ENZYMES

CONTENTS
 DESCRIPTION
 STRUCTURE
 SITES OF ENZYME SYNTHESIS
 CHARACTERESTICS OF ENZYMESEXAMPLES OF
ENZYMES
 CLASSIFICATION OF ENZYMES
 MECHANISM OF ENZYME ACTION
 ENZYME KINETICS
 Factors affecting rate of enzyme catalyzed
reactions
 INHIBITION
 ACTIVATION
 SPECIFICITY
 Enzymes are proteins that
function as biological catalysts
 Catalyst: a substance that
speeds up a chemical reaction
and is not changed by the reaction.
Most enzymes are three
dimensional globular proteins
(tertiary and quaternary structure).
Some special RNA species also
act as enzymes and are called
Ribozymes e.g. hammerhead
Enzymes
Enzymes can break a single
structure into smaller
components or join two or
more substrate molecules
together
Enzymes have a specific region
where the substrate binds and
where catalysis occurs. This is Space filling model of the yeast
called the active site. enzyme hexokinase. Its active
site lies in the groove (arrowed)

Active site has a specific shape

due to tertiary structure of
protein.
 The active site of an enzyme is the region that binds
substrates, co-factors and 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.

 A change in the shape of protein affects the shape of active

site and function of the enzyme.
 Active Site
 A restricted region of an enzyme molecule
which binds to the substrate.

ACTIVE SITE

Substrate
Enzyme

Muhammad Asif Pu 6
substrate
The reactant in biochemical reaction is
termed as substrate
Enzymes are substrate-specific,
although specificity varies from enzyme
to enzyme.
When a substrate binds to an enzyme’s
active site, an enzyme-substrate
complex is formed.
 Enzyme-Substrate Complex

When a substrate binds to an

enzyme’s active site, an enzyme-
substrate complex is formed.

Joins
Substrate
Enzyme

Muhammad Asif Pu 8
 12
The substrate molecules fit the shape of the enzyme

enzyme

substrate A

substrate B
 14

substrates combine temporarily with enzyme

enzyme joins substrates together

 15

enzyme unchanged new compound released

and ready for by enzyme
next reaction
Cofactors
 Cofactors are substances that
are essential to the catalytic
activity of some enzymes.
 Cofactors may alter the shape
of enzymes slightly to make
the active sites functional or
to complete the reactive site.
 Enzyme cofactors include
coenzymes (organic
molecules) or activating ions
(eg. Na+, K+..)
 Vitamins are often coenzymes
(eg. Vit B1, Vit B6…)
 CO FACTOR
 chemical reactions that can not be performed by
standard 20 amino acids.

 Co-factors are of two types:

 Organic co-factors
 Inorganic cofactors
 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
 These are the organic molecules
required for the proper activity of
enzymes.
Example:
 Glycogen phosphorylase requires
the small organic molecule pyridoxal
phosphate.
Prosthetic Group Coenzyme
o A prosthetic group is a o A coenzyme is loosely
tightly bound organic co- bound organic co-factor.
factor e.g. Flavins, heme E.g. NAD
groups and biotin.
 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.
 Enzymes are synthesized by ribosomes which are
attached to the rough endoplasmic reticulum.

 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.
 Enzyme Power!
 All reactants need to have a certain energy before
they will react. This is like an energy barrier that it
has to overcome before a reaction will occur. It is
called the activation energy.

 Enzymes are organic catalysts.

 All catalysts lower the energy barrier, allowing the

reactants (substrates) to react faster forming the
products.

 Enzymes do not participate in the reaction.

 Enzymes

High
Amount of energy stored in the

Without enzyme: The activation

energy required is high.
chemicals

Reactant With enzyme: The activation

energy required is lower.
High energy

Product

Low energy
Low
Start Finish

Direction of reaction
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.

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.
 21

-Enzymes can act on only one type of

substrate (specific)
 They always produce the same end products
 Because enzymes are proteins, they are denatured by
heat or some chemicals

 Denaturing involves a change of shape in the enzyme

molecule so that it cannot combine with the substrate

 Individual enzymes work best at a particular temperature

and pH (acidity or alkalinity)
 22
Enzymes can act on only one type of substrate

cannot combine
this substrate
with this enzyme

this substrate cannot combine

with this enzyme
 23
Because enzymes are proteins, they are
denatured by heat or some chemicals

enzyme
enzyme +
denatured
substrate
by heat

denatured enzyme cannot combine with substrate

 By: AQUIMA HADJIAMAEN
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 such as:

rennin : curding of milk to start cheese-making processor
pepsin : hydrolyzes proteins at acidic pH
trypsin : hydrolyzes proteins at mild alkaline Ph
 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.
 Enzyme Examples
Enzyme Role
Stomach enzyme used to
break protein down into
Pepsin
peptides. Works at very
acidic pH (1.5).
Digestive enzymes which
Proteases act on proteins in the
digestive system
A family of enzymes which
Amylases assist in the breakdown of
carbohydrates

3D molecular structures for A family of enzymes which

Lipases
breakdown lipids
the enzymes pepsin (top)
and hyaluronidase (bottom).
 substrate enzymes products
lactose lactase glucose +
galactose
maltose maltase Glucose

cellulose cellulase Glucose

lipid lipase Glycerol + fatty
acid
starch amylase Maltose
protein protease Peptides +
polypeptide
 Enzyme Examples
 One of the fastest enzymes in the body is
catalase. Catalase breaks down hydrogen
peroxide, a waste product of cell
metabolism, into water and oxygen.
Accumulation of hydrogen peroxide is toxic
so this enzyme performs an important job in
the body.
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 Enzyme
 Enzymes fall into 6 classes based on
function

1. Oxidoreductases: which are involved in oxidation,

reduction, and electron or proton transfer reactions
2. Transferases : catalysing reactions in which groups
are transferred
3. Hydrolases : which cleave various covalent bonds
by hydrolysis
4. Lyases : catalyse reactions forming or breaking
double bonds
5. Isomerases : catalyse isomerisation reactions
6. Ligases : join substituents together covalently.
ENZYME REACTION TYPE EXAMPLES
CLASS
Oxidoreductases Reduction-oxidation Lactate
(redox) dehydrogenase
Transferases Move chemical group Hexokinase
Hydrolases Hydrolysis; bond cleavage Lysozyme
with transfer of functional
group of water

Lysases Non-hydrolytic bond Fumarase

cleavage
Isomerases Intramolecular group Triose phosphate
transfer (isomerization) isomerase

Ligases Synthesis of new covalent RNA polymerase

bond between substrates,
using ATP hydrolysis
 The catalytic efficiency of enzymes is
explained by two perspectives:
 All chemical reactions have energy barriers between
reactants and products.

 The difference in transitional state and substrate is

called activational barrier.
 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.
 CATALYST
 A catalyst is unaltered during the course of a
reaction and functions in both the forward and
reverse directions.
 In a chemical reaction, a catalyst increases the
rate at which the reaction reaches equilibrium.
 For a reaction to proceed from starting material
to product, the chemical transformations of
bond-making and bond-breaking require a
minimal threshold amount of energy, termed
activation energy.
 Generally, a catalyst serves to lower the
activation energy of a particular reaction.
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.
Lock and Key Model
The lock and key model of enzyme action,
proposed earlier this century, proposed that
the substrate was simply drawn into a closely
matching cleft on the enzyme molecule.

Substrate
Products

Enzyme
Induced Fit Model
More recent studies have revealed
Two substrate
molecules are
that the process is much more drawn into the
cleft of the
likely to involve an induced fit. enzyme.

The enzyme or the reactants The enzyme

changes
(substrate) change their shape shape, forcing
slightly. the substrate
molecules to
combine.
The reactants become bound to
enzymes by weak chemical bonds.

This binding can weaken bonds The resulting end

product is
within the reactants themselves, released by the
enzyme which
allowing the reaction to proceed returns to its
normal shape,
more readily. ready to undergo
more reactions.
 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
 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
Energy levels of molecules

Activation energy
of uncatalysed
Initial energy state
Activation energy reactions
of substrates
of enzyme catalysed
reaction

Final energy state of

products

Progress of reaction (time)

 The Michaelis-Menten Equation

 Louis Michaelis and Maude Menten's theory

 It assumes the formation of an enzyme-
substrate complex
 It assumes that the ES complex is in rapid
equilibrium with free enzyme
 Breakdown of ES to form products is assumed
to be slower than 1) formation of ES and 2)
breakdown of ES to re-form E and S
 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.”
k₁ k₂
E + S k₋₁ ES E + P

where:
 S is the substrate
 E is the enzyme
 ES-is the enzyme substrate complex
 P is the product
 K1,K-1 and K2 are rate constants
E + S E S E + P

k1 k2
E+S ES E+P
k-1 k-2
 Michaelis-Menten
Equation
 Michaelis-Menten Equation:

“It is an equation which describes how reaction velocity

varies with substrate concentration.”
Vmax [S]
Vo=
Km+[S]
Where
 Vo is the initial reaction velocity.
 Vmax is the maximum velocity.
 Km is the Michaelis constant = (k₋₁+k₂)/k₁.
 [S] is the substrate concentration.
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.

 However some times heat energy can also increase kinetic

energy to a point that exceed the energy barrier which
results in denaturing of enzymes.
The Effect of Temperature
on Enzyme Action Speeds up all reactions, but

the rate of denaturation of
Optimum
enzymes also increases at
Temperature higher temperatures.
for enzyme Too hot for
Enzyme to  High temperatures break
work
the disulphide bonds
holding the tertiary
structure of the enzyme
Too cold for together thus changing the
Enzyme to shape of the enzyme.
work
 This destroys the active
sites & therefore makes the
enzyme non – functional.
 5- 40oC Temperature
Increase in Activity

40oC - denatures

Rate of Reaction

0 10 20 30 40 50 60

<5oC - inactive
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
 The ph scale measures how acidic or alkaline
a substance is.
 The chemical properties of many solutions
enable them to be divided into 3 categories:
1) Neutral: solutions with a ph of 7.
2) Alkaline: solutions with a ph greater than
7
3) Acidic: solutions with a ph less than 7.
Ph scale
 Effect of pH
 Changing the pH will alter the charge of the enzyme, which
in turn will protein solubility and may change the shape of
the molecule
 Changing the shape or charge of the active site will diminish
its ability to bind to the substrate, halting enzyme function
 Enzymes have an optimum pH and moving outside of this
range will always result in a diminished rate of reaction
 Different enzymes may have a different optimum pH ranges
 trypsin arginase
pepsin
Rate of Reaction (M)

Acidic 2 4 6 8 10
pH Basic
pH and Enzyme Action

Enzymes
• are most active at
optimum pH.

• lose activity in low

or high pH as
tertiary structure is
disrupted.
The Effect of Substrate
Concentration on Enzyme Action
 Assuming that the amount of
enzyme is constant, an
increase in substrate
concentration causes a
diminishing increase in the
reaction rate.

 A maximum rate is obtained

at a certain concentration of
substrate when all enzymes
are occupied substrate (the
rate cannot increase any
further).
 Substrate Concentration
As substrate
concentration increases,
 the rate of reaction
increases (at constant
enzyme concentration).

 the enzyme eventually

becomes saturated, giving
maximum activity.
 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.
Enzymes are used to breakdown In the brewing industry
the starch in grains into biofuels enzymes help a number of
that can be combusted processes including the
Detergents contain clarification of the beer
proteases and lipases to Paper production uses enzymes to
help breakdown protein and helping in the pulping of wood
fat stains Enzymes are widely used in the food
In the textiles industry industry, e.g.
enzymes help in the • fruit juice, pectin to increase the
processing of fibres, juice yield from fruit
e.g. polishing cloth to • Fructose is used as a sweetener, it is
make it appear more converted from glucose by isomerase
shiny • Rennin is used to help in cheese
production
INHIBITION
 Inhibition
o 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

Inhibition

Reversible Irreversible

Non-
Competitive Uncompetitive Mixed
competitive
 REVERSIBLE INHIBITION
o 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).
 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.
 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.
 MIXED INHIBITION
o In this type of inhibition both E.I and E.S.I
complexes are formed.
o Both complexes are catalytically inactive.
Non competitive
inhibition
o It is a special case of inhibition.
o In this inhibitor has the same affinity for either
enzyme E or the E.S complex.
 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.

 SUICIDE INHIBITION :
 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.
 Horse liver dehydrogenase uses Zn- ion for it’s
activation.
 Conversion of an
enzyme precursor

 Specific proteolysis is a common method of

activating enzymes and other proteins in biological
system.

Example:

 The generation of trypsin from trypsinogen leads to

the activation of other zymogens.
ENZYME SPECIFICITY
 ENZYME SPECIFICITY
 Enzymes are highly specific in nature, interacting
with one or few substrates and catalyzing only one
type of chemical reaction.
 Substrate specificity is due to complete fitting of
active site and substrate .
 Example:
 Oxydoreductase do not catalyze hydrolase reactions and
hydrolase do not catalyze reaction involving oxidation and
reduction.
 Types of enzyme specificity
 Enzymes show different degrees of
specificity:

 Bond specificity.
 Group specificity.
 Absolute specificity.
 Optical or stereo-specificity.
 Dual specificity.
 BOND SPECIFICITY
 In this type, enzyme acts on substrates that are
similar in structure and contain the same type of
bond.

Example :
 Amylase which acts on α-1-4 glycosidic ,bond in
starch dextrin and glycogen, shows bond specificity.
 GROUP SPECIFICITY
 In this type of specificity, the enzyme is specific not
only to the type of bond but also to the structure
surrounding it.

Example:
 Pepsin is an endopeptidase enzyme, that hydrolyzes
central peptide bonds in which the amino group
belongs to aromatic amino acids e. g phenyl alanine,
tyrosine and tryptophan.
 SUBSTRATE SPECIFICITY
 In this type of specificity ,the enzymes acts only on
one substrate

Example :

 Maltase , which acts only on maltose, shows

substrate specificity.
 OPTICAL / STEREO-
SPECIFICITY
 In this type of specificity , the
enzyme is not specific to
substrate but also to its optical
configuration
 DUAL SPECIFICITY
 There are two types of dual specificity.
 The enzyme may act on one substrate by two
different reaction types.
Example:
 Isocitrate dehydrogenase enzyme acts on isocitrate
(one substrate) by oxidation followed by
decarboxylation(two different reaction types) .
 Summary: Enzymes
1. Enzymes work very rapidly and help to speed up
biological reactions.
2. Enzymes can be used multiple times (however they do
degrade eventually).
3. Enzymes can work in both directions of a chemical
reaction.
4. Enzymes have optimal temperatures and pH that they
will operate. Beyond these optimum ranges they will
either not work or become denatured
(unfolded/breakdown).
5. Enzymes are usually specific to one particular substrate.