Transcript ENZYMES

Topics covered are : What are enzymes ?
 Cofactors
 General properties
 classification
 Nomenclature
 Regulation of enzyme activity.
 Factors affecting enzymatic
activity
 Inhibitors
What are enzymes?
 Enzymes are large biological molecules responsible for
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thousands of chemical interconversions that sustain life.
Enzymes are soluble ,colloidal organic catalyst ,specific in
action , protein in nature.
They catalyze the hundreds of stepwise reactions that
degrade nutrient molecules ,conserve and transform
chemical energy from simple precursors.
Wilhelm Kühne first used the term enzymes.
For e.g. maltose is the substrate on which the enzyme
maltase acts to form glucose .
Cofactor :-
 Some enzymes require no chemical groups for activity
,others require some additional chemical component
called as COFACTOR
 They are non protein which active the enzyme when
bound with it .
COFACTORS
ORGANIC
INORGANIC
PROSTHETIC
EXAMPLES: INORGANICCu⁺² cytochrome oxidase
Mg⁺² hexokinase, pyruvate kinase
 ORGANICNicotinamide adenine dinucleotide Niacin
Flavin adenine dinucleotide
riboflavin
 PROSTHETICNADH , NADPH
 Enzymes are protein in nature ,when some cofactor
attaches to it then enzyme is known as apoenzyme
 An apoenzyme together with its cofactor is called
a holoenzyme (this is the active form). (e.g. biotin in
the enzyme pyruvate carboxylase).
 Enzyme +cofactor
(apoenzyme)
active enzyme
(holozyme)
PROPERTIES OF ENZYMES : They are proteinaceous in nature.
 They have more catalytic power as compared to
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inorganic catalyst
They have highly specific for their substrate
They accelerate the chemical reactions
Many inhibitors can affect the enzyme activity .
Enzyme have site which is lined with amino acids and
this site is known as ACTIVE SITE.
Classification:The six major classes of enzyme with example are
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as follows :oxidoreductases
Transferases
Hydrolases
Lyases
Isomerases
Ligases
Oxidoreductases:DefinationEnzyme catalyzing oxido-reduction reactions .
E.g.-dehydrogenase
NAD
NADH + H⁺
Transferases:Those enzyme transfering one group to other
compound E.g. hexokinase
ATP + D-Hexose
ADP + D-Hexose-6-phosphate .
Hydrolases: The enzymes which catalyze the hydrolytic cleavage of ester,
ether, peptide, anhydride by addition of water.
E.g.- β-galactosidase
β-galactoside + H₂O
alcohal +D-galactose
Lyases :Enzymes that catalyze removal of groups by leaving double
bond . E.g.- aldolase
ketose -1-phosphate
dihydroxy acetonephosphate+ an
aldehyde.
Isomerases : Enzyme catalyzing inter conversion of optical
,geometrical or positional isomers.
E.g.- triose phosphate isomerase
D-glceraldehyde-3-phosphate
Dihydroxy-acetone
phosphate
Ligases:Enzymes catalyzing the linking together of two
compounds . E.g.- succinate thiokinase
ATP+ acetly –CoA +Co₂
ADP+Pі +Succinyl-CoA
NOMENCLATURE : E.C. no. –
It stands for enzyme commission .
1) the first digit stands for class name
2) the second digit is for sub class
3) third and fourth digit stands for name of enzyme.
For e.g.ATP +D-Glucose
ADP+D-glucose-6-phosphate
enzyme – Glucose phosphotransferase
 Its E.C.no. is 2.7.1.1.
 The fist no. (2) denotes the class name i.e. transferase
 Second no. (7) denotes the sub class
phosphotransferase
 third no. (1) denotes phosphotransferase with
hydroxyl group as acceptor
 Fourth no. (1) denotes D-glucose as the phosphoryl
group acceptor .
Lock and key model : Enzymes are specific for particular substrate and
it was suggested by the Nobel laureate organic
chemist Emil Fischer in 1894 that this was
because both the enzyme and the substrate
possess specific complementary geometric shapes
that fit exactly into one another. This is often
referred to as "the lock and key" model.
How enzymes work ?
 This can be explained with the
help of reaction intermediate
diagram.
Important terms: Transition state – it is the state where maximum
number of bonds breaks and form.
 Activation energy –it is the minimum amount of energy
required for conversion of reactants into products. It is
denoted by Eact .
 The starting point for forward or reverse reaction is
ground state .
 Free energy change denoted by ∆GO which plays
important role.
 In this coordinate diagram the free energy of products
formation is less ,so formation of products is favored in
this reaction i.e. the reaction is forward.
REGULATORY ENZYMES
 These are the enzymes which increase or decrease the
rate of whole of the pathway in response to certain
signals.
 They have multi subunit in which regulatory site and
active site present mostly opposite to each other.
 These are mainly of two types on the basis of their
interactions:
1. Allosteric enzymes
2. Covalent enzymes
1. Allosteric Enzymes:
 These are the enzymes whose activity can be
changed by molecules (effector molecule,
modulator) other than substrate.
These are reversible in nature.
 These are of two types, in response to the
modulator:
a) Negative Allosteric enzyme
b) Positive Allosteric enzyme
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a) Negative Allosteric Enzymes:
These are the enzymes which are
deactivated by the modulator by attaching
with them.
 These are reversible, therefore when the
modulator is removed they are activated.
 In them when modulator binds then the
conformational change at active site doesn’t
allow substrate to bind.
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b) Positive Allosteric Enzymes:
These are the enzymes which are activated
by the modulator by attaching with them.
 These are reversible therefore when the
modulator is removed they are deactivated.
 For example, Phosphorylase-a is the
enzyme which breakdown glycogen into
glucose and Phosphorylase-b is inactive
form.
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• Subunit interactions in an allosteric enzyme, and interactions with inhibitors
and activators.
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Allosteric enzymes does not follow MichaelisMenten graph.
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Allosteric enzymes show more rapid increase in activity,
binding to the enzyme increases the activity much greater
than that observed by Michaelis- Menten graph.
2. Covalent Modifications:
 These are the regulatory enzymes in which
the modulators are bind with the covalent
bonds at regulatory site.
 It is reversible process .
 For example, Phosphorylase-a is the enzyme which breakdown glycogen
into glucose and Phosphorylase-b is inactive form.
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ExampleS: Phosphorylation :- (Tyr,Ser,Thr,His)
ATP ADP O
Enz
Enz P O‾
O‾
 Adenylylation :- (Tyr)
ATP PPі
O
Enz
Enz P O-CH₂
O⁻
H
O
H
OH
adenine
H
OH
 Uridylylation :-
UTP PPi
O
Enz
Enz P O CH₂
O‾
H
O
H
OH
uridine
H H
OH
TEMPERATURE- all enzymes have different optimum
temperature. With increase in temperature, collision
frequency between enzyme and substrate increase.
Therefore there is net increase in activity of enzymes. But
after achieving optimum temperature i.e. temperature in
which activity of enzyme is maximum, further increment in
temperature activity decrease due to denaturation of
protein. That’s why there is bell shaped graph.
pH- Enzyme have an optimum pH(or pH range) at which their
activity is maximal, at higher or lower pH, activity decreases.
Any change in pH above or below the Optimum will quickly ca
use a decrease in the rate of reaction. If we provide acidic or
alkaline condition, ionisation of enzymes occur, this results in
charged changes in its active site and activity decrease.
 CONCENTRATION OF SUBSTRATE- if initially increase
the activity but after the formation of [ES] complex, graph
becomes constant due to formation of [ES] complex which is
denoted as steady state.
NOTE- steady state only occurs when there is excess of
substrate.
 CONCENTRATION OF PRODUCTS- as the product form,
the collision frequency decrease because they interfere with
the collision of substrate and enzyme.
 Increase in product concentration , the enzyme activity
decrease. This is known as FEEDBACK INHIBITION,
occurs only in living system.
MICHAELIS-MENTEN EQUATION
MICHAELIS-MENTEN EQUATION -the rate equation for a
one-substrate enzyme catalyzed reaction.
Vo = initial velocity
Vmax = max. velocity of enzyme which represent the
interaction between enzyme and substrate
Km = concentration of substrate at which Vmax is half
[S]= substrate
LINEWEAVER BURT PLOT/ DOUBLE RECIPROCAL PLOT
TURN OVER NUMBER- it is number of substrate
molecule converting into products per unit time by a
single enzyme molecule. It is represented by K cat.
SPECIFICITY CONSTANT- Kcat/Km
As high as SPECIFICITY CONSTANT more efficient is an
enzyme.
 These are the certain unwanted compounds which
affects the rate of reaction by reacting with the
enzyme molecules.
 On the basis of their nature, they are divided into
two categories:I. Reversible Inhibitors
II. Irreversible Inhibitors
 These inhibitors are reversible in
nature.
 On the basis of site of inhibition, these
are of three types:
i. Competitive inhibitors
ii. Uncompetitive Inhibitors
iii. Mixed Inhibitors
 These inhibitors competes with the substrate for the
active site of an enzyme.
 These inhibitors reduce the efficiency of the enzyme.
 Competitive inhibition can be analyzed quantitatively
by steady-state kinetics.
 In the presence of a competitive inhibitor, the
Michaelis - Menten equation is as follows:
 The Line-Weaverburk plot is as follows:
 From the graph, it is concluded that Km increases and Vmax remains
unaffected.
ii. Uncompetitive Inhibitors:
 These inhibitors binds at a site distinct from the
substrate active site and, unlike a competitive
inhibitor, binds only to ES complex.
 In the presence of a uncompetitive inhibitor, the
Michaelis - Menten equation is as follows:
 The Line-Weaverburk plot is as follows:
 An uncompetitive inhibitor lowers the measured Vmax and apparently Km also
decreases.
iii. Mixed Inhibitors
 These inhibitors also binds at a site distinct from the
substrate active site, but it binds to either E or ES.
 These inhibitors usually affects both Km and Vmax.
 In the presence of a mixed inhibitor, the
Michaelis - Menten equation is as follows:
 From the graph it is concluded that Km increases and Vmax
decreases.
II. Irreversible Inhibitors
 These inhibitors bind covalently with or destroy a
functional group on an enzyme that is essential for the
enzyme’s activity, or those that form a particularly
stable non-covalent association.
 These are irreversible in nature.
 Special class:
• Suicide Inactivator:These compounds are relatively unreactive until they
bind to the active site of a specific enzyme.