ENZYME KINETICS

The study of enzyme reaction rates and how they change

in response to changes in experimental parameters is
known as kinetics.

One of the key factors affecting the enzyme reaction rates

is the concentration of substrate [S].
Effect of Substrate Concentration
V0 : Initial velocity of the reaction
Vmax : Maximum velocity of reaction (i.e. maximum capacity of
enzyme saturated by substrate)
[S] : substrate concentration
Km =1/2 Vmax (Michaelis Menten constant)
(Km is point from which enzyme get activated)
 Significance of Km (Michaelis Constant)
1. Km value is substrate concentration (expressed in moles/L)
at half-maximal velocity.
2. It denotes that 50% of enzyme molecules are bound with
substrate molecules at that particular substrate concentration.
• Km is the signature of the enzyme. Km value is thus a constant for
an enzyme. It is the characteristic feature of a particular enzyme
for a specific substrate.
• Km denotes the affinity of enzyme for substrate. High Km
indicates weak binding and a low Km indicates strong binding with
its substrate.
For ex.
Km of Hexokinase for glucose is 0.05mmol
Km of Glucokinase for glucose is 5.0 mmol

Hexokinase has low Km i.e high affinity and Glucokinase has high
Km i.e. low affinity for Glucose

Km α 1
Affinity of enzyme towards
any substrate
Km is independent of enzyme concentration. If enzyme
concentration is doubled, the Vmax will be double. But the
Km will remain exactly same. In other words, irrespective of
enzyme concentration, 50% molecules are bound to substrate
at that particular substrate concentration.
Effect of enzyme concentration on Km.
Km is independent of enzyme concentration. If enzyme
concentration is doubled, the Vmax will be double. But
the Km will remain exactly same.
 Significance of Vmax

 The Vmax of a reaction is an index of the catalytic

efficiency of an enzyme.
 The Vmax is useful in comparing the activity of one
enzyme with that of another.
REACTION MODEL

where S is the substrate

E is the enzyme
ES is the enzyme-substrate complex
k1, k-1, and k2 are rate constants
MICHAELIS MENTEN EQUATION
ASSUMPTIONS
1. Relative concentrations of E and S
• S >E, so [ES] at any time is small
2. Steady-state assumption
• [ES] does not change in time
• E + S = ES = E + P, the rate of formation of ES is
equal to that of the breakdown of ES
3. Initial velocity
• Used in the analysis of enzyme reactions
• Rate of reaction is measured as soon as E and S
are mixed
• P is very small, the rate of back reaction from P
to S can be ignored
• Describes how reaction velocity varies with substrate
concentration

Vmax S
vo =
Km + S

where Vo = initial reaction velocity

Vmax = maximal velocity
Km = Michaelis constant
S= substrate concentration
When measured velocity (V) is exactly one half of Vmax

Vmax = Vmax [S]

2 Km + [S]
2 Vmax [S]
Km + [S] =
Vmax
Km + [S] = 2 [S]

Km = [S] When V = ½ Vmax

Effect of substrate concentration on reaction
velocities
• Small Km for enzyme
1 reflects a high affinity
of enzyme for the
substrate
• Large Km for enzyme 2
reflects low affinity of
enzyme for the
substrate
At high concentration of
substrate( [S]>>Km), The
At of the reaction is
velocity
zero order – that is, constant
and independent OF
substrate concentration

At low concentration of
substrate( [S]<<Km), The
velocity of the reaction is first
order – that is, proportional
to substrate concentration
 Lineweaver-Burk Plot
• Also called a double-reciprocal plot
• If 1/V0 is plotted VS 1/[S], a straight line is obtained
• The intercept on the x-axis is equal to -1/Km
• The intercept on the y-axis is equal to 1/Vmax
 Lineweaver-Burk plot
(Double reciprocal plot)
 Lineweaver-Burk Plot
• Can be used to calculate Km and Vmax as well as to determine the
mechanism of enzyme inhibitors
• Equation describing the Lineweaver-Burk Plot is:
 ENZYME INHIBITION
A process in which enzyme activity decreases is called as inhibition.
Enzymes are protein in nature, any agent which denatures proteins will
inactivate the enzymes, known as inhibitors or negative modulators.
They can diminish the velocity of an enzyme reaction.
These are inorganic ions like CN which inhabits cytochrome oxidase or
organic substances.
Two general classes of inhibitors are:
1. Reversible inhibitor
2. Irreversible inhibitor.
 REVERSIBLE INHIBITOR
Reversible inhibitors bind to enzymes through non-covalent bonds
and the activity of the enzyme is restored fully when the inhibitor
is removed from the system.

Different types of reversible inhibitors are:

i. Competitive or substrate analogue inhibitor
ii. Non-competitive inhibitor
iii. Uncompetitive inhibitor.
 Competitive or Substrate Analogue Inhibitor
A competitive inhibitor is a structural analogue of the substrate.

Chemical structure of inhibitor (I) resembles that of substrate (S)

and binds to enzyme at active site, forming EI complex rather than
ES-complex.

Degree of competitive inhibition depends on

• The relative concentration of the substrate and inhibitor

• And their affinity towards enzyme

Diagrammatic representation of competitive inhibition

E: Enzyme; S: Substrate; I: Competitive inhibitor; P: Product

The inhibition could be overcome by increasing substrate

concentration
Krebs cycle
Enzyme kinetics of competitive inhibitor

Michaelis Menten Saturation Curve

Lineweaver-Burk Plot
Enzyme kinetics of competitive inhibitor
Vmax is unaltered
Km is increased
 Drugs act as competitive inhibitors
Methotrexate
A structural analog of dihydrofolate (substrate of dihydrofolate
reductase) is used to treat Cancer.
Sulphonamide Folic acid= PABA + Glutamic acid + pteridine ring

Analogue of P- aminobenzoic acid (PABA) and inhibits the

synthesis of folic acid in microorganisms.
Isoniazide [Isonicotinic acid hydrazine (INH)]
It is an anti-tuberculosis drug, inhibits the biosynthesis of NAD
and restrict the growth of the organisms that cause tuberculosis.
Dicumarol
It is an anticoagulant drug structurally similar to vitamin
K. It inhibits the vitamin K activity and inhibits the
formation of prothrombin.
Physostigmine
It inhibits acetylcholinesterase and use to treat glaucoma
and myasthenia gravis.
Drugs such an ibuprofen (anti-inflammatory drug), statin
(cholesterol lowing drug) are competitive inhibitors of
enzymes, that involved in the prostaglandins and cholesterol
synthesis respectively.
Drug Enzyme inhibited Clinical use
Allopurinol Xanthine oxidase Gout
Dicoumarol Vitamin K epoxide reductase Anticoagulant
Penicillin Transpeptidase Antibacterial
Sulfonamide Pteroid synthetase Antibacterial
Trimethoprim FH2-reductase Antibacterial
Pyrimethamine FH2-reductase Antimalarial
Methotrexate FH2-reductase Anticancer
6-mercaptopurine Adenylosuccinate synthetase Anticancer

5-fluorouracil Thymidylate synthase Anticancer

Drug Enzyme inhibited Clinical use

Azaserine Phosphoribosylamidotransferase Anticancer

Cytosine DNA polymerase Anticancer

arabinoside
Acyclovir DNA Polymerase of virus Antiviral

Neostigmine AcetylCholinesterase Myasthenia

Alpha-methyldopa Aromatic L amino acid Hypertension

decarboxylase/DOPA decarboxylase
Mevinolin and (Hydroxy methyl glutaryl)HMGCoA Cholesterol
Lovastatin reductase lowering
Oseltamiver Neuraminidase Influenza
(Tamiflu)
 Non-competitive Inhibitors
 No competition occurs between substrate and inhibitor.
 Inhibitor is usually structurally different from the substrate.
 It binds at a site on the enzyme molecule other than the
substrate-binding site.

 Noncompetitive inhibitor can bind free enzyme (EI) or the

enzyme substrate complex (EIS)
 However, EIS complex does not continue to form product.

 Noncompetitive inhibitor lowers the concentration of

functional enzymes.

 Noncompetitive inhibition cannot be overcome by increasing

the substrate concentration
 Diagrammatic representation of noncompetitive inhibition.

(E = Enzyme; S = Substrate; I = Non-competitive inhibitor; P = Product)

 Examples of non-competitive inhibitors are:

– Ethanol or certain narcotic drugs are non-com­petitive

inhibitor of acid phosphatase.

– Trypsin inhibitors occur in soybean and raw egg

white, inhibit activity of trypsin.

– Ascaris parasites (worm) contain pepsin and trypsin

inhibitors, inhibit action of pepsin and trypsin.

• Cyanide inhibits cytochrome oxidase in ETC
• Fluoride will remove magnesium and manganese ions and so
will inhibit the enzyme, enolase, and consequently the
glycolysis.
• Iodoacetate would inhibit enzymes having -SH group in their
active centers.
• BAL (British Anti Lewisite; dimercaprol) is used as an
antidote for heavy metal poisoning
Michaelis-Menten Saturation Curve
Lineweaver-Burk Plot
For non-competitive inhibition, the Km value is
unchanged while Vmax is lowered
 Comparison of Two Types of Inhibition
Competitive Noncompetitive
inhibition inhibition
Acting on Active site May or may not
Structure of inhibitor Substrate analog Unrelated molecule
Inhibition is Reversible Generally
irreversible
Excess substrate Inhibition relieved No effect
Km Increased No change
Vmax No change Decreased
Significance Drug action Toxicological
 Uncompetitive Inhibitor
 Uncompetitive inhibitor can bind only to the enzyme-
substrate (ES) complex.
 It does not have affinity for free enzyme.
 Enzyme-substrate-inhibitor complex, ESI does not
continue to form any product.
 Uncompetitive inhibition cannot be overcome by the
addition of more substrate.
 Consequently Vmax cannot be attained, even at high
substrate concentration.
Uncompetitive inhibitor binds only to enzyme-
substrate complex.
Uncompetitive inhibitor decreases both Vmax and Km.
 Inhibition of placental alkaline phosphatase (Regan
isoenzyme) by phenylalanine

 The herbicide glyphosate, also known as Roundup, is an

uncompetitive inhibitor of an enzyme in the biosynthetic
pathway for aromatic amino acids in bacteria but it is fairly
nontoxic in animals because they lack the enzyme.
IRREVERSIBLE INHIBITOR
An irreversible inhibitor binds with an enzyme tightly

covalently and forms a stable complex.

An irreversible inhibitor cannot be released by dilution or

dialysis or simply by increasing the concentration of

substrate.
Irreversible inhibitors can be divided into three

categories:

 Substrate analogue inhibitor or affinity labels

 Group specific inhibitors

 Suicide inhibitor or mechanism based

inactivation.
In terms of enzyme kinetics, the effect of an irreversible

inhibitor is like that of the reversible non-competitive

inhibitors resulting in a decreased in Vmax but having

no effect on the Km
Irreversible inhibition
 Substrate Analogue Irreversible Inhibitor or
Affinity Labels

Substrate analogues or affinity labels are molecules

that are structurally similar to the substrate.

These substrate analogues possess a highly reactive

group which is not present in the natural substrate.
The reactive group of substrate analogues covalently
reacts with amino acid residues of the active site of the
enzyme and permanently block the active site of the
enzyme

3-Bromoacetal phosphate (BAP) (structural analogue of

DHAP) covalently modifies glutamic acid residue at the
active site of enzyme phosphotriose isomerase of
glycolysis.
 N-Tosyl-L-phenylalanine Chloromethyl ketone

TPCK react irreversibly with histidine residue present at the

active site of chymotrypsin and inhibit it.

 Group Specific Irreversible Inhibitor

 These inhibitors react with specific R-groups (side chain)

of amino acid residues in the active site of enzyme that

plays an essential role in catalysis, substrate binding or

maintenance of enzyme’s functional conformation.

 Examples of group specific irreversible inhibitors:

• Di-isopropylphosphofluoride (DIPF)

• Iodoacetamide

• Heavy metals
 DIPF can inhibit an enzyme acetylcholine esterase ( an

enzyme important in the transmission of nerve impulses)

by covalently reacting with hydroxyl group of a serine

residue present at the active site of the enzyme

 DIPF has also been found to inhibit trypsin, chymotrypsin,

elastase and phosphoglucomutase

Figure 6.20: Irreversible inhibition of acetylcholinesterase by a
group-specific inhibitor, diisopropylphosphofluoride (DIPF).
 Iodoacetamide and heavy metals like, Pb 2+, Ag+,

Hg2+, etc. which react with sulfhydryl (-SH) group

of cysteine residues present at the active site of the

enzyme and makes them inactive.

 Iodoacetamide also reacts with imidazole group of

histidine residues of the enzymes.

 Importance of Group specific irreversible inhibitors

This type of inhibitors can be used to identify the catalytic

groups present on amino acids of active site such as:
• Thiol group of cysteine
• Hydroxyl group of serine
• Imidazole group of histidine
 Suicide Inhibitor or Mechanism Based inactivation

These compounds are relatively unreactive until they bind to

the active site of a specific enzyme.

On binding to the active site of the enzyme they carry out

the first few catalytic activities of the normal enzyme

reaction.
Instead of being transformed into a normal product, however, the

inhibitor is converted to a very reactive compound that combines

irreversibly with the enzyme leading to its irreversible inhibition

These are also called mechanism based inactivation because they

utilize the normal enzyme reaction mechanism to inactivate the

enzyme
Example Suicide Inhibitor

Penicillin

Inactivates bacterial enzyme glycopeptidyl transpeptidase

involved in the formation of bacterial cell wall.

Allopurinol

In the case of Allopurinol which is oxidized by xanthine

oxidase to alloxanthine that is a strong inhibitor of xanthine
oxidase.
Aspirin
Inactivates an enzyme cyclo-oxygenase required for the synthesis
of prostaglandins
The anti-inflammatory action of Aspirin is also based on the suicide
inhibition. Arachidonic acid is converted to prostaglandin by the enzyme
Cyclo-oxygenase. Aspirin acetylates a serine residue in the active center of
cyclo-oxygenase, thus prostaglandin synthesis is inhibited, and so
inflammation subsides.

Aspirin is also used as antipyretic and analgesic drugs.

It also used to inhibit platelet aggregation and coronary thrombosis
Disulfiram (antabuse)

 Inhibits aldehyde dehydro­genase enzyme resulting in accumulation of

acetaldehyde.

Antabuse, or disulfiram is, a medicine for treatment of alcohol abuse

and alcohol dependence. Antabuse is prescribed to people who want
to quit drinking.
Acetyl CoA TCA cycle

Fatty acid
Clinical Application of Enzyme Inhibitor

 Enzyme inhibitors have therapeutic applications.

 Most antibiotics and anticancer drugs that are used

therapeutically are either competitive inhibitor or mechanism

based suicide inhibitor.

 Enzyme inhibitors as metabolic Poisons
• Many poisons work by inhibiting the action of enzymes
involved in metabolic processes, which hampers the metabolic
pathways.
• This inhibition can be reversible or irreversible.
• Most of them act irreversibly.
Poisons Enzyme inhibited Impaired pathway
Arsenate Glyceraldehyde 3-phosphate Inhibition of
dehydrogenase Glycolysis
Fluoride Enolase
Cyanide Cytochrome oxidase Inhibition of ETC
Rotenone Inhibit transfer of reducing Inhibition of ETC
Amobarbital (I) equivalents in ETC
Piericidin A
Antimycin A (III)
Malonate Succinate dehydrogenase Inhibition of TCA

Diisopropyl Acetylcholinesterase Impair nerve conduction

Fluorophosphate
(nerve gas)
Malathion Acetylcholinesterase Impair nerve conduction
(Organophosphorus
insecticide)
 Regulation of Enzyme activity
• Metabolism is finely regulated according to the needs of
organisms.
• This is achieved by regulating the activity of key enzymes of
metabolic pathway.
• The enzymes which are responsible for fine regulation are
called as rate limiting enzymes
• Step which is catalysed by these enzymes is called as rate
limiting steps
 Feedback regulation:
The inhibition of the first step by the end product, in a
series of reactions of a metabolic pathway is called
feedback regulation/inhibition
 ALLOSTERIC REGULATION

Allosteric enzyme is a regulatory enzyme.

The term allosteric derives from Greek word, allo

means other and steros means space or site.

Allosteric enzymes are having additional site other

than active site for binding of modulator (regulatory
metabolites).
Allosteric enzymes may be inhibited or stimulated by
their modulators
Modulators that inhibit enzyme activity are termed
negative modulators. Whereas those that increase
enzyme activity are called positive modulators.
 Examples of allosteric enzymes
Enzyme Metabolic Pathway Allosteric Allosteric
inhibitor activator
ALA synthase Heme synthesis Heme --
Aspartate Pyrimidine syn. CTP ATP
transcarbamoylase
HMGCoA reductase Cholesterol syn. Cholesterol --
Phosphofructokinase Glycolysis ATP, citrate AMP, F-2,6-bisp.
Pyruvate carboxylase Gluconeogenesis ADP Acetyl CoA
Acetyl CoA carboxylase Fatty acid synthesis Acyl CoA Citrate
Citrate synthase Krebs cycle ATP --
Carbamoyl phosphate synthetase I Urea cycle -- NAG
Carbamoyl phosphate synthetase II Pyrimidine syn. UTP --
 Covalent Modulation

• In this type of regulation, stimulation or inhibition of

enzyme activity is achieved by the addition of group
(phosphate group) to the enzyme by covalent bond or the
removal of group by cleaving of covalent bond
• Some of the regulatory enzymes are occur in the body as
active or inactive form which are interconvertible time to
time
 ISOENZYME

Isoenzymes or isozymes are multiple forms

(isomers) of the same enzyme that catalyze the same
biochemical reaction.

Isoenzymes show different chemical and physical

properties like electrophoretic mobility and kinetic
properties.

Only those enzymes, which are in polymeric form

demonstrate isoenzyme.
For example:

1. Lactate dehydrogenase (LDH)

2. Creatine kinase (CK)

 Lactate Dehydrogenase (LDH)

Lactate dehydrogenase is a tetrameric enzyme that

catalyzes the oxidation of L-lactate to pyruvate.

LDH is made up of two types of polypeptide M (muscle)

type and H (heart) type.

LDH has five isoenzymes:
– LDH1
– LDH2
– LDH3
– LDH4
– LDH5.
Five isoenzymes of LDH can be detected by
electrophoresis as they have different electrophoretic
mobilities.
LDH1 is the fastest moving fraction towards the anode

and LDH5 is the slowest moving isoenzyme of LDH.

LDH1 predominates in cells of cardiac muscle, and

erythrocytes and LDH5 is the most abundant form in

the liver and in skeletal muscle
 Clinical Applications of LDH

1. Significant elevation of LDH1 and LDH2 occurs within

24 to 48 hours after myocardial infarction.

2. Predominant elevation of LDH2 and LDH3 occur in

leukaemia.

3. LDH3 elevated in malignancy of many tissues.

4. Elevation of LDH5 occurs after damage to the liver or

skeletal muscle.
 Creatine Kinase (CK)

Creatine kinase isoenzymes are dimer that are made up of

two types of polypeptide chains, which may be either M
(muscle) type or B (brain) type, generating three
isoenzymes.

1 CK1 (BB) : present in the brain

2 CK2 (MB) : present only in Cardiac tissue

3 CK3 (MM) : present in skeletal muscle

 Clinical Application
1. CK1 may be elevated in neonates particularly in damaged brain
or very low birth weight new-born

2. Increased level of CK2 occurs in myocardial infarction Cardiac

tissue is the only tissue which has mixed MB (CK2) isoenzyme.

3. CK-MB isoenzyme starts to increase within 4 hours after an acute

myocardial infarction (AMI) and reaches a maximum within 24 hrs.

4. Elevated levels of CK3 in serum occur in dystrophies and

myopathies.