Dr Abdul Lateef
Assistant Professor
Dept of Biochemistry
Definition
 Enzymes are biocatalysts synthesized by living cells.
 They are protein in nature.
 (Except Ribozyme which is RNA).
 They are colloidal
 Thermolabile in character and
 Specific in their action.
Cofactor
 The protein part of the enzyme on its own is not
always adequate to bring about the catalytic activity.
Many enzymes requires certain non protein small
additional factors collectively referred to as cofactor for
catalysis.
 The cofactor may be organic or inorganic in nature.
Coenzyme:
 The non protein organic low molecular weight and
dialysable substance associated with enzyme function is
known as coenzyme.
Coenzyme as second substrate:
 Coenzyme are regarded as second substrate or co-substrate
since they have affinity with the enzyme comparable with
that of the substrates.
 Coenzymes undergo alterations during the enzymatic
reactions which are later regenerated.
 This is in contrast to substrate which is converted to
product.
Activator:
 The term activator is referred to the inorganic cofactor
(Mg, Ca, Mn etc) necessary to enhance enzyme
activity.
Prosthetic group:
 The functional enzyme is referred to as holoenzyme
which is made up of a protein part(apoenzyme) and a
non protein part(coenzyme).
 The term prosthetic group is used when a non protein
moiety is tightly bound to the enzyme which is not
easily separated by dialysis.
IUBMB
 IUBMB - International Union of Biochemistry and
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Molecular Biology.
Used for nomenclature of enzymes.
A four digit enzyme commission (EC) number is assigned
to each enzyme representing the class (first digit), sub class
(second digit), sub sub class (third digit) and the individual
enzyme (fourth digit)
The nomenclature was determined by the Enzyme
Commission in 1961 (with the latest update having
occurred in 1992)
All enzymes are assigned an “EC” number
Classification of enzymes
 EC 1. Oxidoreductases
 EC 2. Transferases
 EC 3. Hydrolases
 EC 4. Lyases
 EC 5. Isomerases
 EC 6. Ligases
Class 1: Oxidoreductases
 Catalyze the transfer of hydrogen or oxygen atoms or
electrons from one substrate to another.
 Also called oxidases, dehydrogenases, or reductases
 Catalyzes redox reactions, therefore an electron
donor/acceptor is also required to complete the reaction.
 Ex: Alcohol dehydrogenase. (1.1.1.1)
Example:
RCH2-OH 
RCH=O
Class 2: Transferases
 Transfer chemical groups from one molecule to another or
to another part of the same molecule
 Catalyze group transfer reactions.
 Ex: Hexokinase (2.7.1.1)
 These are of the general form:
A-X + B ↔ BX + A
Classs 3: Hydrolases
 Catalyze hydrolytic reactions
 Cleave a bond using water to produce two
molecules from one
 Ex: Lipase (3.1.1.3)
 These are of the general form:
A-X + H2O ↔ X-OH + HA
Class 4: Lyases
 Catalyze non-hydrolytic removal of functional groups from
substrates
 Remove a group from or add a group to double bonds
 Often creating a double bond in the product
 Or the reverse reaction, ie, addition of function groups
across a double bond.
Ex:Aldolases. (4.1.2.7)
Class 5: Isomerases
 Catalyzes isomerization reactions.
 Interconvert isomeric structures by molecular
rearrangements
 Ex: Triose phosphate isomerase (5.3.1.1)
Class 6: Ligases
 Catalyzes the synthesis of various (mostly C-X) bonds
 Coupled with the breakdown of energy containing
substrates, usually ATP
 Join two separate molecules by the formation of a new
chemical bond usually with energy supplied by the
cleavage of an ATP
 Ex:Glutamine synthetase (6.3.1.2)
Mechanism of enzyme action
Mechanism of enzyme action
 Fischer’s Lock and key model
 Koshland’s Induced Fit Model
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The Lock and Key Hypothesis
1.
2.
3.
4.
5.
6.
7.
Fit between the substrate and the active site of the
enzyme is exact
Like a key fits into a lock very precisely
The key is analogous to the enzyme and the substrate
analogous to the lock.
Temporary structure called the enzyme-substrate
complex formed
Products have a different shape from the substrate
Once formed, they are released from the active site
Leaving it free to become attached to another substrate
The Induced Fit Hypothesis
 Some proteins can change their shape
(conformation)
 When a substrate combines with an enzyme, it
induces a change in the enzyme’s conformation
 The active site is then moulded into a precise
conformation
 Making the chemical environment suitable for the
reaction
 The bonds of the substrate are stretched to make
the reaction easier (lowers activation energy)
MODE OF ACTION OF ENZYMES
 After binding takes place, catalysis generates transition
state complexes leading to the formation of reaction
products.
 Enzymes accelerate reaction rates, but do not
alter the equilibrium point of the reaction.
Enzymes increase reaction rates by decreasing the
amount of energy required to form the transition
state.
Activation energy is the energy required to reach the
transition state
 Energy of activation is the energy difference between the
reactants and a high energy intermediate (transition state) that
occurs during the formation of product.
A ↔ T* ↔ B
All chemical reactions have this energy barrier separating
reactants and products.
 Because of the high energy of activation, the rates of un-catalyzed
reactions are slow, because only a small proportion of molecules
may possess enough energy to achieve the transition state.
 The lower the energy of activation, more molecules have sufficient
energy to pass through the transition state at a given temperature.
 Enzymes lower the energy of activation and thus allow a reaction
to proceed rapidly.
Mechanism of Catalysis
Acid - Base catalysis
Enzyme gives or takes H+ to bring about catalysis.
(At physiological pH, histidine is the most
important amino acid)
Substrate strain
Substrate strain increases energy level of substrate
leading to a transition state
Covalent catalysis
The covalent binding of the substrate to the enzyme
Factors affecting enzyme activity
 Important factors that affect enzyme activity are
1. Enzyme concentration
2. Substrate concentration
3. Temperature
4. pH
5. Product concentration
6. Presence of activators or inhibitors
7. Availability of coenzymes
Effect of Substrate Concentration
 The rate of an enzyme
catalyzed reaction increases
with substrate
concentration in a
rectangular hyperbolic
curve.
 This increase in rate occurs
until maximum velocity
(Vmax) is reached
Vmax reflects the saturation of all the available
binding sites on the enzyme with substrate
Effect of Enzyme Concentration
 At a given substrate
concentration, the initial
velocity of an enzyme
catalyzed reaction is
proportional to the enzyme
concentration
 Property made use of in
determining the level of
particular enzyme in
plasma, serum or tissues
Effect of Temperature
 Reaction velocity increases with
an increase in temperature till a
peak is reached, often at 40-60oC.
 This is the result of the increased
number of molecules having
sufficient energy to pass the
energy barrier & form products.
 A further increase in
temperature causes denaturation
of enzyme & decreases the
reaction velocity.
 An important exception is the
Taq polymerase from
thermophilic bacteria that is
active at very high temperatures
and is used for PCR (polymerase
chain reaction).
Effect of pH
• Most enzymes in the
human body function
optimally in the
physiological pH range
(around pH 7.4).
• Some exceptional
enzymes include pepsin
with a pH optimum of
1.5-2.0, secreted in gastric
juice.
• Changes in pH affect
ionic charge of amino
acid side chains of
enzymes (histidine,
glutamate, cysteine) and
dramatically affect
catalytic function.
Effect of product concentration
 The accumulation of reaction products generally
decreases the enzyme velocity.
 For certain enzymes the product combines with the
active site of enzyme and forms a loose complex and
thus inhibits the enzyme activity.
Effect of activators:
 ↑ enzyme activity.
 Metal activated enzymes: metal not held tightly. Ex:
ATPase (Mg and Ca) and Enolase (Mg)
 Metalloenzymes: metals are held tightly. Ex: Alcohol
dehydrogenase (Zn)
Availability of Coenzymes:
 ↑ enzyme activity.
Enzyme Kinetics
Enzyme Kinetics
Michaelis and Menten, in 1913, developed a simple
model for examining the kinetics of enzyme catalyzed
reactions.
• The model assumes that the enzyme [E] reversibly
combines with its substrate [S] to form an
intermediate enzyme-substrate complex [ES] that
subsequently breaks down to product. ES is
relatively stable.
• The series of events can be shown thus:
k1
k2
E + S ↔
ES ↔ E + P
k-1
k-2
• The Michaelis-Menten equation describes how reaction
velocity varies with substrate concentration
Where:
V1 = initial reaction velocity
Vmax = maximal velocity
Km = Michaelis constant = (k-1 + k2)/k1
[S] = substrate concentration
Michaelis Constant (Km)
 The Km is a constant and is characteristic of an enzyme
and its particular substrate
 It is a measure of the affinity of the enzyme for that
substrate. (Low values indicate high affinity.)
 It is also numerically equal to substrate concentration at
which the reaction velocity is equal to ½ Vmax.
A small or low Km:
• Reflects a high affinity of
enzyme for substrate.
• A very low concentration
of substrate is needed to
reach a velocity that is, say,
half maximal.
A large or high Km:
 Reflects low affinity of
enzyme for the substrate
 A high concentration of
substrate is needed to
saturate one half the enzyme.
 At [S] near point A the rate is
directly proportional to
substrate concentration and the
reaction rate is said to be first
order (i.e., dependent upon
[S]).
 At [S] near point C and at very
high substrate concentrations,
the rate is nearly independent of
substrate concentration and the
reaction rate is said to be zero
order (i.e., not dependent upon
[S]).
 At [S] near point B, the rate is ½
Vmax. The substrate
concentration at point B is by
definition equal to Km.
Enzyme Inhibition
 Enzyme inhibitor is defined as a substance which
binds with the enzyme and brings about a decrease in
catalytic activity of that enzyme.
 The inhibitor may be organic or inorganic in nature.
 There are three broad categories of enzyme inhibition:
 1. Reversible inhibition.
 2.irreversible inhibition.
 3.Allosteric inhibition.
Inhibitors
 Inhibitors are chemicals that reduce the rate of
enzymic reactions
 The are usually specific and they work at low
concentrations
 They block the enzyme but they do not usually destroy
it
 Many drugs and poisons are inhibitors of enzymes in
the nervous system
Types of enzyme inhibition
 Reversible inhibitors: These can be washed out of
the solution of enzyme by dialysis.
There are two categories:
Competitive and non competitive.
Competitive Inhibition
A competitive inhibitor:
 Has a structure similar to substrate
 Occupies active site
 Competes with substrate for active site
 Inhibition is reversed by increasing substrate
concentration
Vmax is unchanged.
Km is increased
Malonate as an Example of a Competitive Inhibitor
 Succinate Dehydrogenase is an
important enzyme of the TCA cycle
that converts succinate to fumarate
 Malonate is structurally very
similar to succinate and acts as a
competitive inhibitor of the
enzyme
Malonate cannot form a C=C
 Inhibition by malonate can be
reversed by increasing the
concentration of succinate
NoncompetitiveInhibition
A noncompetitive inhibitor
 Not a structural analogue
 Binds to the enzyme but not at active site
 Changes the shape of enzyme
 Substrate binds but catalysis does not occur in the
presence of inhibitor
 Effect is not reversed by adding substrate
 Km is unaltered.
 Vmax is decreased proportionately to inhibitor
concentration.
Lead Poisoning as an Example of a
Noncompetitive Inhibitor
 Lead binds with the sulfhydryl group of cysteine
residues in enzymes. The cysteine residues are not
part of the active site.
 Lead causes noncompetitive inhibition of enzymes
like the Ferrochelatase (required in the synthesis
of heme for incorporation into heme proteins such
as hemoglobin).
Uncompetitive inhibitors
 Inhibit mainly multisubstrate enzymes.
 Inhibitor binds only after
first substrate forms ES
complex.
 ESI complex cannot form
products.
Irreversible inhibition
 Inhibitor binds covalently with the enzyme
to form a stable complex
 Covalent modification (usually) of the active
site
 Examples, di-isopropylfluorophosphate
(“DIFP” - binds to serine residues),
iodoacetate, heavy metal ions
 Drug aspirin; acetylates serine residues in
active site of cyclooxygenase
The switch: Allosteric inhibition
Allosteric means “other site”
Active site
E
Allosteric
site
Switching off
 These enzymes have
two receptor sites
 One site fits the
substrate like other
enzymes
 The other site fits an
inhibitor molecule
Substrate
cannot fit
into the
active site
Inhibitor
molecule
Inhibitor fits
into allosteric
site
The allosteric site the enzyme “on-off”
switch
Active
site
Substrate
fits into
the
active
site
E
Allosteri
c site
empty
The
inhibitor
molecule is
absent
Conformational
change
Substrate
cannot fit
into the
active
site
E
Inhibitor
molecule
is
present
Inhibitor fits
into
allosteric
site
A change in shape
 When the inhibitor is present it fits into its site and
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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
Negative feedback is achieved
 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
Examples of Enzyme Inhibition –
Drug Therapy: Enzymes Used for Therapeutic Purpose
 Methotrexate, an inhibitor of dihydrofolate reductase, is used
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in cancer chemotherapy to inhibit DNA synthesis in rapidly
growing cells (i.e., tumors).
Aspirin is used to inhibit the synthesis of prostaglandins (by
cyclooxygenases 1 and 2) which are at least partly responsible
for the aches and pains of arthritis.
Sulfonamides (folate synthase) are used to inhibit folic acid
synthesis, essential for the metabolism and growth of diseasecausing bacteria.
Allopurinol is used to inhibit xanthine oxidase in treatment of
hyperuricemia and gout.
Statins are used to inhibit HMG-CoA reductase and lower
blood cholesterol levels.
Dicumarol is a structural analog of vitamin K and is used as an
anticoagulant.
Regulation of Enzyme Activity
Long Term Regulation of Enzyme Activity
Two principal mechanisms include:
1. Regulation of gene expression: By increasing (induction)
or decreasing (repression) the rate of gene transcription, the
quantity and rate of enzyme synthesis is controlled. Its a slow
control process that may take hours to days.
2. Regulated enzyme degradation can be slowed down or
speeded up by ubiquitin/proteosome pathway and lysosomal
pathway.
Short Term Regulation of Enzyme Activity
Short term regulation does not affect the concentration of
enzyme. It is reversible and rapid in action and actually carries
out most of the moment-to-moment physiological regulation of
enzyme activity.
These mechanisms include:
• Changes in substrate concentration
• Product inhibition (hexokinase is strongly inhibited by its product glucose 6phosphate whereas glucokinase is not
• Feedback inhibition (product of a pathway often inhibits its own synthesis
back up at the first step.)
• Activation of pre-existing pools of inactive pro-enzymes to produce active
enzymes (protease activation)
• Regulation by reversible covalent modification (phosphorylationdephosphorylation)
• Allosteric regulation. PFK-1 regulation by F2,6BP
Allosteric Regulation of Enzymes
 Some multimeric enzymes bind small, physiologically
important molecules that modulate their activity
 These are known as allosteric enzymes and the small
regulatory molecules to which they bind are known as
allosteric effectors
 Allosteric effectors bind to the enzyme at a site
different from the catalytic site
 Binding of effector causes conformational changes to
the catalytically active site(s)
 The hallmark of effectors is that when they bind to
enzymes, they alter the catalytic properties of an
enzyme's active site.
 Those that increase catalytic activity are known as positive
effectors.
 Effectors that reduce or inhibit catalytic activity are negative
effectors
 Most allosteric enzymes are generally located at or near
branch points in metabolic pathways, where they are
influential in directing substrates along one or another of
the available metabolic paths
Isoenzymes
Isoenzymes
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Isoenzymes are different forms of same enzyme,
catalyzing same chemical reactions but present
at different tissues exhibiting different physical
and kinetic properties
Produced by different combination of
polypeptide subunits
Coded by different genes but expressed
differentially in different tissues
Useful in clinical diagnosis and monitoring
E.g., Creatine kinase (CK) and
Lactate dehydrogenase (LDH)
Importance of isoenzymes measurements in
serum:
 Presence of
disease
 Organs involved
 Aetiology /nature of
disease: differential diagnosis.
disease  more damaged cells-more
leaked enzymes in blood
 Extent of
 Time course of
disease.
Measurement of enzyme activity
 Enzyme activity is expressed in
International unit (IU)
It corresponds to the amount of enzymes that
catalyzes the conversion of one micromole
(mol) of substrate to product per minute
Diagnostic importance of LDH
Type
Compositi
on
Location
Importance
LDH1 30% HHHH
Heart, RBC,
Myocardial infarction
LDH2 35% HHHM
White cells
Megaloblastic anemia
Leukemia, malignancy
LDH3 20% HHMM
Lung
Pulmonary infarction
LDH4 10% HMMM
Kidney,
placenta,
pancreas
Kidney and pancretic
disease.
LDH5 5%
Liver, skeletal
muscle
Liver disease, muscle
injury
MMMM
Diagnostic importance of CK
Type
Compostion Location Importance
CK1 80%
MM
Skeletal
muscle
Muscular dystrophy
CK2 5%
MB
Heart
Myocardial infarction
CK3 1%
BB
Brain
Brain disorders
M. Zaharna Clin. Chem. 2009
Thank you