ENZYMES

INTRODUCTION
 INTRODUCTION
• Enzymes are protein catalysts for chemical reactions
in biological systems.
• They increase the rate of chemical reactions taking
place within living cells without changing themselves.
• Catalyst is a substance, which accelerates the rate of
a chemical reaction without being consumed in the
reaction, or affects the end point.
• Enzymes are the active substances that are in the
yeast (en = in, zyme = yeast).
 Cont--------
• Enzymes are Biological Catalysts
• Are (almost) always proteins
• Are extraordinarily efficient better than chemical
catalysts –
• Are remarkably specific
• Need mild physiological conditions to function
(temperature, salt, pH, etc.)
• Often require a cofactor (metal ion, prosthetic group,
or coenzyme)
• May be modified for regulatory purposes
 Why Study Enzymes?

• They catalyze stereo-specific reactions with virtually

no undesirable “side-products”
• Many disease states are related to altered or absent
enzyme activity
• Many vitamins, drugs, and toxins are enzyme
modulators
• Measurement of activity is diagnostic for many
diseases
• Enzymes are important practical tools in industry,
medicine, and agriculture
 NATURE
• Enzymes are highly specific in their action and
act inside the cells (e.g., metabolic enzymes)
and outside cells (e.g., the digestive enzymes
and blood clotting factors).
• Most enzymes belong to simple proteins.
• Some enzymes require the help of certain
non-protein factors that may be loosely
attached to them(a coenzyme or cofactor)
• or firmly (a prothestic group) attached to the
protein part of the enzyme (apoenzyme).
 Classification of Enzymes

Enzymes are classified into 6 classes as follows:

1. Oxidoreductases:
•These are enzymes catalyzing oxidation-reduction
reactions,
•Act on many chemical groupings to add or remove
hydrogen atoms.

e.g., oxidases, oxygenases, reductases,

dehydrogenases and peroxidases.
 Continue------
2. Transferases: These are enzymes catalyzing
the transfer of a chemical group from one
compound to the other,
• They are subdivided into:
• (a) Amino transferases: These bring about the
exchange of amino and keto group between
an amino acid and a keto acid.
 Cont-----
• (b) Kinases: These bring about the transfer of
a phosphate radical using ATP as the donor or
ADP acceptor.
• © Glycosyltransferases: These bring about the
transfer of a glycosyl group.
• (d) C1-Transferases: These bring about the
transfer of an acyl group.
 Continue------
3. Hydrolases: These are enzymes catalyzing the
process of hydrolysis (breakdown of the compound by
addition of water)
• AB + H2O AOH +HB

e.g. thiolases, amidases, ribonucleases,

deoxyribonucleases, hydrolytic deaminases,
phospholipases, phosphatases, glycosidases, esterase
and peptidases.
 Continue------
4. Lyases: these are enzymes, which catalyze
breakdown of substrates by mechanisms other
than hydrolysis and oxidation.
• They include desulfhydrases and dehydratases
which reversibly remove or add H2S or water
from substrate.
 Cont-----
• Non-oxidative decarboxylases: which remove
CO2, e.g. pyruvic decarboxylase, aldolases,
lyases
• cleavage enzymes are lyases. Phosphorylases
cut the substrate by adding phosphate, e.g.,
glycogen phosphorylase, e.g., (Glycogen)n +
H3PO4  (Glycogen)n-1 + Glucose-1-phoaphate
 Continue------
5. Isomerases: these are enzymes catalyzing
isomerization, e.g.,
• Epimerases, e.g., UDP-Glucose  UDP-Galactose.
• Cis-trans-isomerase, e.g., trans-Retinol  cis-
Retinol.
• Mutase, e.g., glucose-1-phosphate  glucose-6-
phosphate.
• Aldo-keto-isomerase, Glucose-6- phosphate 
Fructose-6- phosphate.
 Continue------
6. Ligases or synthetases:
• These are enzymes catalyzing the process of
ligation or binding of 2 molecules together in
the presence of ATP.
• Glycogen synthase, DNA Ligase, Fatty acid
synthase
e.g., Fatty acid + CoASH + ATP  Acyl-CoA +
AMP + PPi.
 Systems of enzyme nomenclature

1. Substrate-dependent naming:
The name is derived from the substrate with -
ase as a suffix,
e.g., urease (for urea), lipase (for lipids) and
protease (for protein).
 Continue------
2. Chemical nature of the reaction-dependent
naming:
• The name depends on nature of chemical
reaction with -ase as a suffix,
E.g., hydrolase (for hydrolysis) and
dehydrogenase (for removal of hydrogen).
 Continue------
3. Systemic naming:
• The International Biochemical Union give
every enzyme a code digital name.
• The digital name is of four digits, the first
refers to the enzyme class, the second refers
to the subclass, the third refers to the sub-
subclass and the fourth refers to the name of
the individual enzyme itself.
 Functional sites in the enzymes system:

Active site:
Substrate-binding site at which substrate
specifically binds and it is the site that carries
out the chemical action.
Allosteric site:
The term allosteric site means “the other site”
and allostery means “a change in shape”.
 Enzyme-substrate combination:
• During the enzyme action, there is a
temporary combination between the enzyme
and its substrate.
• The enzyme combines with its substrate to
give an enzyme substrate complex.
• The enzyme is liberated in a free state to
combine with new substrate(s) and so on. So
the enzyme acts only as a catalyst for the
proceeding of the reaction.
Continue------
 Cont-----
• LOCK-AND-KEY MODEL proposed by Emil
Fischer in 1894,
• the shape of the substrate and the active site
of the enzyme are thought to fit together like
a key into its lock.
• The two shapes are considered as rigid and
fixed, and perfectly complement each other
when brought together in the right alignment.
 Cont-----
• Induced-Fit model:
• The enzyme structure is flexible, not rigid.
• The active site adjusts to fit the shape of the
substrate more closely. At the same time the
substrate adjusts its shape to better adapt to
the geometry of the active site. As a result,
the reacting section of the substrate becomes
aligned exactly with the groups in the
active site that catalyze the reaction.
 Structural components of the enzyme system (holoenzyme):

The apoenzyme
• the protein part of the enzyme that
determines the substrate specificity and the
major determinant of the nature of the
chemical reaction.
 The coenzyme
• is an organic mostly vitamin-derived
compound or a free nucleotide, e.g., ATP,
cAMP, UTP.
• Examples of vitamin coenzymes are CoASH,
TPP, NAD, and FAD.
• The coenzyme is loosely (i.e., non-covalently)
attached to the enzyme and is not consumed
in the reaction.
 The activator
• It is an inorganic metal or non-metal ion, e.g.,
Ca2+, Mg2+, Fe2+, Zn2+ or Cu2+.
• Like coenzyme, the activator is loosely (i.e.,
non-covalently) attached to the enzyme and is
not consumed in the reaction.
• Both the coenzymes and activators are known
as cofactors.
 The prosthetic group
• It is an inorganic metal or an organic
compound or both, firmly (i.e., covalently)
bound to the apoenzyme.
• e.g., a heme group in hemoglobin.
Continue------
 Activation Energy

• It is the amount of energy required to raise all

the molecules in one mole of a substance to
the transition state at which it is ready to
cleave or form a product.
 How Enzymes Work

• For a molecule to be stable, it must have a high

activation energy for conversion to product (that’s
what “stable” means!)
• Even though equilibrium (thermodynamic) may
hugely favor product (remember coal and
diamond?)
• So, to convert stable molecules into other stable
molecules (even where equilibrium is unfavorable),
requires selective reduction of the activation
energy – the job of enzymes!
 Mechanism of enzyme action

• Enzymes increase the rate of reactions by

decreasing the activation energy required by
substrates to reach a transition state.
Factors affecting the rate of enzyme catalyzed reaction

1. Temperature:
• Increase of temperature increases the rate of
enzyme reaction until optimum temperature
at which the enzyme acts maximally
• This optimum temperature is 37 - 40 oC for
human enzymes
 Cont……
• The velocity is almost doubled for every 10 oC
increased.
• Since enzymes are protein in nature,
temperature increase above the optimum
temperature denatures and irreversibly
inactivates the enzyme.
 2. PH:
• It affects the state of ionization of amino acid residues
at the active sites of the enzyme and ionization of the
substrate.
• Extreme pH caused by strong acids or strong alkalis
lead to denaturation and destroy the enzyme due to
conformational and/or ionization change that also
affects the substrate.
• Starting from a specific pH, increase in pH leads to
increase of enzyme activity to certain pH at which the
enzyme acts maximally. Such pH is called optimum pH.
 3. Concentration of substrate:

• The rate of enzyme reaction is increased with

the increase of substrate concentration till a
certain point at which any increase in the
substrate concentration will cause no further
increase in the rate of enzyme reaction.
• This is due to saturation of all of the active
sites of the enzyme by the substrate and so
the excess substrate will find no free enzyme
to interact with.
 4. Concentration of enzyme:
• The rate of enzyme reaction directly
proportionate with the concentration of
enzyme provided that all other conditions are
constant and product is withdrawn.
• This is true at the beginning of the reaction
because the presence of some impurities and
accumulation of the product at the end of the
reaction inhibit the enzyme activity.
 Enzyme kinetics
• A mathematical and graphical study of the
rates of enzyme-catalyzed reactions.
• It studies reaction rates and how they change
in response to changes in experimental
parameters.
 Michaelis-Menten equation
• For enzyme-catalyzed reactions:
E + S < -----> ES -----> E + P
• The rate or velocity is dependent upon both [enzyme] and
[substrate].
k1 k3
E + S < -----> ES -----> E + P
k2
• k1 and k2 govern the rates of association and dissociation of ES

• k3 is the turnover number or catalytic constant

 Cont-----
• Vo = Vmax[S]
Km + [S] Michaelis-Menten equation
Vo = Initial reaction velocity( the rate of reaction
as soon as ES is formed.
Vmax = Maximum velocity ( when all the active sites are

occupied) it is the measure of the efficiency of

enzymes.
 Cont-----
• Km = the substrate concentration that produces
half-maximum velocity of the reaction (½Vmax)
that is termed Km value or Michaelis constant,
is constant and specific for every enzyme.
• Different enzymes reach Vmax at different [S]
because enzymes differ in their affinity for the
substrate or Km.
 Cont-----
• 1) The greater the tendency for an enzyme and
substrate to form an ES, the
higher the enzyme’s affinity for the substrate --->
lower Km.
• 2) At a given [S], the more enzyme will be in ES for
an enzyme with a higher affinity
• i.e. the greater the affinity, the lower the [S]
needed to saturate the enzyme or to reach Vmax.
 Cont-----

1/V
Vmax

1/2 Vmax 1/Vmax

- 1/Km

Km 1/S
Substrate concentration Lineweaver-Burk plot
 Cont----
• Example: Hexokinase and glucokinase are
isoenzymes that catalize convertion of glucose into
glucose-6-phosphate.
• Hexokinase is more active than glucokinase because
the amount of glucose (substrate) needed to reach ½
Vmax in case of hexokinase is less than in case of
glucokinase, i.e., Km of hexokinase is less than
glucokinase.
• This difference is of utmost physiological importance
to give each of them a distinct physiological role.
 Cont----

Hexokinase
Vmax

1/2 Vmax Glucokinase

Km Km
glucose concentration
 Enzyme inhibitors

1. Non-specific Inhibitors
They are irreversible enzyme poisons that
affect all enzymes, e.g.,
All denaturing factors, e.g., high temperature,
strong acids, strong alkalis, high pressure,
ultraviolet rays, Heavy metals
• All these non-specific inhibitors cause
irreversible destruction of the enzyme.
 2. Specific inhibitors

• Coenzyme inhibitors: e.g., cyanide, hydrazine

and hydroxylamine inhibit pyridoxal
phosphate (coenzyme form of vitamin B6),
anti-vitamins such as dicumarol against
vitamin K and sulfanilamide against p-amino
benzoic acid.
 Cont…..
2. Inhibitors of specific ion cofactor: e.g., Fluoride
inhibits the Mg2+ of enolase enzyme.

3. Prosthetic group inhibitors: e.g., cyanide inhibits

the heme prosthetic group of cytochrome oxidase.

• The coenzyme inhibitors, prosthetic group inhibitors

and inhibitors of specific ion cofactor cause
irreversible and non-competitive enzyme inhibition
 Apoenzyme inhibitors
Competitive inhibitors:
• The inhibitor has a structural similarity to the
substrate. So, it binds to the active site
(substrate-binding site) of in competition with
the substrate depending on their relative
concentration.
• Increasing substrate concentration or lowering
inhibitor concentration reverses this enzyme
inhibition.
 Conti…..
• The reaction is summarized as follows,
Without inhibitor, E + S  ES  E + P,
With inhibitor, E + I  EI  No product
Examples:
• Classic example is inhibition of succinic acid
dehydrogenase ( that oxidizes succinate into
malate) by malonic acid. This inhibition
prevents energy production and so malonic
acid is toxic to cells.
 Conti…..

COOH CH2 COOH

CH2
COOH CH2 COOH
malonic acid succinic acid
 Conti…
• Allopurinol is a competitive inhibitor for
xanthine oxidase that synthesizes uric acid
from purines. Therefore, allopurinol is used for
treating Gout.
• Methotrexate is competitive inhibitor of
tetrahydrofolate reductase that produces
tetrahydrofolate essential for cell division.
Therefore, methotrexate is used as anticancer
agent.
 Cont-----
• Sulfonamides are competitive inhibitors for
conversion of p-aminobenzoic acid into folic
acid in bacteria. Therefore, sulfonamides are
used as bacteriostatic antibiotics.
• In competitive inhibition Vmax is unaltered and
Km is increased.
 Non competitive inhibitors:
• The inhibitor is not structurally similar to the
substrate and does not bind its binding site.
• The inhibitor binds either to the free enzyme
or the enzyme-substrate complex (i.e.,
inhibitor does not prevent substrate binding)
but it decreases the enzyme catalytic rate.
• This inhibition can not be reversed by
increasing substrate concentration, but by
special measures such as dialysis.
 Conti…
• Examples of noncompetitive inhibitors is the
inhibition of an enzyme by hydrogen ion at acidic
side and by the hydroxyl ion at the alkaline side of its
optimum pH. The reaction is summarized as follows,
• Without inhibitor, E + S  ES  E + P.
• With inhibitor, E + S + I  EI or ESIno product
• In noncompetitive inhibition Vmax is decreased and
Km is unaltered.
 Uncompetitive inhibitors
• This inhibitor binds at a site that only becomes
available after the substrate has bound to the
enzyme.
• This is encountered in multi-substrate
enzymes, where the inhibitor compete with
one substrate (e.g., S2) of them and is
uncompetitive for the another (e.g., S1). The
reaction is summarized as follows,
 Cont-----
• Without inhibitor, E + S1  ES1 + S2  ES1S2 
E + Ps.
• With inhibitor, E + S1  ES1 + I  ES1I  no
product
• In uncompetitive inhibition both Vmax and Km
decreases.
 Regulation of enzyme activity

A. End products or feedback regulation

Feedback inhibition refers to the phenomenon
whereby a late product in a cascade of catabolic
or anabolic reactions inhibits an enzyme used in
the early steps of the pathway.

inhibits

enzyme 1 enzyme 2 enzyme 3

A B C D
 B. Allosteric modulation
• Allosteric effectors are positive if they
enhance the catalytic rate by increasing the
enzyme-substrate affinity and negative if they
decrease the catalytic rate
 Conti…

Allosteric Catalytic/Substrate-binding site No substrate binding,

site No product

+ +
Allosteric
inhibitor Conformational substrate Substrate binding
Allosteric enzyme Change site unfit

+ + +
Allosteric
activator Products
Allosteric enzyme Conformational substrate Perfect binding
Change
 C. Covalent modification
• It means the addition by a covalent bond of a
foreign group, e.g., phosphate group, into the
enzyme or removal of such group.
• Some enzymes such as glycogen synthase is
inactivated upon phosphorylation, whereas,
glycogen phosphorylase is activated by
phosphorylation.
 D. Proenzymes (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.
 Cont-----
• After excess polypeptide chain is removed,
the enzyme becomes active. For example,
trypsin (an important catalyst for the
digestion of the proteins) is manufactured
as the inactive molecule trypsinogen (a
zymogen). When a fragment containing six
amino acid residues is removed from the N-
terminal end of trypsinogen, the molecule
becomes a fully active trypsin molecule.
 ISO-ENZYMES
• Iso-enzymes are physically distinct forms of the
same enzyme activity.
• Higher organisms have several physically distinct
versions of a given enzyme, each of which catalyzes
the same reaction.
• Isozymes arise through gene duplication and
exhibit differences in properties such as sensitivity
to particular regulatory factors or substrate affinity
that adapts them to specific tissues or
circumstances.
 Cont-----
• Some isozymes enhance survival by providing a
“backup” copy of an essential enzyme. Isozymes
are expressed only in specific cell types.
• The expression of isozymes in specific cells occurs
during certain periods of development, or in
response to specific physiologic or
pathophysiologic changes.
• Thus analysis of the presence and distribution of
enzymes and isozymes are often useful in
diagnosis.
 Cont-----
• Isoforms of Lactate dehydrogenase is useful in
diagnosis of myocardial infarction.
• While study of alkaline phosphatase isoforms
are helpful in diagnosis of various bone
disorder and obstructive liver diseases.
 CLINICAL SIGNIFICANCE OF ENZYMES
• The measurement of enzymes level in serum is
applied in diagnostic application.
• Detection of certain enzymes in the serum
indicates that tissue or cellular damage has
occurred resulting in the release of intracellular
components into the blood.
• Hence, when a physician indicates that he/she is
going to assay for liver enzymes, the purpose is
to ascertain the potential for liver cell damage.
 Cont------
• Commonly assayed enzymes are the amino
transferases: alanine transaminase, ALT (sometimes
still referred to as serum glutamate-pyruvate
aminotransferase, SGPT) and aspartate
aminotransferase, AST (also referred to as serum
glutamate- oxaloacetate aminotransferase, SGOT);
lactate dehydrogenase, LDH; creatine kinase, CK
(also called creatine phosphokinase, CPK); gamma-
glutamyl transpeptidase, GGT. Other enzymes are
assayed under a variety of different clinical situations