Enzymology

 Life is short and thus has to be catalyzed.

 Self replication and catalysis are believed

to be the two fundamental conditions for

life to be evolved
 The Nobel Prize in Chemistry
1946
“ For his discovery “For their preparation of enzymes
that enzymes & virus proteins in a pure
can be form"
crystallized"

James Batcheller John Howard Wendell

Sumner Northrop Meredith
Stanley
1/2 of the prize 1/4 of the prize 1/4 of the prize
Cornell University Rockefeller Institute for Rockefeller Institute for
Ithaca, NY, USA Medical Research Medical Research
Princeton, NJ, USA Princeton, NJ, USA
1887-1955 1891-1987 1904-1971
Leonor Michaelis Maud Menten
(1875-1949) (1879-1960)
German Canadian
 Definition:
 Defined as organic biocatalysts synthesized by living
cells. They are protein in nature (exception - RNA
acting as ribozyme), colloidal and thermolabile in
character, and specific in their action.
 Enzyme catalysis is very rapid; usually 1 molecule of
an enzyme can act upon about 1000 molecules of
the substrate per minute.
 Lack of enzymes will lead to block in metabolic
pathways causing inborn errors of metabolism.
 Characteristics of Enzymes

 Almost all enzymes are proteins.

 Enzymes follow the physical and chemical
reactions of proteins.
 They are heat labile.
 They are water-soluble.
 They can be precipitated by protein precipitating
reagents (ammonium sulfate or trichloroacetic
acid).
 They contain 16% weight as nitrogen.
 Naming of Enzymes

 According to the reaction they carry out.

 Suffix - ase is added to the name of the substrate
(e.g., lactase is the enzyme that cleaves lactose) or
the type of reaction
 e.g., DNA polymerase forms DNA polymers).
 Systematic names – based on IUBMB - EC.
 International Union of biochemistry and molecular
biology form system for nomenclature and
classification
 IUBMB classification of enzymes
 Based on the reaction they catalyze – grouped
into 6 major classes - (OTHLIL)
1. Oxidoreductase
2. Transferase
3. Hydrolase
4. Lyase
5. Isomerase
6. Ligase
1. Oxidoreductases:
 This group of enzymes will catalyse oxidation of
one substrate with simultaneous reduction of
another substrate or co-enzyme.
 Catalyze oxidation/reduction reactions.
 They catalyze the addition of oxygen, transfer of
hydrogen & transfer of electrons.
 AH2 + B → A + BH2
 Subclasses:
 Oxidases & dehydrogenases
 Oxidases
 Oxidases catalyse the transfer of hydrogen or
electrons from donor, using oxygen as hydrogen
acceptor - E.g. cytochrome oxidase
 Dehydrogenases:
 Dehydrogenases catalyse the transfer of hydrogen
(or electrons), but the hydrogen acceptor is a
molecule other than oxygen.
 The hydrogen acceptors are usually NAD or NADP &
FAD or FMN - E.g. LDH
Oxido-reductases
2. Transferases:
 This class of enzymes transfers one group (other
than hydrogen) from the substrate to another
substrate.
 Transfer a functional group (e.g. a methyl, alcoholic,
aldehyde, ketone, acyl, sulphur or phosphate group).
 A–X + B → A + B–X
 Subclass:
 Transferases (amino transaminases) - amino group
 Kinases - phosphate group
Transaminases
3. Hydrolases:
 This class of enzymes can hydrolyse ester, ether,
peptide or glycosidic bonds by adding water and
then breaking the bond.
 Phosphatases, Esterases, Peptidases, Lipases

A–B + H2O → A–OH + B–H

4. Lyases:
 These enzymes can remove groups from substrates
or break bonds by mechanisms other than
hydrolysis to form double bonds and addition of
groups to break double bonds.
 Addition or removal of groups to form double
bonds.
 Elimination and addition reactions.
 Decarboxylases, Synthases
 A -B + X-Y → AX - BY
5. Isomerases:
 Catalyze intra-molecular group transfer (transfer
of groups within the same molecule).
 These enzymes can produce optical, geometric or
positional isomers of substrates.
 E.g. Epimerases, Mutases, Racemases,
epimerases, cis-trans isomerases
 Interconversion of isomers.
 A → A'
6. Ligases:
 These enzymes link two substrates together,
usually with the simultaneous hydrolysis of ATP,
(Latin, Ligare = to bind).
 IUBMB - EC numbers
 Each enzyme is described by a sequence of four
numbers preceded by "EC"
 First digit represents the class - classifies the
enzyme based on its reaction.
 Second digit stands for the subclass - indicates the
type of group involved in the reaction.
 Third digit is the sub-subclass or subgroup -
indicates substrate on which group acts.
 Fourth digit gives the number of the particular
enzyme in the list- indicates - serial number of
individual enzyme.
 Lactate dehydrogenase
(lactate:NAD+ oxidoreductase)
 Co-
enzymes
 Enzymes may be simple proteins, or complex
enzymes, containing a non-protein part, called the
prosthetic group.
 The prosthetic group is called the co-enzyme.
 It is heat stable.
 Salient features of co-enzymes:
 The protein part of the enzyme gives the necessary
three dimensional infrastructure for chemical reaction;
but the group is transferred from or accepted by the
co-enzyme
 Essential for the biological activity of the enzyme
 It is a low molecular weight organic substance
 The co-enzymes combine loosely with the enzyme
molecules & separated easily by dialysis
 When the reaction is completed, the co-enzyme is
released from the apo-enzyme, and can bind to
another enzyme molecule
 Most of them are derivatives of B complex vitamin
 Non – Vitamin Coenzymes

ATP Donates Phosphate, adenosine, AMP moieties

CDP Required in phospholipid synthesis as a

carrier of choline, ethanolamine

UDP Carrier of glucose – glycogen synthesis

galactose

SAM Methyl group donor

PAPS Sulfate group donor in mucopolysaccharide

synthesis
 Cofactors
• Enzymes may be simple proteins or Compound.
• Many enzymes require small molecules or metal ions to
participate directly in substrate binding or catalysis.
• Active enzyme / Holoenzyme.
• Polypeptide portion of enzyme (apoenzyme)
• Nonprotein prosthetic group (cofactor)
• They can be -
• inorganic metal ions - cofactors or activators.
• complex organic or metallo-organic – coenzymes
• Cofactors are bound to the enzyme to maintain the
correct configuration of the active site .
• Prosthetic groups:
• Some cofactors bind to the enzyme protein very
tightly (non-covalently or covalently).
e.g – FMN, PLP, Biotin, Cu, Mg, Zn
• Metalloenzymes:
• Enzymes with tightly bound metal ions.
• Some metal ions (Fe2+, Cu2+) participate in redox
reactions.
• Others stabilize either the enzyme or substrate
over the course of the reaction.
• Metal-activated enzymes - Enzymes that require
a metal ion cofactor.
• Apoenzyme + cofactor = Holoenzyme
• A holoenzyme also refers to the assembled form
of a multiple subunit protein.
• Holoenzyme:
• A complete, catalytically active enzyme together
with its bound cofactors.
Figure 5.3
 Mechanism of Enzyme Action

• Catalysis is the prime function of enzymes

• For any chemical reaction to occur, the reactants
have to be in an activated state or transition state.
Generation of transition state complexes & formation
of products:
• Binding of the substrate to the active site of the
enzyme causes bonding rearrangements that leads
to an intermediate state called “transition-complex”
• This is an activated form of substrate immediately
preceding the formation of products.
• An enzyme speeds a reaction by lowering the
activation energy
• Less energy is needed to convert reactants to
products.
• This allows more molecules to form product.
Activation free energy (G):
• The energy required to convert substrates from
ground state to transition state.
• Substrates need a large amount of energy to
reach a transition state, which then decays into
products.
• The enzyme stabilizes the transition state,
reducing the energy needed to form products
• The enzyme does not affect the equilibrium
position of the reaction
Enzyme-Substrate Binding
 Steps of Enzyme Catalysis

• Formation of enzyme – substrate complex.

• Generation of Transition-state complexes
• Formation of Reaction Products
ES Complex
ES Complex
 Theories to explain ES Complex

Lock and key model or Fischer's template theory

• The active site has a rigid shape.
• Only substrates with the matching shape can fit.
• The substrate is a key that fits the lock of the active site.
• Fails to explain the stabilization of the transition state, action
of allosteric modulators.
• Active site of unbound enzyme is complementary in
shape to substrate
 Induced-fit Model
• The active sites of some enzymes assume a shape that is
complementary to that of the transition state only after the
substrate is bound.
• The active site is flexible, not rigid.
• Substrate binding brings conformation changes in active site
– nascent active site
• Enables strong binding site - improves catalysis.
• There is a greater range of substrate specificity.
• Active site forms a shape complementary to
substrate only after it is bound
 Substrate strain theory

• As the substrate flexes to fit the active site, bonds in

the substrate are flexed and stressed.
• This causes changes/conversion to product.
• Induced fit and substrate strain combinedly operate
in enzyme action.
 Mechanism of enzyme catalysis

• The formation of an enzyme-substrate complex (ES) is very

crucial for the catalysis to occur, and for the product
formation.

The enhancement in the rate of the reaction is mainly due to

four processes:
• Acid-base catalysis
• Substrate strain
• Covalent catalysis
• Entropy effects
 Acid-base catalysis

• Role of acids and bases is quite important in enzymology.

• At the physiological pH, histidine is the most important
amino acid, the protonated form of which functions as an
acid and its corresponding conjugate as a base.
• The other acids are –OH group of tyrosine, -SH group of
cysteine, and e-amino group of lysine.
• The conjugates of these acids and carboxyl ions (COO-)
function as bases.
 Substrate strain

• During the course of strain induction, the energy

level of the substrate is raised, leading to a
transition state.
• The mechanism of lysozyme (an enzyme of tears,
that cleaves β -1,4 glycosidic bonds) action is
believed to be due to a combination of substrates
strain and acid-base catalysis
 Covalent catalysis

• In the covalent catalysis, the negatively charged

(nucleophilic) or positively charged (electrophilic)
group is present at the active site of the enzyme.
• This group attacks the substrate that results in the
covalent binding of the substrate to the enzyme.
• In the serine proteases (so named due to the
presence of serine at active site), covalent catalysis
along with acid-base catalysis occur, e.g.
chymotrypsin, trypsin etc
 Entropy effect

• Entropy is a term used in thermodynamics.

• It is defined as the extent of disorder in a system
• The enzymes bring about a decrease in the entropy of the
reactants.
• This enables the reactants to come closer to the enzyme
and thus increase the rate of reaction.
• In the actual catalysis of the enzymes, more than one of
the processes acid-base catalysis, substrate strain, covalent
catalysis and entropy are simultaneously operative.
• This will help the substrate (s) to attain a transition state
leading to the formation of products.
 Thermodynamics of enzymatic reactions

• The enzyme catalysed reactions may be broadly

grouped into three types based on thermodynamic
(energy) considerations.
1. lsothermic reactions:
• The energy exchange between reactants and
products is negligible. e.g. glycogen phosphorylase

Glycogen + Pi Glucose 1-
phosphate
2. Exothermic (exergonic) reactions:
• Energy is liberated in these reactions. E.g. urease

Urea NH3 + CO2 + energy

3. Endothermic (endergonic) reactions:

• Energy is consumed in these reactions e.g. glucokinase

Glucose + ATP Glucose 6-phosphate +

ADP
 Active site

• The active site (or active centre) of an enzyme

represents as the small region at which the

substrate(s) binds and participates in the catalysis

• Active site is due to tertiary structure of protein.

• Clefts / crevices – provide suitable environment for

reaction
 Salient features of active site

• The existence of active site is due to the tertiary

structure of protein resulting in three dimensional
native conformation
• The active site is made up of amino acids (known as
catalytic residues) which are far from each other in
the linear sequence of amino acids (primary
structure of protein).
• For instance, the enzyme lysozyme has 129 amino
acids.
• Lysozyme has 129 amino acids.
• The active site is formed by the contribution of amino
acid residues numbered - 35, 52, 62, 63 and 101.
• Active sites are regarded as clefts or crevices or
pockets occupying a small region in a big enzyme
molecule
• The active site is not rigid in structure and shape.
• It is rather flexible to promote the specific substrate
binding.
• The active site possesses a substrate binding site and
a catalytic site.
• The latter is for the catalysis of the specific reaction.
• The coenzymes or cofactors on which some enzymes
depend are present as a part of the catalytic site.
• The substrate (s) binds at the active site by weak non-
covalent bonds.
• Enzymes are specific in their function due to the
existence of active sites.
• The commonly found amino acids at the active
sites are serine, aspartate, histidine, cysteine,
lysine, arginine, glutamate, tyrosine.
• Among these amino acids, serine is the most
frequently found.
• The substrate (s) binds the enzyme (E) at the
active site to form enzyme-substrate complex (ES)
• The product (P) is released after the catalysis and
the enzyme is available for reuse
• The commonly found amino acids at the active
sites are serine, aspartate, histidine, cysteine,
lysine, arginine, glutamate, tyrosine.
• Among these amino acids, serine is the most
frequently found.
• The substrate (s) binds the enzyme (E) at the
active site to form enzyme-substrate complex (ES)
• The product (P) is released after the catalysis and
the enzyme is available for reuse
Factors affecting enzyme activity
The contact between the enzyme and substrate is
the most essential pre-requisite for enzyme
activity

1. Enzyme concentration
2. Substrate concentration
3. Temperature
4. Hydrogen ion concentration (pH)
5. Product concentration
6. Presence of activators
7. Time
8. Light & radiation
 Enzyme concentration
• Rate of a reaction or velocity (V) is directly proportional to the
enzyme concentration, when sufficient substrate is present.
• Substrate is a molecule on which enzyme acts.
• Velocity (Reaction rate) refers to change in the concentration
of substrate or reaction product (s) per unit time.
• It is expressed as moles/liter/sec.
• Maximum velocity (Vmax):
• It refers to maximum change in the product or substrate
concentration at a given enzyme concentration.
Effect of enzyme concentration
 Effect of Substrate Concentration
• Increase in the substrate concentration gradually increases
the velocity of enzyme reaction within the limited range of
substrate levels.
• A rectangular hyperbola is obtained when velocity is
plotted against the substrate concentration
• Three distinct phases of the reaction are observed in the
graph (A-linear; B-curve; C-almost unchanged.
• The maximum velocity obtained is called Vmax.
• It represents the maximum reaction rate attainable in
presence of excess substrate (at substrate saturation
level).
Effect of Substrate
Concentration
 Michaelis-Menten Equation
• Michaelis-Mention equation is a rate equation for reaction
kinetics in enzyme catalysed reaction
• Written as
V max (S)
__________
V=
Km + S

• The velocity of enzyme catalysed reactions is altered as the

substrate concentration is increased.
• First order reaction:
• At low substrate concentration, velocity increases
proportionally as the concentration of the substrate is
increased.
• Mixed order reaction:
• When the concentration of the substrate is further
increased (at mid substrate concentration), the
velocity increases, but not proportionally to
substrate concentration.
• Zero order reaction:
• At high substrate concentration, the velocity is
maximum & is independent of substrate
concentration.
 Enzyme kinetics & Km value

• The enzyme (E) reacts with substrate (S) to form

unstable enzyme-substrate (ES) complex.
• The ES complex is either converted to product (P)
or can dissociate back to enzyme (E) & substrate
Substrate (S) + Enzyme (E)
(S). Enzyme substrate (ES)

Enzyme substrate (ES) Product (P) + Enzyme (E)

 K1
K3
E+S ES E+P
K2

• K1,K2 & K3 are velocity constants.

• Km, Michaelis-menten constant is given by the
formula…

K2 + K3
Km =
K1
• Michaelis-menten set up mathematical expressions
for the rate of all the three reactions in the
equation.
• V as the initial rate of reaction (velocity)
• S as the initial concentration of the substrate
• V max as the maximum velocity attained with high
substrate concentration when all the enzyme
molecules are occupied.
• Km as Michaelis-menten constant
V max (S)
V=
Km + (S)
• Measured velocity (V) is equal to ½ Vmax.
• So,

V max (S)
½ V max =
Km + (S)

Km + (S) = 2V max (S)

V max

Km + (S) = 2 (S)

Km = (S)

K stands for constant & M stands for Michaelis

 Michaelis constant
• The formation of enzyme - substrate complex is a reversible
reaction, while the breakdown of the complex to enzyme +
product is irreversible.
• 50% velocity in Y axis is extrapolated to the corresponding
point on X-axis, which gives the numerical value of Km.
• The lesser the numerical value of Km, the affinity of the
enzyme for the substrate is more.
• E.g: Km of glucokinase is 10 mmol/L and hexokinase is 0.05
mmol/L.
• 50% molecules of hexokinase are saturated even at a lower
concentration of glucose.
• Hexokinase has more affinity for glucose than glucokinase.
Effect of enzyme concentration on Km
 Salient features of Km
• Km value is substrate concentration (expressed in
moles/L) at half-maximal velocity.
• It denotes that 50% of enzyme molecules are
bound with substrate molecules at that particular
substrate concentration.
• Km is independent of enzyme concentration.
• If enzyme concentration is doubled, the Vmax will
be double.
• But the Km will remain exactly same irrespective of
enzyme 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.
• The affinity of an enzyme towards its substrate is
inversely related to the dissociation constant, Kd
for the ES complex.
• Km denotes the affinity of enzyme for substrate.
• The lesser the numerical value of Km, the affinity
of the enzyme for the substrate is more.
 Double reciprocal plot

• Sometimes it is impractical to achieve high substrate

concentrations to reach the maximal velocity
conditions.
• So, ½Vmax or Km may be difficult to determine.
• The experimental data at lower concentrations is
plotted as reciprocals.
• The straight line thus obtained is extrapolated to get
the reciprocal of Km.
• Called as Lineweaver–Burk Plot or Double Reciprocal
Plot which can be derived from the Michaelis-Menten
equation
Lineweaver-Burk plot
 Effect of Temperature

• The velocity of enzyme reaction increases when

temperature of the medium is increased; reaches a
maximum and then falls (Bell shaped curve).
• The temperature at which maximum amount of the
substrate is converted to the product per unit time is
called the optimum temperature.
• Temperature is increased, more molecules get
activation energy, or molecules are at increased rate
of motion.
• Their collision probabilities are increased and so the
reaction velocity is enhanced.
Effect of Temperature
 Temperature coefficient Q10
• The temperature coefficient (Q10) is the factor by which the rate
of catalysis is increased by a rise in 10°C.
• Generally, the rate of reaction of most enzymes will double by a
rise in 10°C.
• When temperature is more than 50°C, heat denaturation and
consequent loss of tertiary structure of protein occurs.
• Activity of the enzyme is decreased.
• Most human enzymes have the optimum temperature around
37°C.
• Certain bacteria living in hot springs will have enzymes with
optimum temperature near 100°C.
 Effect of pH
• Each enzyme has an optimum pH (usually pH
between 6 and 8).
• On both sides of which the velocity will be drastically
reduced.
• The graph will show a bell shaped curve
• The pH decides the charge on the amino acid
residues at the active site.
• The net charge on the enzyme protein would
influence substrate binding and catalytic activity.
• Optimum pH may vary depending on the
temperature, concentration of substrate, presence of
ions etc.
• Pepsin (optimum pH 1-2); ALP (optimum pH 9-10) &
acid phosphatase (4-5)
Effect of pH
 Effect of product concentration

• The accumulation of reaction products generally

decreases the enzyme velocity.
• For certain enzymes, the products combine with the
active site of enzyme and form a loose complex and,
thus, inhibit the enzyme activity.
• In the living system, this type of inhibition is
generally prevented by a quick removal of products
formed
 Effect of activators
• Some of the enzymes require certain inorganic
metallic cations like Mg2+, Mn2+, Zn2+, Ca2+, Co2+, Cu2+,
Na+, K+, for their optimum activity
• Anions are also needed for enzyme activity e.g.
chloride ion for amylase
• Metals function as activators of enzyme velocity
through various mechanisms combining with the
substrate, formation of ES-metal complex, direct
participation in the reaction and bringing a
conformational change in the enzyme.
• Two categories of enzymes requiring metals for their
activity
1. Metal-activated enzymes:
• The metal is not tightly held by the enzyme and can be
exchanged easily with other ions.
• e.g. ATPase (Mg2+ and Ca2+) & Enolase (Mg2+)
2. Metalloenzyme:
• These enzymes hold the metals rather tightly which are
not readily exchanged.
• e.g. Alcohol dehydrogenase, carbonic anhydrase,
alkaline phosphatase, carboxypeptidase and aldolase
contain zinc.
• Phenol oxidase (copper)
 Effect of time
• Under ideal and optimal conditions (like pH, temperature
etc.), the time required for an enzyme reaction is less.

• Variations in the time of the reaction are generally related

to the alterations in pH and temperature.

Effect of light and radiation

• Exposure of enzymes to ultraviolet, beta, gamma & X-rays

inactivates certain enzymes due to the formation of
peroxides. e.g. UV rays inhibit salivary amylase activity
 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.

• They are usually specific and they work at low

concentrations

• block the enzyme but they do not usually destroy

it

• Many drugs and poisons are inhibitors of enzymes

in the nervous system
 Type of Enzyme Inhibitors
Type of
Inhibitors

Reversible

Irreversible
Competitive

Active Site Uncompetitive

Directed

Suicide / kcat
Non- Competitive
Inhibitors
 Reversible inhibition

• The inhibitor binds non-covalently with enzyme and

the enzyme inhibition can be reversed if the inhibitor
is removed.
• Binding is weak and thus, inhibition is reversible.
• Do not cause any permanent changes in the enzyme
• Subtypes:
• Competitive & Non-competitive Inhibition
 Competitive inhibition
• The inhibitor (I) molecules resembles the substrate
(S)
• Also called as substrate analogue inhibition
• Binds to active site – forms EI complex.
• EI complex cannot rive rise to product formation.
• As long as the competitive inhibitor holds the active
site, the enzyme is not available for the substrate to
bind.
• Relative concentrations of S, I determine inhibition.
ES E+P
E +S +I
No product
EI
formation
 Binding of S & I in different
Situations

• Classical Competitive Inhibition (S & I

compete for the same binding site)

S I

Enzyme
• Binding of I to a distinct inhibitor site
causes a conformational change in the
enzyme that distorts or masks the S binding
site or vice versa.
I S

Enzyme

S I
I S

Enzyme Enzyme
• A competitive inhibitor diminishes the rate of catalysis
by reducing the proportion of enzyme molecules
bound to a substrate.
• Competitive inhibition can be relieved by increasing
the substrate concentration & maximum velocity is
regained.
• A higher substrate concentration is therefore needed
to achieve a half maximum rate, Km increases
• High concentrations of the substrate displace the
inhibitor again.
• The V max, not influenced by this type of inhibition.
In the presence of a competitive inhibitor
Km increases

V max unchanged

Vmax

v
No inhibitor
½ Vmax
+ C Inhibitor

Km Kmapp [s]
 Lineweaver Burk plot

• In the presence
[I]2
[I ]
of a competitive K i )
( 1+
Km [I]1
inhibitor Km ma
x
= V
pe
increases Slo

• V max unchanged

1
Km

1
Kmapp
• E. g. Malonate – structural analog of succinate-inhibits
succinate dehydrogenase.
Antimetabolites:
• These chemical compounds that block the metabolic
reactions by their inhibitory action on enzymes.
• Antimetabolites are usually structural analogues of
substrates and thus are competitive inhibitors.
• They are in use for cancer therapy, gout etc.
 Examples of competitive inhibition

Enzyme Substrate Competitive inhibitor

Succinate Succinate Malonate

Dehydrogenase
Dihydrofolate 7,8-dihydrofolate Aminopterin
Reductase
Xanthine Oxidase Hypoxanthine Allopurinol

Acetyl cholinesterase Acetylcholine Succinylcholine

Lactate Lactate Oxamate

Dehydrogenase
HMG CoA Reductase HMG Co A HMG
 Non-Competitive Inhibition

• The inhibitor binds at a site other than the active

site on the enzyme & causes conformational
changes on enzymes or some times it may react
with functional group at the active site &
inactivates the enzyme.
• Inhibitor has no structural resemblance with the
substrate.
• There is no competition for the active site of the
enzyme molecule.
• There usually exists a strong affinity for the inhibitor to
bind at the second site.
• The inhibitor does not interfere with the enzyme-substrate
binding.
• The inhibitor generally binds with the enzyme as well as
the ES complex.
• Km value is unchanged & V max is lowered.
• Heavy metal ions (Ag+, Pb2+, Hg2+ etc.) can non-
competitively inhibit the enzymes by binding with
cysteinyl sulfhydryl groups & inactivates the
enzymes.
• Non-competitive inhibition is also called as enzyme
poisons

E+S ES E+P
+ +
I I

EI + S EIS
Non-Competitive Inhibition

Enzyme Enzyme

S
I I
Enzyme Enzyme
 Non-Competitive Inhibition

Vmax Vmax = Decreases.

Km = Unchanged
Vmax i
v No inhibitor
½ Vmax
+ NC
½ Vmax Inhibitor
i

K [s]
m
 Lineweaver – Burk Plot

• Km value is [I]2
unchanged [I]1
1/v
• V max is 1 No
lowered Vmaxi
Inhibitor

1
Km 1
Vmax
1/
[s]
 Comparison between competitive & Non-
competitive inhibition
Competitive Inhibition Non-competitive
Inhibition
Acting on Active site May or may not

Structure of Substrate analogue Unrelated molecule

inhibitor
Excess Substrate Inhibition Relieved No effect

Km Increased No Change

V max No Change Decreased

Significance Drug Action Toxicological

 Uncompetitive Inhibition

• Here inhibitor does not have any affinity for the active
site of enzyme.
• Inhibitor binds only with enzyme-substrate complex; but
not with free enzyme.
• Both V max and Km are decreased
• UC Inhibition is rare in single-substrate
reactions.
• E.g. Phenylalanine inhibits alkaline
phosphatase in intestinal cells
• It is common in multi-substrate reactions
E+S ES E+
P
+
I

ESI
 Uncompetitive
Inhibition

Enzyme
Enzyme

S
I
Enzyme
Uncompetitive Inhibition

Vmax
Vmax = Decreases
Km = Decreases
Vmax i
v ½ Vmax No inhibitor

½ Vmax + UC
Inhibitor
i

K [s]
Kmapp
m
 Irreversible inhibition
• In this type, Inhibitor binds at or near the active
site of the enzyme irreversibly, usually by covalent
bonds, so that it can’t subsequently dissociate
from the enzyme
• As a result the enzyme is permanently inactive
• Compounds which irreversibly denature the
enzyme protein or cause non-specific inactivation
of the active site are not usually regarded as
irreversible inhibitors.
 Examples
These inhibitors are toxic poisonous substances.
• Iodoacetate:
It is an irreversible inhibitor of the enzymes like papain
and glyceraldehyde 3-phosphate dehydrogenase
Iodoacetate combines with sulfhydryl (-SH) groups at the
active site of these enzymes and makes them inactive.
• Diisopropyl fluorophosphate (DFP) is a nerve gas
developed by the Germans during Second World War.
DFP irreversibly binds with enzymes containing serine at
the active site, e.g. serine proteases, acetylcholine
esterase.
• Disulfiram (Antabuse)s a drug used in the treatment of
alcoholism.
• lt irreversibly inhibits the enzyme aldehyde dehydrogenase.
• Alcohol addicts, when treated with disulfiram become sick
due to the accumulation of acetaldehyde, leading to alcohol
avoidance
 Suicidal inhibition
• This is a special type of irreversible inhibition.
• Also called as mechanism based inactivation.
• In this case, the original inhibitor (the structural
analogue/competitive inhibitor) is converted to a more
effective inhibitor with the help of same enzyme that
ought to be inhibited.
• The formed inhibitor binds irreversibly with the
enzyme.
• Allopurinol, an inhibitor of xanthine oxidase, gets
converted to alloxanthine, a more effective inhibitor of
• The use of certain purine and pyrimidine

analogues in cancer therapy is also explained on

the basis suicide inhibition.

• 5-fluorouracil gets converted to

fluorodeoxyuridylate which inhibits the enzyme

thymidylate synthase, and thus nucleotides

synthesis
 During thymidylate synthesis, N5,N10-
methyleneTHF is converted to 7,8-dihydrofolate;
methyleneTHF is regenerated in two steps
 Allosteric regulation
• Allosteric enzyme has one catalytic site where the
substrate binds and another separate allosteric site
where the modifier binds (allo = other)
• Allosteric and substrate binding sites may or may not
be physically adjacent.
• The binding of the regulatory molecule can either
enhance the activity of the enzyme (allosteric
activation), or inhibit the activity of the enzyme
(allosteric inhibition).
• The binding of substrate to one of the subunits of the
enzyme may enhance substrate binding by other
subunits.
• This effect is said to be positive co-operativity
• If the binding of substrate to one of the subunits
decreases the activity of substrate binding by other
sites, the effect is called negative co-operativity.
• In most cases, a combination is observed, resulting
in a sigmoid shaped curve
 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 Substrate Inhibitor
cannot fit molecule
enzymes into the
active Inhibitor fits into
• The other site fits an
site allosteric site
inhibitor molecule
Allosteric inhibition
 Salient Features, Allosteric Inhibition

• The inhibitor is not a substrate analogue

• It is partially reversible, when excess substrate is
added.
• Km is usually increased & V max is reduced.
• The effect of allosteric modifier is maximum at or
near substrate concentration equivalent to Km.
• Allosteric enzymes are made up of subunits, e.g.
Aspartate transcarbamoylase has 6 subunits and
pyruvate kinase has 4 subunits
 Allosteric enzymes
Enzyme Allosteric Inhibitor Allosteric
Activator

ALA synthase Heme

Aspartate transcarbamoylase CTP ATP

HMGCoA-reductase Cholesterol
Phosphofructokinase ATP, citrate AMP, F-2,6-P
Pyruvate carboxylase ADP AcetylCoA
Acetyl CoA carboxylase AcylCoA Citrate
Citrate synthase ATP
Carbamoyl phosphate NAG
synthetase I

Carbamoyl phosphate UTP

synthetase II
 Importance of Enzyme Inhibition
• For understanding the regulation of enzyme activity
within the living cells
• To elucidate the kinetic mechanism of an enzyme
catalyzing a multi-substrate reaction
• Useful in elucidating the cellular metabolic
pathways by causing accumulation of intermediates
• Identification of the catalytic groups at the active
site
• Provide information about substrate specificity of
the enzyme
 Regulation of enzyme activity

• Allosteric regulation
• Activation of latent enzymes
• Compartmentation of metabolic pathways
• Control of enzyme synthesis
• Enzyme degradation
• lsoenzymes
 Allosteric Regulation or Allosteric
Inhibition

• Enzymes possess additional sites, known as allosteric

sites besides the active site. Such enzymes are
known as allosteric enzymes.

• Allosteric effectors:
• The catalytic activity of certain regulatory enzymes is
modified by certain low molecular weight substances
or molecules known as allosteric effectors or
modifiers bind at the allosteric site and regulate the
enzyme activity.
• Classes of allosteric enzyme:
• They are divided into two classes based on the influence of
allosteric effector on Km and V max.
• K-class of allosteric enzymes:
• The allosteric inhibitor increases the Km and not the V max.
• Double reciprocal plots, similar to competitive inhibition are
obtained e.g. phosphofructokinase.
• V-class of allosteric enzymes:
• The allosteric inhibitor decreases the V max and not the
Km.
• Double reciprocal plots resemble that of non-competitive
inhibition e.g. acetyl CoA carboxylase
 Feedback regulation
• The process of inhibiting the first step by the final
product, in a series of enzyme catalysed reactions of a
metabolic pathway is referred to as feedback
regulation.
• The very first step (A to B) by the enzyme is the most
effective for regulating the pathway, by the final end
product D.
• This type of control is often called negative feedback
regulation A B C D
 Feedback regulation

Carbamoyl phosphate +
Aspartate

Aspartate
transcarbamylase
Carbamoyl
Feedback Aspartate + Pi
control

Cytidine
triphosphate (CTP)
 Activation of latent enzymes
• Some enzymes are synthesized as Proenzymes or
zymogens which undergo irreversible covalent activation
by the breakdown of one or more peptide bonds
• Chymotrypsinogen pepsinogen and plasminogen, are
respectively- converted to the active enzymes
chymotrypsin, pepsin and plasmin.
• Certain enzymes exist in the active and inactive forms
which are interconvertible
• The inter-conversion is brought about by the reversible
covalent modifications, namely phosphorylation and
dephosphorylation, and oxidation and reduction of
disulfide bonds
 Examples

• There are some enzymes which are active in

dephosphorylated state and become inactive
when phosphorylated e.g. glycogen synthase,
acetyl CoA carboxylase.
• A few enzymes are active only with sulfhydryl (-
SH) groups
• E.g. succinate dehydrogenase, urease.
 Compartmentation

• Generally, the synthetic (anabolic) and breakdown

(catabolic) pathways are operative in different
cellular organelle.

• E.g. Enzymes for fatty acid synthesis are found in

the cytosol whereas enzymes for fatty acid
oxidation are present in the mitochondria
 Induction and repression

• Induction is used to represent increased synthesis of enzyme

while repression indicates its decreased synthesis.
• Induction or repression which ultimately determines the enzyme
concentration at the gene level through the mediation of
hormones or other substance.
• E.g of Induction: The hormone insulin induces the synthesis of
glycogen synthetase, glucokinase, phosphofructokinase and
pyruvate kinase.
• All these enzymes are involved in the utilization of glucose.
• The hormone cortisol induces the synthesis of many enzymes
e.g. pyruvate carboxylase, tryptophan oxygenase and tyrosine
aminotransferase
• Examples of repression:
• In many instances, substrate can repress the
synthesis of enzyme.
• Pyruvate carboxylase is a key enzyme in the
synthesis of glucose from non-carbohydrate
sources like pyruvate and amino acids.
• lf there is sufficient glucose available, there is no
necessity for its synthesis.
• This is achieved through repression of pyruvate
carboxylase by glucose.
 Enzyme degradation

• Every enzyme has half-life.

• It is in days while for others in hours or in minutes,
• e.g. LDH4 - 5 to 6 days;
• LDH1 - 8 to 12 hours;
• Amylase -3 to 5 hours
• The key and regulatory enzymes are most rapidly
degraded.
• lf not needed, they immediately disappear and,
when required, they are quickly synthesized
Isoenzymes & Clinical
Enzymology
 Classification of Plasma enzymes

• Enzymes present in plasma are grouped

into functional & non-functional enzymes
Functional enzymes Non-functional enzymes

Pseudocholinesterase Aspartate transaminase

Lipoprotein lipase Creatinine kinase

Ceruloplasmin Amylase

Blood coagulation enzymes Alkaline phosphatase

 Functional enzymes
• Few enzymes in plasma are functionally important &
they are involved in blood clotting, lipoprotein
metabolism & drug metabolism.
• Synthesized in liver & released into plasma.
• E.g. Pseudocholinesterase, Lipoprotein lipase,
Ceruloplasmin, Blood coagulation enzymes
• Clinical significance:
• These are present in higher concentrations in plasma
than cells.
• These are clinically significant when the serum level is
decreased below the reference range.
 Non-functional enzymes
• Most of the enzymes present in plasma serve no
function in the plasma.
• Non-functional enzymes are derived from cells of
organs & tissues.
• Present in high concentrations within cells
• E.g. AST, ALT, LDH,CK,ALP, amylase
• Clinical significance:
• Non-functional enzymes in plasma are present in low
concentrations.
• These are clinically important when the serum level is
increased above the reference range.
 Causes for increased level of
non-functional enzymes in plasma

Increased level of non-functional enzymes in

plasma include
• Hypoxic or infective insults
• Disease states of tissues
• Excessive synthesis or induction of enzymes
by cells with overflow into plasma
• Vigorous exercise
• Decreased renal clearance
 Isoenzymes

• Isoenzymes or isozymes are multiple forms of

same enzyme that catalyze the same chemical
reaction
• Different chemical and physical properties:
• Electrophoretic mobility
• Kinetic properties
• Amino acid sequence
• Amino acid composition
Clinical significance of enzymes &
iso-enzymes in different disease
conditions
 Markers for cardiac diseases

• Creatine kinase (CK-MB)

• Cardiac troponin I (CTI) & Cardiac troponin
(CTT)
• CTI & CTT are not true enzymes
• Brain natriuretic peptide (BNP)
• BNP is a reliable marker of ventricular function
• Lactate dehydrogenase
• Aspartate transferase
Cardiac biomarkers are used to detect cardiac
diseases, include

Any chest pain

Unstable angina
Suspicious ECG changes
History suggestive of myocardial infarction
Following surgical coronary revascularization
Patients with hypotension and dyspnea
 Creatinine phosphokinase (CPK)
• It catalyses creatine to creatine phosphate.
• Normal serum value:
• 15-100 U/L for males & 10-80 U/L for females.
• CPK consists of 3 isoenzymes.

Three Iso-enzymes are separated by electrophoresis.

• CPK-1 (also called CPK-BB) is found mostly in the
brain & lungs.
• CPK-2 (also called CPK-MB) is found mostly in the
heart.
• CPK-3 (also called CPK-MM) is found mostly in
skeletal muscle.
 Creatine phosphokinase isoenzymes

ISOENZYMES SUB- TISSUE % IN SERUM

UNIT

CK1 BB Brain 1
Fast moving

CK2 MB Heart 5
2% of total

CK3
Slow moving MM Skeletal muscle 80
 Clinical significance of CK

• CPK & heart attack:

• CPK2 isoenzymes is very small, (2% of total

CPK activity) & undetectable in plasma.

• In myocardial infarction (MI), CPK2 levels are

increased within 4 hrs, then falls rapidly.

• Total CPK level is elevated upto 20-folds in

MI.
 CPK & Muscle diseases

• CPK level is elevated in muscular dystrophy

(500-1500U/L)

• CPK level is highly elevated in crush injury,

fracture & acute cerebrovascular accidents.

• Estimation of total CPK is employed in

muscular dystrophies & CPK-MB isoenzyme is
estimated in myocardial infarction.
 Cardiac troponins (CTI/CTT)

• They are not enzymes.

• Troponins are now accepted as reliable markers for
MI
• Cardiac troponins have become one of the main tests
in early detection of an ischemic episode and in
monitoring the patient
• The troponin complex consists of 3 components
• Troponin C (calcium binding subunit),
• Troponin I (actomyosin ATPase inhibitory subunit) &
• Troponin T (tropomyosin binding subunit)
• Troponin I (TnI) is encoded by 3 different genes, giving
rise to 3 isoforms; the "slow" and "fast" moving forms
are skeletal variety.
• Cardiac isoform is specific for cardiac muscle; the
amino acid sequence is different in skeletal muscle
isoform.
• Cardiac isoform of CTnT and CTnI are mainly (95%)
located in myofibrils and the remaining 5% is
cytoplasmic.
• They are identified and quantitated by immunological
(ELISA or immuno turbidimetric) reactions.
• Troponin I is released into the blood within 4 hours after the
onset of symptoms of myocardial ischemia; peaks at 14-24
hours and remains elevated for 3-5 days post-infarction.
• CTI is very useful as a marker at any time interval after the
heart attack.
• It is not increased in muscle injury; whereas CK2 may be
elevated in some muscle injury.
• The initial increase is due to liberation of the cytoplasmic
fraction and sustained elevation is due to the release from
myofibrils.
• Serum level of Troponin T (TnT) increases within 6 hrs of
myocardial infarction, peaks at 72 hours and then remains
elevated up to 7-14 days.
 Brain Natriuretic Peptide (BNP)
• The natriuretic peptide family consists of three
peptides:
• Atrial natriuretic peptide (ANP),
• Brain natriuretic peptide (BNP)
• C-type natriuretic peptide (CNP).
• The clinical significance of CNP is not clear.
• ANP is produced primarily in the cardiac atria.
• BNP is present in human brain, but more in the
cardiac ventricles.
• Human pro–BNP contains 108 amino acids.
• It is cleaved by enzymes within cardiac myocytes
into the active C-terminal BNP (32 amino acids)
and an inactive peptide (proBNP 1–76).
• Both are seen in circulation.
• The active BNP is secreted by the ventricles of
the heart in response to excessive stretching of
heart muscle cells (cardiomyocytes).
 Clinical Significance
• Patients with congestive heart failure have high
plasma concentrations of ANP and BNP.
• The concentrations are correlated with the extent of
ventricular dysfunction.
• High concentrations of BNP predict poor long-term
survival.
• In breathlessness, BNP test helps in the
differentiation of the cause as heart failure or
obstructive lung disease.
• The best marker of ventricular dysfunction is pro-
 Lactate Dehydrogenase
• Lactate dehydrogenase, reversibly converts lactate
to pyruvate, in different tissues.
• LDH consists of 5 iso-enzymes –
LDH1,LDH2,LDH3,LDH4 & LDH5
• These isoenzymes are separated by cellulose
acetate electrophoresis at pH 8.6
• Normal values:
• Serum -100 -200 U/L
• CSF - 7 -30 U/L
• Urine - 40 -100 U/L
LDH reaction
Isoenzyme Compositi Electrophoret Present in Elevated in
name on
LDH isoforms
ic migration

LDH 1
Heat ( H4) Fastest Myocardium myocardial infarction
resistant moving , RBC,
kidney
LDH2 Myocardium Kidney disease,
Heat (H3M1) , RBC, megaloblastic anemia
resistant kidney

LDH3 (H2M2) brain Leukemia, malignancy

LDH4 (H1M3) Lung, Pulmonary infarction

Heat labile spleen

LDH5
Heat labile (M4) Slowest Skeletal Skeletal muscle and
Inhibited by moving muscle, liver diseases
urea Liver
 Aspartate aminotransferase (AST)
• It was also called as serum glutamate oxaloacetate
transaminase (SGOT).
• AST needs pyridoxal phosphate (vitamin B6) as co-enzyme.
• Normal serum level: 8 to 20 U/L.
• It is a marker of liver injury and shows moderate to drastic
increase in parenchymal liver diseases like hepatitis and
malignancies of liver.
• AST was used as a marker of myocardial ischemia in olden
days.
• The level is significantly elevated in myocardial infarction.
• But troponins have replaced AST as a diagnostic marker in
IHD
 Enzyme profiles in liver diseases

• Enzymes commonly studied for diagnosis of

liver diseases are:

• Alanine amino transaminase (ALT)

• Alkaline phosphatase (ALP)

• Nucleotide phosphatase (NTP)

• Gamma glutamyl transferase (GGT)

 Alanine aminotransferase (ALT)
• It was called as serum glutamate pyruvate transaminase
(SGPT)
• The enzyme needs pyridoxal phosphate as coenzyme.
• Normal serum level: male is 13-35 U/L & female is 10-30
U/L.
• Very high values (300 to 1000 U/L) are seen in acute
hepatitis, either toxic or viral in origin.
• Both ALT and AST levels are increased in liver disease, but
ALT > AST.
• Rise in ALT levels may be noticed several days before
clinical signs such as jaundice are manifested.
• Moderate increase (50 to 100 U/L) of ALT may be seen in
chronic liver diseases such as cirrhosis, hepatitis C and
non-alcoholic steatohepatitis (NASH).
 Alkaline Phosphatase (ALP)
• ALP is nonspecific enzyme.
• It hydrolyses aliphatic, aromatic or heterocyclic
compounds.
• Optimum pH-9 &10 & it is activated by Mg2+ &Mn.
• Zn is a constituent of ALP.
• It is produced by osteoblasts of bone, and is
associated with the calcification process.
• It is localised in cell membranes -ecto-enzyme.
• It is associated with transport mechanisms in liver,
kidney & intestinal mucosa.
• Normal range-40-125 U/L.
• In children, Increased levels are seen, due to
increased osteoblastic activity.
• Moderate (2-3) increase in ALP level is seen in
hepatic diseases such as infective hepatitis,
alcoholic hepatitis or hepatocellular carcinoma.
• Very high levels of ALP (10-12 times of upper
limit) may be noticed in extrahepatic obstruction
(obstructive jaundice) caused by gallstones or by
pressure on bile duct by carcinoma of head of
pancreas
• Intrahepatic cholestasis may be due to virus
(infective hepatitis) or by drugs (chlorpromazine).
• ALP is produced by epithelial cells of biliary
canaliculi & obstruction of bile with consequent
irritation of epithelial cells leads to secretion of ALP
into serum.
• Drastically high levels of ALP (10-25 times of upper
limit) are seen in bone diseases where osteoblastic
activity is enhanced such as Paget's disease
(osteitis deformatis), rickets, osteomalacia,
osteoblastoma, metastatic carcinoma of bone and
hyperparathyroidism.
 Isoenzymes of ALP

• Alpha-1 ALP moves in alpha-1 position, it is

synthesized by epithelial cells of biliary canaliculi.

• It is about 10% of total activity and is increased in

obstructive jaundice.

• Alpha-2 heat labile ALP is stable at 56oC; but loses its

activity when kept at 65oC for 30 minutes.

• It is produced by hepatic cells.

• This liver iso-enzyme forms about 25% of total ALP.

• Alpha-2 heat stable ALP will not be destroyed at 65oC,
but is inhibited by phenylalanine.
• It is of placental origin, which is found in blood in
normal pregnancy.
• An isoenzyme closely resembling the placental form
is characteristically seen in circulation in about 15%
cases of carcinoma of lung, liver and gut and named
as Regan iso-enzyme or carcinoplacental iso-enzyme.
• Normal level is only 1% of the total ALP.
 Nucleotide Phosphatase (NTP)
• It is also known as 5' nucleotidase.
• This enzyme hydrolyses 5' nucleotides to
corresponding nucleosides at an optimum pH of 7.5.
• Nickel ions inhibit NTP but not ALP.
• It is a marker enzyme for plasma membranes and it
is ecto-enzyme
• Normal NTP level in serum is 2-10 IU/L.
• It is moderately increased in hepatitis and highly
elevated in biliary obstruction.
• Unaffected by bone diseases.
 Gamma Glutamyl Transferase (GGT)
• It can transfer gamma glutamyl residues to substrate.
• In the body it is used in the synthesis of glutathione
• GGT has 11 iso-enzymes.
• It is seen in liver, kidney, pancreas, intestinal cells &
prostate gland.
• Normal serum value of GGT is 10-30 U/L.
• It is moderately increased in infective hepatitis and
prostate cancers
• GGT is clinically important because of its sensitivity to
detect alcohol abuse.
• GGT is increased in alcoholics.
• Increase in GGT level is generally proportional to the
amount of alcohol intake.
 Enzyme profiles in muscle diseases

• Enzymes commonly studied for diagnosis of muscle

diseases are:
• AST
• CPK
• Aldolase
• In muscular dystrophies, probably due to increased
leakage of enzymes from damaged cells.
• CPK is most reliable indicator of muscular diseases.
 Aldolase

• It is a tetrameric enzyme with A and B subunits; so

there are 5 iso-enzymes.
• It is a glycolytic enzyme.
• Normal range of serum is 1.5-7 U/L.
• It is drastically elevated in muscle damages such as
progressive muscular dystrophy, poliomyelitis,
myasthenia gravis and multiple sclerosis.
• It is a very sensitive early index in muscle wasting
diseases.