Enzymes

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ENZYMES

CONTENTS
 Chemistry
 Classification
 Mechanism of Enzyme Action
 Enzyme Kinetics
 Inhibition
 Activation
 Specificity
CHEMISTRY
INTRODUCTION
 Enzymes are biological catalysts
that speed up the rate of the
biochemical reaction.

 Most enzymes are three


dimensional globular proteins
(tertiary and quaternary structure).

Hammerhead Enzyme
STRUCTURE OF ENZYMES
 The active site of an enzyme is the region that binds substrates,
co-factors / prosthetic groups and contains residue that helps to
hold the substrate.

 Active sites generally occupy less than 5% of the total surface


area of enzyme.

 Active site has a specific shape due to tertiary structure of protein.

 A change in the shape of protein affects the shape of active site


and function of the enzyme.
ACTIVE SITE
 Active site can be further divided into :
CO-FACTORS

 Co-factors is the non protein molecule which carries out chemical reactions that can
not be performed by standard 20 amino acids.

 Co-factors are of two types:


 Organic co-factors
 Inorganic co-factors
INORGANIC CO-FACTORS

These are the inorganic molecules required for the proper activity of
enzymes.
Examples:
 Enzyme carbonic anhydrase requires Zn for it’s activity.
 Hexokinase has co-factor Mg
ORGANIC CO-FACTORS
These are the organic molecules required for the proper activity of
enzymes.
Example:
 Glycogen phosphorylase requires the small organic molecule
pyridoxal phosphate.
TYPES OF ORGANIC CO-FACTORS
Prosthetic Group A prosthetic Coenzyme
group is a tightly bound organic A coenzyme is loosely bound
co- factor e.g. Flavins, heme organic co-factor. E.g. NAD+ +
groups and biotin.
TYPES OF ORGANIC CO-FACTORS continuation…

 An enzyme with it’s co-factor removed is designated as apoenzyme.

 The complete complex of a protein with all necessary small organic


molecules, metal ions and other components is termed as holoenzyme of
holoprotein.
Substrate
 The reactant in biochemical reaction is termed as substrate.

 When a substrate binds to an enzyme it forms an enzyme- substrate


complex.
SITES OF ENZYMES SYNTHESIS
 Enzymes are synthesized by ribosomes which are attached to the R.E.R
Information for the synthesis of enzyme is carried by DNA.
 Amino acids are bonded together to form specific enzyme according to the
DNA’s codes.
Intracellular and Extracellular Enzymes
Intracellular enzymes are synthesized and retained in the cell for the use of cell
itself.
They are found in the cytoplasm, nucleus, mitochondria and chloroplast.
Example :
 Oxydoreductase catalyses biological oxidation.
 Enzymes involved in reduction in the mitochondria.

Extracellular enzymes are synthesized in the cell but secreted from the cell to
work externally.
Example :
 Digestive enzyme produced by the pancreas, are not used by the cells
in the pancreas but are transported to the duodenum.
CHARACTERISTICS

 Enzymes speed up the reaction by lowering the activation energy of the reaction.
 Their presence does not effect the nature and properties of end product.
 They are highly specific in their action that is each enzyme can catalyze one kind of
substrate.
 Small amount of enzymes can accelerate chemical reactions.
 Enzymes are sensitive to change in pH, temperature and substrate concentration.
 Turnover number is defined as the number of substrate molecules transformed per
minute by one enzyme molecule.
NOMENCLATURE OF ENZYMES
 An enzyme is named according to the name of the substrate it catalyses.

 Some enzymes were named before a systematic way of naming enzyme was
formed.
Example:
 pepsin, trypsin and rennin

 By adding suffix -ase at the end of the name of the substrate, enzymes are named.

 Enzyme for catalyzing the hydrolysis is termed as hydrolase.

Example:
CLASSIFICATION
CLASSIFICATION OF ENZYMES
 A systematic classification of enzymes has been developed by International
Enzyme Commission.

 This classification is based on the type of reactions catalyzed by enzymes.

 There are six major classes.

 Each class is further divided into sub classes, sub sub-classes and so on, to
describe the huge number of different enzyme- catalyzed reactions.
CLASSIFICATION OF ENZYMES
MECHANISM OF
ENZYME ACTION
MECHANISM OF ENZYME ACTION

 The catalytic efficiency of enzymes is explained by two perspectives:


THERMODYNAMIC CHANGES
 All chemical reactions have energy barriers between reactants and products.

 The difference in transitional state and substrate is called activational barrier.


THERMODYNAMIC CHANGES
 Only a few substances cross the activation barrier and change into products.

 That is why rate of uncatalyzed reactions is much slow.

 Enzymes provide an alternate pathway for conversion of substrate into products.

 Enzymes accelerate reaction rates by forming transitional state having low


activational energy.

 Hence, the reaction rate is increased many folds in the presence of enzymes.

 The total energy of the system remains the same and equilibrium state is not
disturbed.
LOCK AND KEY MODEL

 Proposed by EMIL FISCHER in 1894.

 Lock and key hypothesis assumes the active site of an enzymes are rigid in
its shape.

 There is no change in the active site before and after a chemical reaction.
INDUCED FIT MODEL

 More recent studies have revealed that the process is much more
likely to involve an induced fit model(proposed by DANIAL KOSH
LAND in 1958).
 According to this exposure of an enzyme to substrate cause a change
in enzyme, which causes the active site to change it’s shape to allow
enzyme and substrate to bind.
INDUCED FIT MODEL
ENZYMES KINETICS
INTRODUCTION

‘‘It is a branch of biochemistry in which we study the rate of enzyme catalyzed


reactions.”

 Kinetic analysis reveals the number and order of the individual steps by which
enzymes transform substrate into products.

 Studying an enzyme's kinetics in this way can reveal the catalytic mechanism of
that enzyme, its role in metabolism, how its activity is controlled, and how a
drug or an agonist might inhibit the enzyme
RATES OF REACTION AND THEIR DEPENDENCE ON
ACTIVATION ENERGY

 Activation Energy (Ea): “The least amount of energy needed for a chemical
reaction to take place.”
 Enzyme (as a catalyst) acts on substrate in such a way that they lower the
activation energy by changing the route of the reaction.
 The reduction of activation energy (Ea) increases the amount of reactant
molecules that achieve a sufficient level of energy, so that they reach the
activation energy and form the product.
Example:
 Carbonic anhydrase catalyses the hydration of 10⁶ CO₂ molecules per
second which is 10⁷x faster than spontaneous hydration.
ENZYMES LOWER THE ACTIVATION
ENERGY OF A REACTION
KINETICS OF ENZYMES CATALYSIS
 Enzymes catalysis:
“ It is an increase in the rate of reaction with the help of enzyme(as
catalyst).”

 Catalysis by enzymes that proceed via unique reaction mechanism, typically


occurs when the transition state intermediate forms a covalent bond with the
enzyme(covalent catalysis).

 During the process of catalysis enzymes always emerge unchanged at the


completion of the reaction.
FACTORS AFFECTING RATE OF ENZYME
CATALYZED REACTIONS
 Temperature

 Hydrogen ion concentration(pH)

 Substrate concentration
EFFECT OF TEMPERATURE
 Raising the temperature increases the rate of enzyme catalyzed reaction by increasing
kinetic energy of reacting molecules.

 Enzymes work maximum over a particular temperature known as optimum


temperature. Enzymes for humans generally exhibit stability temperature up to 35-45
ᵒC.

 The temperature coefficient is a factor Q₁₀ by which the rate of biological processes
increases for a 10 ᵒC increase in temperature.

 For most biological processes Q₁₀ = 2.

 However some times heat energy can also increase kinetic energy to a point that
exceed the energy barrier which results in denaturing of enzymes.
EFFECT OF PH
 Rate of almost all enzymes catalyzed reactions depends on pH

 Most enzymes exhibit optimal activity at pH value between 5 and 9

 High or low pH value than optimum value will cause ionization of enzyme
which result in denaturation of enzyme
A change in pH can alter the ionization of
the R groups of the amino acids. When
the charges on the amino acids change,
hydrogen bonding within the protein
molecule change and the molecule
changes shape. The new shape may not
be effective.
PH AFFECTS THE FORMATION OF HYDROGEN
BONDS AND SULPHUR BRIDGES IN PROTEINS AND SO
AFFECTS SHAPE
MICHAELIS-MENTEN MODEL & EFFECTS OF
SUBSTRATE CONCENTRATION
Michaelis-Menten Model:
“According to this model the enzyme reversibly combines with
substrate to form an ES complex that subsequently yields product,
regenerating the free enzyme.”
PHARMACEUTICAL IMPORTANCE
 Enzymes are virtually involved in all physiological processes
which makes them the targets of choice for drugs that cure or
ameliorate human disease.

 Applied enzyme kinetics represents the principal tool by which


scientist identify and characterize therapeutic agents that
selectively inhibit the rates of specific enzymes catalyzed
processes.

 Enzymes kinetics thus play a critical role in drug discovery as


well as elaborating the mode of action of drugs.
TEMPERATURE AND ENZYME ACTION
Enzymes
are most active at an
optimum temperature
(usually 37 °C in humans).
show little activity at low
temperatures.
lose activity at high
temperatures as
denaturation occurs
PH AND ENZYME ACTION
Enzymes
are most active at optimum pH.
contain R groups of amino acids
with proper charges at optimum
pH.
lose activity in low or high pH
as tertiary structure is disrupted.
SUBSTRATE CONCENTRATION
As substrate concentration
increases;

the rate of reaction increases (at


constant enzyme concentration).

 the enzyme eventually becomes


saturated, giving maximum
activity.
INHIBITION
INHIBITION
The prevention of an enzyme process as a result of interaction of inhibitors
with the enzyme.

 INHIBITORS:
Any substance that can diminish the velocity of an enzyme
catalyzed reaction is called an inhibitor.
TYPES OF INHIBITION
REVERSIBLE INHIBITION
 It is an inhibition of enzyme activity in which the inhibiting molecular
entity can associate and dissociate from the protein‘s binding site.

TYPES OF REVERSIBLE INHIBITION


There are four types;
 Competitive inhibition.
 Uncompetitive inhibition.
 Mixed inhibition.
 Non-competitive inhibition.
COMPETITIVE INHIBITION
In this type of inhibition, the inhibitors compete with the substrate for
the active site. Formation of E.S complex is reduced while a new E.I complex
is formed.
EXAMPLES OF COMPETITIVE INHIBITION

Statin Drug As Example Of Competitive Inhibition:

 Statin drugs such as lipitor compete with HMG-CoA(substrate) and inhibit


the active site of HMG CoA-REDUCTASE (that bring about the catalysis of
cholesterol synthesis).

β-Hydroxy β-methylglutaryl-CoA (HMG-CoA) reductase


inhibitors, more popularly known as statins, work by reducing
the cholesterol levels in the body2. HMG-CoA inhibitors interfere
with the ability of the body to build cholesterol from dietary fat
UNCOMPETITIVE INHIBITION

In this type of inhibition, inhibitor does not compete with the substrate
for the active site of enzyme instead it binds to another site known as allosteric
site.

An allosteric site is a place on a protein


where an effector molecule binds,
resulting in a conformational change or
altered protein dynamics
EXAMPLES OF UNCOMPETITIVE INHIBITION

 Drugs to treat cases of poisoning by methanol or ethylene glycol act as


uncompetitive inhibitors.

 Tetramethylene sulfoxide and 3- butylthiolene 1-oxide are uncompetitive


inhibitors of liver alcohaldehydrogenase.

Alcohol dehydrogenase is our primary defense against


alcohol, a toxic molecule that compromises the function of
our nervous system.
MIXED INHIBITION
 In this type of inhibition both E.I and E.S.I complexes are formed.
 Both complexes are catalytically inactive.

NON COMPETITIVE INHIBITION


 It is a special case of inhibition.
 In this inhibitor has the same affinity
 A noncompetitive inhibitor binds to the enzyme away from the
active site, altering the shape of the enzyme so that even if the
substrate can bind, the active site functions less effectively.

IRREVERSIBLE INHIBITION
 This type of inhibition involves the
covalent attachment of the inhibitor
to the enzyme.

 The catalytic activity of enzyme is


completely lost.

 It can only be restored only by


synthesizing molecules.
EXAMPLES OF IRREVERSIBLE INHIBITION

Aspirin which targets and covalently modifies a key enzyme involved in


inflammation is an irreversible inhibitor.
Aspirin, which inhibits cyclooxygenase 1 and 2 enzymes
The prostaglandinds are a group of lipids made at sites of tissue
SUICIDE INHIBITION : damage or infection that are involved in dealing with injury and
illness

 It is an unusual type of irreversible inhibition where the enzyme converts


the inhibitor into a reactive form in its active site.
ACTIVATION
ACTIVATION
Activation is defined as the conversion of an inactive form of an enzyme
to active form which processes the metabolic activity.

TYPES OF ACTIVATION
 Activation by co-factors.

 Conversion of an enzyme precursor.


ACTIVATION BY CO FACTORS

Many enzymes are activated by co-factors

Examples;
 DNA polymerase is a holoenzyme that catalyzes the polymerization of de -
oxyribonucleotide into a DNA strand. It uses Mg- ion for catalytic activity.

Polymerization is a process in which small molecules (monomers)


combine chemically to produce large chainlike or network
molecules (polymers)
DIAGNOSTIC SIGNIFICANCES OF ENZYMES
DIAGNOSTIC SIGNIFICANCES OF ISOENZYMES
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