compounds that resemble the
substrate and combine with the enzyme to form an EI
complex, but without leading to catalysis.
Clinical Significance:

 Medical therapy based on competitive inhibition is used to

treat patients who have ingested methanol, a solvent found in
gas-line antifreeze.
 The liver enzyme alcohol dehydrogenase converts methanol to
formaldehyde, which is damaging to many tissues especially
eyes.
 Ethanol competes effectively with methanol as an alternative
substrate for alcohol dehydrogenase converting it to
acetaldehyde.
 This slows the formation of formaldehyde, lessening the
danger while the kidneys filter out the methanol to be excreted
harmlessly in the urine.
 Pyrazole is a inhibitor of alcohol dehydrogenase.

 Allopurinol is a drug used to treat gout. Uric acid is formed in

the body by oxidation of hypoxanthine by the enzyme xanthine
oxidase. Allopurinol acts as a competitive inhibitor of xanthine
oxidase reducing uric acid formation.

 Methotrexate is used in cancer therapy. It’s a structural analog

of folic acid. It inhibits folate reductase and prevents formation
of FH4, which in turn inhibits DNA synthesis.
 Succinylcholine is used as a muscle relaxant. It is structurally
similar to acetylcholine. It competitively binds to post-synaptic
receptors. Acetylcholinesterase cannot hydrolyse them which
causes continued depolarization resulting in muscle relaxation.

 Dicoumarol is used as an anticoagulant, structurally similar to

vitamin K and competitively inhibits vitamin K epoxide
reductase, an enzyme that recycles vitamin K.
• An noncompetitive inhibitor binds reversibly to both the
free enzyme and the ES complex.

• Non-competitive inhibition is observed only for enzymes with

two or more substrates.

• These inhibitors bring about changes in the 3D structure of the

enzyme inactivating.

• Compounds that are these types of inhibitors are more useful

as drugs since they inhibit the enzyme independent of the
concentration if the substrate.
• Example; alanine noncompetitively inhibits the enzyme
pyruvate kinase.
Irreversible inhibition

• The irreversible inhibitors are those that bind covalently with

or destroy a functional group on an.
• These inhibitors chemically modify and inactivate the
enzymes.
• If non-competitive inhibitor can be removed only at the loss of
enzyme activity, it is known as irreversible non-competitive
inhibition.
• Examples of non-competitive inhibitor ;
- iodoacetate inhibiting enzymes like glyceraldehyde 3-
phospahte and papin
- heavy metals like silver, mercury
Clinical Significance

 British anti Lewesite (BAL) is used as antidote for heavy

metal poisoning. Heavy metals inhibit enzymes by reacting
with –SH groups. BAL provides –SH for the heavy metals to
act on.
 Antabuse (disulfiram) used in treatment of alcoholism. It
inhibits aldehyde dehydrogenase preventing oxidation of
acetaldehyde which accumulates producing sickening effect
leading to aversion to alcohol.
• Suicide inhibition is a special type of irreversible non-
competitive inhibition in which the substrate analog is
converted to a more effective inhibitor with the help of the
enzyme to be inhibited.
• The new inhibitor formed binds irreversibly with the enzyme.
• Examples include allopurinol, 5-fluorouracil.
• Uncompetitive inhibitor can bind to the ES complex rather
than the free enzyme, binding to an alternative binding site.

• In contrast to competitive inhibition, increasing the

concentration of substrate will not overcome the effect of the
inhibitor because [I] binds to ES complex and not the free
enzyme.

Allosteric inhibition
• It is a kind of inhibition when the inhibitor binds to the
enzyme at a site other than the active site, sometimes on a
different region in the enzyme called allosteric site.
 REGULATION OF ENZYME ACTIVITY

• Regulatory enzymes exhibit increased or decreased catalytic

activity in response to certain signals.

• These enzymes allow the cell to meet changing needs for

energy and for biomolecules required in growth and repair.

• Allosteric enzymes function through reversible, non-covalent

binding of regulatory compounds called allosteric modulators
or allosteric effectors, which are generally small metabolites or
cofactors.
• Binding of the allosteric effector brings about conformational
changes of the enzyme so the affinity for the substrate or other
ligands also changes.
• Positive (+) allosteric effectors increase the enzyme affinity for
the substrate. The reverse is true for negative (-) effectors.
• Allosteric site at which the positive effector binds is called
activator site, negative effector binds at an inhibitory site.
• Feedback regulation is when the product inhibits or activates
the enzyme activity in response to stimuli.
• If the end product becomes available in the environment, it is
unnecessary and wasteful for the cells to continue to produce
the product. Cells have the ability to shut down a pathway
when it is not needed.