ENZYME

Enzyme
Enzyme are the biological catalysts produced
by living cells.
 Important characteristic features of enzymes:
 Catalytic power(ratio of enzyme catalyzed
rate of a reaction to the uncatalyzed rate)
 Specificity (selectivity of enzyme to their
substrate)
 Regulation (Control of enzymatic reaction)
 Enzyme Catalytic Power
Catalysts are substances that speed up the
rate of a chemical reaction.
 Enzymes cause cellular reactions to occur
millions of times faster than corresponding
uncatalyzed reactions.
 Enzymes can increase the rate of a reaction by
a factor of up to 1020 over uncatalyzed
reactions.
 Activation energy: an input
of energy before molecules
will react together

 Enzymes Lower a
Reaction’s Activation
Energy
 Enzymes are not consumed
(reusable)
Activation Energy Profile

 An enzyme alters the rate of a

reaction, but not its free energy
change or position of equilibrium
 G = Gfinal -Ginitial
 Importance
Enzymes play an important role in
•Metabolism
- Enzymes are the biological catalysts that speed up the
metabolic reactions that occur in the body.
•Diagnosis
- Level of enzyme in blood
•Therapeutics
- Digestive enzymes
 Carbohydrate Digestion

Absorption/t
ransport

Digestion

3-4
Metabolic
pathway
 Enzymes assayed for diagnostic purposes
Enzyme Location Cause of elevated plasma level

Acid phosphatase - ACP Prostate Prostatic cancer

Alkaline phosphatase – ALP Bone, liver Rickets, hypoparathyroidism,

osteomalacia, obstructive
jaundice, cancer of bone/liver

Alanine aminotransferase – ALT Liver (muscle, Hepatitis, jaundice, circulatory

heart, kidney) faillure with liver congestion

Aspartate aminotransferase – AST Heart, muscle, Myocardial infarction, muscle

red cells, liver damage, anemia, hepatitis,

circulatory faillure with liver
congestion

Amylase - AM Pancres Acute pancreatitis, peptic ulcer

-Glutamyl transferase – GMT Liver, kidney, Hepatitis, alcoholic liver

pancreas damage, cholestasis
 Enzymes in therapy

• Substitution of missing production of digestive enzymes

– digestive enzymes – pepsin trypsin…

• Removal of deposits of death tissue or fibrin (e.g. in

lungs, eyes), treatment of skin defects – proteinases,
nucleases, collagenase

• Acceleration of fibrinolysis in lungs embolization

(activation of plasmin
and plasminogen) – streptokinase, urokinase
 ACTIVE SITES
 Enzyme molecules contain a special pocket or cleft
called the active sites.
 A relatively small part of an enzyme that is actually
involved in catalysis, i.e., where the substrate or
substrates attach to by noncovalent forces, e.g.,
hydrogen bonding, electrostatic attractions, van der
Waals attractions
 SUBSTRATE
 Reactants in biochemical reactions - substrate
 When a substrate binds to an enzyme it forms an enzyme-
substrate complex.
 Composition of Enzyme
 Non-protein enzymes: A few enzymes are made of
ribonucleic acids and catalyze cellular reactions
involving nucleic acids.
 Ribozyme – catalytic RNA involved in the removal
of parts to form a functional RNA molecule

 Most enzymes are large protein molecules with

complex three-dimensional shapes
 Simple/Globular: only protein
 Conjugated protein/Complex or holoenzymes
 Composition of Enzyme
 Simple/Globular: only protein
 consist entirely of amino acid units
 the 3o structure of the simple proteins (enzymes)
that makes it biologically active
 Examples: pepsin, trypsin, ribonuclease

 Conjugated protein/Complex or holoenzymes

 protein part and non-protein part
 Protein part – apoenzyme
 Non-protein part – cofactor/coenzyme
 Conjugated Protein/Holoenzymes

 Apoenzyme  Holoenzyme
 protein part  the resulting complete,
 inactive in itself active enzyme when the
 Cofactor or Coenzyme apoenzyme has been
 nonprotein organic moiety activated by the coenzyme
 the activator
 NON-PROTEIN

 Co-factor- metal ion moiety

 Coenzymes – small organic molecule
 Cosubstrates –only transiently /loosely associated
or bound to the enzymes
 Prosthetic groups – permanently/tightly bound to
the enzymes
Cofactor

a) The apoenzyme is unable to bind to its substrate.

b) When the required cofactor, in this case a copper
ion, Cu2+, is available, it binds to the apoenzyme.
c) Now the active site takes on the correct
configuration, the enzyme-substrate complex
forms, and the reaction occurs.
 Cofactor
 Inorganic substances (Zn, Fe, Cu, Metal Ion Enzyme

Mg) needed for proper enzymatic Fe2+ or Fe3+ Peroxidase

Cu2+ Cytochrome oxidase
activity.
activity
Zn2+ DNA polymerase
 Iron must be present in the Mg2+ Hexokinase

quaternary structure - hemoglobin Mn2+ Arginase

K+ Pyruvate kinase
in order for it to pick up oxygen. Ni2+ Urease
Mo Nitrate reductase
Se Glutathione peroxidase
 Coenzyme
 Coenzyme
 Small organic molecules acting as carrier molecule
 Examples : vitamins or compound derived from
vitamins.
 Types
 Cosubstrates: transiently/loosely bound to apoenzyme
Example: NAD+ - dissociate from the enzyme in an
altered state
 Prosthetic group: tightly bound to the apoenzyme
 Example: FAD
Coenzyme
Coenzyme
 Coenzyme

Role of Vitamin C in collagen formation

Collagen contains hydroxylysine and hydroxylproline.

Hydroxylation of lysine and proline in collagen formation

are catalyzed by enzymes and require ascorbic acid (Vit. C).

In Vit. C deficiency, hydroxylation is impaired, and the triple

helix of the collagen is not assembled properly.
 Nomenclature of enzymes
1. The suffix –ase identifies a substance as an enzyme.
Ex.: urease, sucrase, lipase are all enzyme designations.
The suffix –in is still found in many of the digestive enzymes.
Ex.: trypsin, pepsin.

Enzymes are most commonly named by using a system that

attempts to provide information about the function (rather than
the structure) of the enzyme.

2.The type of reaction catalyzed (function) by an enzyme is often

noted with a prefix.
Ex.: Oxidase catalyzes oxidation reaction
Hydrolase catalyzes hydrolysis reaction
 Nomenclature of enzymes
Important aspects in naming enzymes are as follows:

3.The identity of the substrate is often noted in addition to the

type of reaction/function
Ex.: alcohol dehydrogenase oxides ethanol
glucose oxidase, pyruvate carboxylase, succinate
dehydrogenase

4.Infrequently, the substrate but not the reaction type is given

Ex.: urease, lactase
 Enzymes are classified into six functional Classes
(EC number Classification) by the
International Union of Biochemists (I.U.B.).
on the Basis of the Types of Reactions that they Catalyze

• EC 1. Oxidoreductases
• EC 2. Transferases
• EC 3. Hydrolases
• EC 4. Lyases
• EC 5. Isomerases
• EC 6. Ligases
EC (enzyme commission)
 Principle of the international
classification

Each enzyme has classification number

consisting of four digits:
Example:
EC:   (2.7.1.1) HEXOKINASE
 Enzyme Nomenclature and Classification
 A series of four number serves to specify a particular enzyme.
 The numbers are preceded by the letters EC (enzyme commission).

First number: class

Second number: subclass (electron donors, type of substrate, etc.)
Third number: characteristics of the reaction (functional groups, etc.)
Fourth number: order of the individual entries

Carboxipeptidase A (peptidyl-L-amino acid hydrolase)

EC 3.4.17.1
Class: 3  Hydrolases.
Subclass: 4  peptide bond
17  metallocarboxypeptidases.
Entry number: 1
EC: (2.7.1.1) these components indicate the
following groups of enzymes:
2: Class (transferase)
7: Subclass (transfer of phosphate)
1: Sub-sub class (alcohol is phosphate acceptor)
1 : entry number

Specific name
ATP,D-HEXOSE-6-PHOSPHOTRANSFERASE (Hexokinase)
1 : Specific name
ATP,D-HEXOSE-6-PHOSPHOTRANSFERASE (Hexokinase)

6 CH 2 O H 6 CH O PO 2 
2 3
ATP ADP
5 O 5 O
H H H H
H H
4 1 4 H 1
OH H OH
M g 2+
OH OH OH OH
3 2 3 2
H OH H ex ok inase H OH
glu cose glucose -6 -ph osp hate

Hexokinase catalyzes:
Glucose + ATP  glucose-6-P + ADP
 Oxidoreductases

A + B:  A: + B
a. Oxidases – oxidation of a substrate
-involved in oxidizing or taking away electrons from a
substrate
b.Reductases – reduction of a substrate
-involved in reducing or giving electrons to a substrate
 Oxidoreductases
c. Dehydrogenase – removal or addition of hydrogen atoms
 Transferases
Transfer of functional
groups between two
substrates
The basic reaction that transferases catalyzed is when a functional
X group is moved from Molecule B to another Molecule A:
A + BX  AX + B
a. Kinases – transfer of a phosphate group between
substrates

ATP,D-HEXOSE-6-PHOSPHOTRANSFERASE (Hexokinase)
 Transferases
b. Transaminase – transfer of an amino group
between substrates

ALT: Alanine transaminase

 Hydrolases

Addition of a water
molecule to a bond
causing the bond to
break

A + H 2O  B + C

• Catalyze hydrolysis reactions where water is the

acceptor of the transferred group
 Hydrolases
a. Proteases – hydrolysis of peptide linkages in proteins

b. Carbohydrases – hydrolysis of glycosidic bonds in

carbohydrates: sucrase, lactase, cellulase
 Hydrolases
c. Lipases – hydrolysis of ester linkages in lipids
 Hydrolases
d. Nucleases
– hydrolysis of sugar
phosphate ester
bonds in nucleic acid

e. Phosphatases – hydrolysis of phosphate ester bonds

 Lyases

Cleave various bonds by

means other than
hydrolysis and oxidation

A  B + C

Catalyze the dissociation of a molecule

without using water.
 Lyases
a.Dehydratases – removal of water from substrate

 ACP (Acyl carrier protein)

b. Decarboxylases – removal of carbon dioxide from

substrate
 Lyases
c. Deaminases – removal of ammonia from
substrate
 Isomerases

• catalyze the conversion of a substrate into another compound

that is isomeric with it.
• facilitate intramolecular rearrangements in which bonds are
broken and formed or they can catalyze  conformational
changes.

A  B
 Isomerases
a. Racemases – conversion of D to L isomer or vice versa

b. Mutases – transfer of a functional group within a

molecule
 Isomerases
c. Epimerases – conversion of one sugar epimer into another
 Ligases/Synthetases

• Catalyze ligation, or joining of two substrates

• Require chemical energy (e.g. ATP)
A + B  AB
a. Synthetases – formation of new bond between two
substrates with participation of ATP
 Ligases/Synthetases
b. Carboxylases – formation of new bond between
substrate and carbon dioxide with
participation of ATP
 The Models of Enzyme Action
Lock and key theory
(Fisher, 1890)
• In the lock-and-key model of enzyme action, the
shape of the substrate and conformation of the
active site are complementary to one another.

Enzyme-substrate complex interactions

 Lock-and-Key Model

Lock-and-key model explains:

enzyme specificity
the loss of activity when enzymes denature
 The Models of Enzyme Action
Induced fit theory (Koshland , 1958)
• In induced induced-fit model of enzyme action, the enzyme
undergoes a conformational change upon binding to
substrate.
• The shape of the active site becomes complementary to the
shape of the substrate only after the substrate binds to the
enzyme.
• This is a more thorough
explanation for the active-site
properties of an enzyme because
it includes the specificity of the
lock-and-key model coupled with
the flexibility of the enzyme
protein. Enzyme-substrate
complex interactions
 Specificity of Enzymes
 Ability of an enzyme to choose exact substrate from a group
of similar chemical molecules.
 A molecular recognition mechanism which operates through
the structural and conformational complementary between
enzyme and substrate.
 Enzymes show different degrees of specificity towards their
substrate.
 Bond Specificity of Enzymes
 Relative or linkage specificity
 Enzymes are specific to substrates having similar bonds and
similar structure. Enzymes attack a particular kind of chemical
bond, irrespective of the structural features in the vicinity of the
linkage
 Enzymes are specific only to certain types of bonds
 Peptide bonds are hydrolyzed by proteinases (peptidases
member of the group of proteinases)
 Bond Specificity of Enzymes
 -amylases which hydrolyze -1-4 glycosidic bond linkage in
starch and glycogen ( the enzyme is specific only to the -1-4
glycosidic bond -1-4 glycosidic bond and not to the substrate
 Group Specificity of Enzymes
 The enzyme is specific to a bond and groups surrounding the bonds.
 Moderate specificity (More than that of bond specificity)
 Pepsin, a digestive enzyme, can hydrolyze a peptide bond in which
the amino group is contributed by an aromatic amino acid such as
phenyl alanine, tyrosine and tryptophan.

 Trypsin, a serine protease of digestive system, can hydrolyze a

peptide bond in which the amino group is contributed by any basic
amino acid such as lysine, arginine and histidine.
 Group Specificity of Enzymes
 Aminopeptidase and carboxypeptidase can hydrolyze both a
protein from N-terminal and C-terminal, respectively.
 Stereo-specificity of enzymes
 Stereochemical or Optical specificity
 The enzyme is specific not only to substrate but also to its optical
configuration.
 The highest specificity shown by any class of enzymes

L-amino acid oxidase acts -amylase hydrolyzes only the

only on L-amino acid but -glycosidic bonds but not the
not on D-amino acid β-glycosidic bonds
 Geometric specificity of enzymes
 Single enzyme can act on different substrates having
similar molecular geometry
 Specificity is less.
 Alcohol dehydrogenase can oxidase both ethanol and
methanol to yield corresponding aldehydes since both
alcohols have similar molecular geometry.
Ethanol
Methanol
 Substrate Specificity of Enzymes
 Absolute specificity
 Specificity is very high.
 Enzymes showing substrate specificity are specific
only to one substrate and one reaction.
 Maltase can only act on the -1-4 glycosidic linkage
of two glucose molecules in maltose
 Substrate Specificity of Enzymes
 Lactase can only hydrolyze the β-1-4 glycosidic bond
of lactose to yield galactose and glucose.
 Co-factor Specificity of Enzymes
 Co-factors are nonprotein part of enzyme required for
the functioning of some enzymes.
 Enzymes which requires co-factors for their activity
shows co-factor specificity.
 Only correct combination of substrates and co-factor
allows enzymatic reactions.
 In the absence of specific co-factor, the enzymes will be
inactive even if there are plenty of substrates.
 Enzyme Specificity
 (5th) Bond (Relative) specificity – specific to bonds
 (4th) Group specificity – specific to bonds and groups surrounding
the bonds
 (3rd) Substrate (Absolute) specificity – specific to only one
substrate and reaction
 (1st, highest) Stereo (Stereochemical) specificity – specific to
substrate and its optical conformation
 (6th, least) Geometrical specificity – substrate having similar
molecular geometry
 (2nd) Co-factor specificity – specific to exact substrate, co-factor
combination

 Ascending order of specificity

Geometrical< Bond < Group < Substrate < Co-factor < Stereo
Factors Affecting Enzyme Activity
 Enzyme activity is a measure of the rate at which an
enzyme converts substrate to products.

 Factors affecting enzyme activity:

(1)Effect of Temperature
(2)Effect of pH
(3)Concentration of Enzyme
(4)Concentration of Substrate
 Factors Affecting Enzyme Activity
1) Temperature
• A rise in temperature
increases the kinetic energy
of enzyme and substrate
molecules molecules are
moving faster and colliding
more frequently.

• As the temperature of an
enzymatically catalyzed
reaction increases, so does
the rate of the reaction.
• Optimum temperature The temp at which
enzymatic reaction occur fastest.

The temperature that produces maximum activity for an

enzyme is known as the optimum temperature for that
enzyme.
 Factors Affecting Enzyme Activity
Temperature…
o Optimum temperature for enzymes is variable:

• The optimum temperature for most human enzymes is

between 35 and 40°C. Human enzymes start to
denature at temperatures above 40°C

• Thermophilic bacteria found in the hot springs have

optimum temperatures of 70°C.
 Factors Affecting Enzyme Activity
Temperature…
 Further elevation of the
temperature results in a
decrease in reaction velocity
as a result of temperature-
induced denaturation of the
enzyme leading to
. derangement in the native
(tertiary) structure of the
protein and active site.
o Enzymes become inactive.
 Factors Affecting Enzyme Activity
2) pH

• Small changes in pH (less than one unit) can result in enzyme

denaturation and subsequent loss of catalytic activity)
 Factors Affecting Enzyme Activity
pH (Effect of pH on the ionization of the active site)

 The catalytic process usually requires that the enzyme

and substrate have specific chemical groups in either
an ionized or un-ionized state in order to interact.

 For example, catalytic activity may require that an

amino group of the enzyme be in the protonated form
(–NH3+). At alkaline pH, this group is deprotonated,
and the rate of the reaction, therefore, declines.
pH (Effect of pH on enzyme denaturation)

 Extremes of pH can also lead to

denaturation of the enzyme, because the
structure of the catalytically active protein
molecule depends on the ionic character
of the amino acid side chains.
 • Optimum pH is the pH at which an enzyme has maximum
activity; biochemical buffers help maintain the optimum pH
for an enzyme.
• Each enzyme has a characteristic optimum pH, which usually
falls within the physiological pH range of 7.0-7.5 , except
digestive (stomach) enzymes pepsin (functions best at pH 2.0)
and intestinal trypsin (functions best at pH 8.0)
 Factors Affecting Enzyme Activity
3) Concentration of the enzyme
• If the amount of substrate present is kept constant and the
enzyme concentration is increased, the reaction rate
proportionately increases because more substrate
molecules can be accommodated in a given amount of time

Initially there is a reaction

(although very slow)
 Factors Affecting Enzyme Activity
4) Concentration of substrate

• The rate of reaction

increases as substrate
concentration increases (at
constant enzyme
concentration) and
thereafter remains constant
– this activity pattern is
called a saturation curve

• Maximum activity occurs

when the enzyme is
saturated (when all enzymes
are binding substrate)
 Enzyme Kinetics
 It is the analysis of the velocity (or rate) of a chemical
reaction catalyzed by an enzyme, and how the velocities
can change on the basis of environmental parameters
modifications.

WHAT DO YOU HAVE TO KNOW?

Velocities
The rate of an enzyme-catalysed reaction as defined in
mathematical way
The order of a reaction (zero order/ first-order reaction)
 Enzyme Kinetics
Steady-state
 Under experimental conditions [S]>>>[E].
 The [ES] quickly reaches a constant value in such dynamic
system, and remains constant until complete P formation: Steady
State assumption
Concentration

E = Enzyme S = Substrate P = Product

ES = Enzyme-Substrate complex
k1 = rate constant for the forward
reaction
k-1 = rate constant for the breakdown of
0 the ES to substrate
Time
Early stage Steady state k2 = rate constant for the formation of
ES formation [ES] is constant the products
Substrate Concentration and Reaction Rate

Steady-state
[ES]
 Michaelis-Menten Equation

The velocity/rate of the product formation is: v  k 2 [ES]

[ES] depends on:
 the velocity of ES formation from E + S
 the velocity of its dissociation to regenerate E+S or to form E
+ P.

d[ES]
 k1 [E][ S ]  k 1 [ ES ]  k 2 [ ES ]
dt
 Assumption of steady state
Transient phase where in the course of a reaction
the concentration of ES does not change

d  ES 
0
dt
 Assumption of equilibrium

d[ES]
 k1 [E][ S ]  k 1 [ ES ]  k 2 [ ES ]
dt
d [ ES ]
Steady-state  0, so k1 [ E ][ S ]  k1 [ ES ]  k2 [ ES ]
dt

when k2<<k-1 k1[E][S] = [ES] (k-1 + k2)

k 1  E  S
Ks  
k1  ES
Ks is the dissociation constant for the ES complex.
 k1[E][S] = [ES] (k-1 + k2)
The total enzymes includes
the free and those complexed  E  T   E    ES
with subtrates [E] = [E]T –[ES]

k1   E  T -  ES   S   k -1  k 2   ES
k1 [ E ]T [ S ]  k1 [ ES ][ S ]  ( k 1  k 2 )[ ES ]

k1 [ E ]T [ S ]  ( k1[ S ]  k 1  k 2 )[ ES ]
k1 [ E ]T [ S ]
[ ES ] 
k1 [ S ]  k  1  k 2
[ E ]T [ S ]
[ ES ] 
[ S ]  ( k  1  k 2 ) / k1
 [ E ]T [ S ]
[ ES ] 
[ S ]  ( k  1  k 2 ) / k1
k 1  k 2
Let KM, Michaelis constant KM 
k1
[ E ]T [ S ]
[ ES ] 
[S]  K M

The velocity/rate of the product formation is: v  k 2 [ES]

k 2 [ E ]T [ S ]
v [ES] = v/k2
[S]  K M
Maximal velocity is obtained when the enzyme is saturated:
[E]T=[ES] V  k [E] max 2 T

Vmax [ S ] [E]T = Vmax

Michaelis-Menten v
Equation [S]  K M k2
• Each substrate must occupy an enzyme active site for a finite
amount of time, and the products must leave the site before
the cycle can be repeated.
• When each enzyme molecule is working at full capacity, the
incoming substrate molecules must “wait their turn” for an
empty active site. At this point, the enzyme is said to be under
saturating conditions.
 Michaelis Menten Equation
The Michaelis-Menten equation describes how reaction velocity
varies with substrate concentration:

where:
vo = initial reaction velocity
Vmax = maximal velocity
Km = Michaelis constant = (k-1 + k2)/k1
[S] = substrate concentration

The rate or velocity of a reaction (v) is the number

of substrate molecules converted to product per unit time.
 k 1  k 2
Important conclusions about KM 
k1
Michaelis-Menten kinetics
1. Km—the Michaelis constant—
 indicates how efficient in an
enzyme selecting substrates
(specificity)
 reflects the affinity of the
enzyme for that substrate
  (low) Km reflects a high
affinity of the enzyme for
substrate, because a low
concentration of substrate is
needed to reach a velocity that
is ½ Vmax
  large (high) Km reflects a low
affinity
 If Km = [S], Vo = ½ Vmax.
Important conclusions about Michaelis-Menten kinetics

2) Relationship of velocity to enzyme concentration

 The rate of the reaction is directly proportional to the enzyme
concentration at all substrate concentrations.
 For example, if the enzyme concentration is halved, the initial
rate of the reaction (vo), as well as that of Vmax, are reduced to
half that of the original.
Important conclusions about
Michaelis-Menten kinetics

3. Order of reaction:
 When [S] <<< Km, the
velocity of the reaction is
approximately [S] <<< Km
proportional to the
substrate concentration.

vo  [S]
Michaelis-Menten

The rate of a enzymatic reactions depends on the

substrate concentration
 The rate of
reaction is
first order
with respect
to substrate.

1st order
 Important conclusions about
Michaelis-Menten kinetics [S] >>> Km

3. Order of reaction:
 When [S] >>> Km, the velocity is
constant and equal to Vmax.

vo = Vmax
 The rate of reaction is then zero order
independent of substrate
concentration, and is said to be
zero order with respect to
substrate concentration
[S] = Low → High
 y = mx + b = b + mx

https://www.slideshare.net/MUBOSScz/enzymes-2
Michaelis-Menten Experimental determination
Equation
of KM and Vmax
Several rearrangements of the
Michaelis-Menten equation transform
it into a straight-line equation.
Lineweaver-Burk
double-reciprocal plot:

1 KM 1 1
 
v Vmax [ S ] Vmax

 In Vo vs [S] plot, it is Y = mx + b
not always possible to m – slope
b= Y-intercept
measure Vmax
because of the gradual
upward slope.
 Inhibition of Enzymes
Inhibition: Velocity of an enzymatic reaction is
decreased or inhibited by some agent (inhibitors)
 Inhibitors
• Any molecule that acts directly on an enzyme to lower
its catalytic rate or reduce the rate of enzymatic
reactions.
• Usually specific and work at low concentrations
• Block the enzyme but they do not usually destroy it.
• These can be cellular metabolites, or foreign
substances such as drugs or toxins that have either a
therapeutic or toxic (can be lethal) effect.
 Types of Inhibition
 Reversible inhibition
 Inhibitor interact with the enzyme through
noncovalent association/dissociation reactions.
a) Competitive
b) Un-competitive
c) Non-competitive/Mixed

 Irreversible inhibition
 Inhibitor causes stable, covalent alterations in the
enzyme
 Reversible Inhibitor

Inhibitor binds non-covalently

(weak interaction) with enzyme
If inhibitor is removed, then the
action of enzyme is fully restored
(Reversible)
The activity of enzyme is fully
restored on removing the inhibitor by
dialysis
 Reversible Inhibitor
Competitive Inhibitor
Competitive inhibitors are those which mimics the shape of
the actual substrate and binds to the active site.
 Reversible Inhibitor
Competitive Inhibitor
 Competitive Inhibitor

Without competitive
Inhibitor

Competitive inhibitor
present

The affinity of enzyme towards

substrate is reduced in the presence
of inhibitor. Km increases.
No change in Vmax.
The Vmax can be reached even
in the presence of inhibitor, but
at much higher concentrations
of [S] that have to overcome the
competing inhibitor
concentration..
Succinate binds to the enzyme succinate dehydrogenase.
A dehydrogenation reaction occurs, and the product—fumarate—is
released from the enzyme.

Malonate (competitive inhibitor) also binds to the active site of succinate

dehydrogenase. In this case, however, no subsequent reaction occurs
while malonate remains bound to the enzyme.
 Reversible Inhibitor
Uncompetitive Inhibitor
 Uncompetitive inhibitors do not bind to the free
enzyme.
 They bind only to the enzyme-substrate complex to
yield an inactive E.S.I. complex.
 Inhibitor cause structural distortion of the active site,
thus, enzyme become catalytically inactive.
 Reversible Inhibitor
Uncompetitive Inhibitor
 Inhibitor can not be reversed by increasing the substrate
concentration since inhibitor does not compete with the
substrate for the same binding site
Km lower. The affinity of enzyme towards
substrate is increased in the presence of
inhibitor. (More formation of ES complex)

Vmax lower due to the inactive

ESI (enzyme-substrate-inhibitor) Inactive
complex, lesser substrate Complex
converted to product (cannot react)
 Reversible Inhibitor
Uncompetitive Inhibitor
 Inhibitor can not be reversed by increasing the substrate
concentration since inhibitor does not compete with the
substrate for the same binding site
Vmax lower due to the inactive ESI
(enzyme-substrate-inhibitor) complex,
lesser substrate converted to product

Km lower. The affinity of enzyme towards

substrate is increased in the presence of
inhibitor. (More formation of ES complex)
 Noncompetitive (Mixed) Inhibitor
Combination of competitive and uncompetitive
Non-competitive inhibitors do not compete for the
active site with substrate but does not allow substrate
to bind at the active site.
 Noncompetitive (Mixed) Inhibitor
.
Allosteric inhibitors bind at different position but
actually causes change in the active site so new
substrate moiety cannot bind.
 Noncompetitive (Mixed) Inhibitor
The substrate is sterically hindered, blocking the active site so
as substrate can not interact with the enzyme
 Noncompetitive (Mixed) Inhibitor
Adding more substrate will not affect the reaction
because the active site is unavailable.
Noncompetitive inhibitors decreases Vmax without
any change in Km.
Reversible
Inhibitors
 Irreversible Inhibition
 Inhibitor binds tightly, often covalently (strong), to the
enzyme, so it can not dissociate from the enzyme
 Irreversible Inhibition

 Enzyme activity is not regained by increasing the

concentration of substrate.
Irreversible Inhibition
Irreversible Inhibition
 Irreversible Inhibition
 Aspirin: causes covalent modification in a
cyclooxygenase involved in inflammation
Irreversible inhibition is aspirin’s mode of operation. Aspirin irreversibly
inhibits the cyclooxygenase (COX) enzyme, which catalyzes one of the
reactions involved in prostaglandin production. Prostaglandins have a
wide range of biological effects, including causing pain, inflammation, and
fever. In order to prevent pain, inflammation, and fever, we use aspirin (or
other nonsteroidal anti inflammatory drugs (NSAIDs)).
In this reaction, an acetyl group from aspirin is exchanged for a hydrogen
atom (H) from a particular side-chain in the COX enzyme's active site.
When an acetyl group is bonded to the enzyme’s active site, it is no
longer possible for substrates to bind to the enzyme, and therefore the
enzyme is permanently inactivated.
 Suicide inhibitors/inactivators
 a special class of irreversible inhibitors
 also called as mechanism-based inactivators
because they hijack the normal enzyme
reaction mechanism to inactivate the
enzyme.
- relatively unreactive until they bind to the
active site of a specific enzyme.
- The inhibitor is converted to a more
effective inhibitor with the help of the
enzyme to be inhibited
Irreversible (Suicide) Inhibition
 Regulation of Enzyme Activity
Living systems must regulate the enzymatic catalytic activity to:

Coordinate metabolic processes

Promote adaptations to environmental changes
Growth and complete the living cycle in the correct way

Two mechanisms of regulation:

1. Control of the enzyme availability

2. Control of the enzymatic activity, by means of modifications of
the conformation or structure
 Allosteric Regulation
Some enzymes have receptor sites away from the active
site called ALLOSTERIC SITES.
These enzymes are usually proteins made of several
subunits each with an active site.
The presence of an allosteric effector can alter the
affinity of the enzyme for its substrate, or modify the
maximal catalytic activity of the enzyme, or both.

Allosteric effectors are substances that bind at allosteric site

and regulate enzyme activity
Positive allosteric effectors – enzyme activity is increased
Negative allosteric effector – enzyme activity is decreased;
allosteric inhibitors
 Allosteric Inhibition
• Binding of an allosteric inhibitor stabilizes the
inactive form of the enzyme.
 Allosteric Activators
Binding an ACTIVATOR to an allosteric site
stabilizes the protein conformation
This keeps all the active sites available for the
substrates to bind to them.
 Applications of inhibitors

• Negative feedback: end point or end product

inhibition
• Medicine antibiotics, sulphonamides, sedatives and
stimulants.
• Poisons snake bite, plant alkaloids and nerve gases
 Feedback Inhibition
 Feedback inhibition is a cellular control mechanism in
which an enzyme’s activity is inhibited by the enzyme’s
end product. This mechanism allows cells to regulate
how much of an enzyme’s end product is produced.
Negative feedback inhibition is like a thermostat.
When it is cold, the thermostat turns on the heater to produce heat.
When it is too warm, the heat will cause the thermostat to turn off the
heater. Heat has a negative effect on the thermostat.

Cold Thermostat Heater Heat

turning off
 Feedback
Inhibition
 As the enzyme’s
end product
accumulates, it
inhibits the enzyme
by binding to the
first enzyme in the
pathway.
 This shuts down
the entire
sequence.
Feedback inhibition occurs when an end product synthesized
after a chain of anabolic pathways becomes an inhibitor that binds
at allosteric site of the first enzyme that made this end product
and affects the shape of the enzyme. Thus the enzyme no longer
can bind the substrate at its active site. The metabolic pathway is
then switch off and can no longer produce the end products that
were the same as the inhibitor that bind to the allosteric site. This
can be used as a method of metabolic control.
 CLINICAL APPLICATIONS OF ENZYMES
 In healthy individuals, the levels of these enzymes are
fairly constant, and represent a steady state in which the
rate of release from damaged cells into the plasma is
balanced by an equal rate of removal of the enzyme
protein from the plasma.
 The activities of many enzymes are routinely determined
for diagnostic purposes in diseases of the heart, liver,
skeletal muscle, and other tissues.
 Increased plasma levels of these enzyme may indicate
tissue damage.
 Thus, determining the degree of elevation of a particular
enzyme activity in the plasma is often useful in evaluating
the prognosis for the patient.
CLINICAL APPLICATIONS OF ENZYMES

Necrosis – localized death of living tissue

 CLINICAL APPLICATIONS OF ENZYMES
Isoenzymes in Medical Diagnosis

 Enzymes that catalyze the same reactions but vary slightly in

structure are called ISOENZYMES.

Example:
 There are five isoenzymes for lactate dehydrogenase (LDH), an
enzyme that converts lactic acid to pyruvic acid.
Each LDH consists of a quaternary structure containing four
subunits.
•In heart muscle, the most prevalent subunit is designated as H.
•In skeletal muscle, the major subunit is designated as M.
 CLINICAL APPLICATIONS OF ENZYMES
Isoenzymes in Medical Diagnosis

• In healthy tissues, these enzymes are contained within cellular

membranes.
• If the cells of a particular organ are damaged, the contents
including the enzymes spill into the blood.
• By identifying the isoenzyme that becomes elevated in the blood
serum, it is possible to determine which type of tissue has been
damaged.
o Example: Liver diseases can be detected by a rise in the serum
LDH5 level.
CLINICAL APPLICATIONS OF ENZYMES