ENZYME

Prepared by
Sourav Debnath
Associate Professor
Department of Biochemistry and Food Analysis
Patuakhali
12/20/2022 Science and Technology University 1
Biomolecules
An organic compound normally present as an essential
component of living organism.

Biomolecules include macromolecules like proteins,

carbohydrates, lipids and nucleic acids.

Biomolecules consists mainly of carbon and hydrogen with

nitrogen, oxygen, sulphur, and phosphorus.

Biomolecules are very large molecules of many atoms, that

are covalently bound together.

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Classes of Biomolecules
•Carbohydrates
•Proteins
•Lipids
•Enzyme
•Nucleic acids

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 ENZYME
Definition
Enzymes (enG = in; zymeG = yeast) may be defined as
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.

Enzymes are protein biocatalysts which are produced

by living cell but are capable of acting independently
in the cell.

E+S ES E+P
 Enzyme Action

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 ENZYME
Properties of enzyme as catalyst
1. Enzymes are very active positive catalyst. They have a
extraordinary catalytic power which is generally greater
than synthetic catalyst. This catalytic reaction is 107
times faster than uncatalysts reaction.
2. Enzymes have a high degree of specificity for their
structure. A specific enzyme always acts on specific
structure.
3. Enzyme can not start any reaction. It can speed up or
increase the reaction.
4. They accelerate specific reaction without any
formation of by product.
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Properties of enzyme as protein
In 1926 the truth that “All enzymes are protein” was
established by James sumner. The following properties of
enzymes proof that all enzymes are protein.
1. Enzymes are nondializable and molecular weight of enzyme
is high.
2. Like protein the fundamental structure of enzyme is amino
acids.
3. Enzymes give biuret colour test like protein.
4. Enzymes show primary, secondary and tertiary structures
like protein.
5. Enzymes like protein denatured by protein denaturants like
urea, heat, strong acid etc.
6. Enzymes contain peptide bond like protein.
So, we can say all enzymes are protein.
Enzymes have extraordinary catalytic power but proteins do
not have. So, all proteins are not enzyme.
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 CLASSIFICATION OF ENZYME
The International Union of Biochemistry (lUB) system of enzyme
classification

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1. Oxidoreductases : Enzymes involved in oxidation-reduction
reactions.
AH2 + B A + BH2

Malic acid +NAD oxaloacetic acid +NADH + H+

dehydrogenase
2. Transferases: Enzymes that catalyze the transfer of functional
groups.
AX + B A + BX

D- Glucose + ATP Glucose-6- phosphate + ADP

hexokinase
3. Hydrolases : Enzymes that bring about hydrolysis of various
compounds.
AB + H2O AH + BOH

Fat Glycerol + Fatty acid

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4. Lyases:
Enzymes specialized in the addition or removal.
AB + XY AX - BY
Oxaloacetate + acetyl CoA Citrate
Citrate synthase
5. lsomerases: Enzymes involved isomerization reactions.
A A'
Glucose-6- phosphate Fructose -6- phosphate
Hexose phosphate isomerase

6. Ligases: Enzymes catalyze the synthetic reactions (Greek: ligate-

to bind) where two molecules are joined together and ATP is used.
ATP ADP
A+B AB

Glutamic acid +ATP +NH3 Glutaminne + ADP + Pi

Glutamin synthetase

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 ENZYME SPECIFICITY
Enzyme specificity
Enzymes are highly specific in their action when
compared with the chemical catalysts. The
occurrence of thousands of enzymes in the
biological system might be due to the specific
nature of enzymes. Three types of enzyme
specificity are well-recognized
1. Stereospecificity or Optical specificity
2. Reaction specificity
3. Substrate specificity
3a. Absolute substrate specificity
3b. Relative substrate specificity
3c. Broad specificity
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Stereospecificity or optical specificity:
Stereoisomers are the compounds which have the same
molecular formula, but differ in their structural
configuration. If an enzyme is specific towards a
particular sterio-chemical configuration of substrate,
this enzyme is called Stereospecificity or optical
specificity.

e.g. L-amino acid oxidase and D-amino acid oxidase act

on L- and D-amino acids respectively.

Hexokinase acts on D-hexoses

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Reaction specificity: The same substrate can undergo
different types of reactions, each catalyzed by a separate
enzyme and this is referred to as reaction specificity.
An amino acid can undergo transamination
(Amionotransferase),oxidativedeamination,(Dehydrogenese)
decarboxylation,(Decarboxylase) etc. The enzymes
however, are different for each of these reactions

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3. Substrate specificity: The substrate
specificity varies from enzyme to enzyme. lt
may be either absolute, relative or broad.

3a. Absolute substrate specificity

3b. Relative substrate specificity
3c. Broad specificity

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3a. Absolute substrate specificity: If an enzymes act
only on one substrate it is then said to exhibit absolute
substrate specificity.
e.g. glucokinase acts on glucose to give glucose -6 -
phosphate, urease cleaves urea to ammonia and carbon
dioxide.

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3b. Relative substrate specificity: If an enzyme is specific
towards the specific group or a bond of the substrate then it is said
to exhibit relative substrate specificity.

The action of trypsin is a good example for group specificity.

Trypsin hydrolyses peptide linkage involving arginine or lysine.
Chymotrypsin cleaves peptide bonds attached to aromatic amino
acids (phenylalanine, tyrosine and tryptophan).

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3c. Broad specificity:
Some enzymes act on closely related substrates which is
commonly known as broad substrate specificity.

e.g. Hexokinase acts on glucose, fructose and not on

sucrose.

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FACTORS AFFECTING ENZYME ACTIVITY

1. Concentration of enzyme
2. Effect of temperature
3. Effect of pH
4. Effect of activators
5. Effect of time
6. Concentration of substrate

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Concentration of enzyme
As the concentration of the enzyme is increased, the
velocity of the reaction proportionately increases.

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Effect of temperature
Velocity of an enzyme reaction increases with increase in
temperature up to a maximum and then declines. A bell-
shaped curve is usually observed. The optimum
temperature for most of the enzymes is between 400C-
450C. However, a few enzymes (e.g. venom
phosphokinases) are active even at 1000C. Some plant
enzymes like urease have optimum activity around 600C.

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Effect of pH
Increase in the hydrogen ion concentration (pH)
considerably influences the enzyme activity and a bell-
shaped curve is normally obtained. Each enzyme has an
optimum pH at which the velocity is maximum. Below
and above this pH, the enzyme activity is much lower
and at extreme pH, the enzyme becomes totally
inactive.

Most of the enzymes of higher organisms show

optimum activity around neutral pH (6-8). There are,
however many exceptions like pepsin (1-2), acid
phosphatase (4-5) and alkaline phosphatase (10-11).
Enzymes from fungi and plants are most active in acidic
pH (4-6).
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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 activators
Some of the enzymes require certain inorganic
metallic cations like Mg2+, Mn2+,zn2+, ca2+, co2+, cu2+,
Na+, K+ etc for their optimum activity. Rarely, anions
are also needed for enzyme activity e.g. chloride ion
(Cl- for amylase.
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 Concentration of substrate
Increase in the substrate concentration, gradually
increases the velocity of enzyme reaction within
the limited range of substrate levels. A rectangular
hyperbolic curve is obtained when velocity is
plotted against the substrate concentration

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Michaelis-Menten equation
The velocity of enzyme reaction is significantly affected by
substrate concentration. So there is close relationship between
them. The first significance explanation of the relationship is
provided by Michaelis-Menten equation. This is a mathematical
equation and quantitative relationship which fulfill the
requirement of rectangular hyperbolic curve.

The equation is:

Vmax × [S]
V=
Km +[S]
Where,
V = Measured velocity,
Vmax = Maximum velocity,
S = Substrate concentration,
K = Michaelis- Menten constant.
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Km: km is the Michaelis- Menten constant. It is defined as the
substrate concentration (expressed in moles/l) to produce half-
maximum velocity in an enzyme catalyzed reaction. It indicates
that half of the enzyme molecules (i.e. 50%) are bound with the
substrate molecules when the substrate concentration equals the
Km value.
Significance of km
1. Km is the substrate concentration at which the velocity of the reaction is half
of its maximum velocity. So km is a measured of substrate concentration.
2. Km indicates the affinity of ES (enzyme-substrate) complex.
3. A high km indicates the weak affinity between the enzyme and substrate.
4. A low km indicates the strong affinity between enzyme and substrate.
5. Km is a measure of the strength of ES (enzyme substrate) complex.
6. It is important to the mode of action of catalyzed reaction.

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Proof that, Km is the substrate concentration at which the velocity of the
reaction is half of its maximum velocity. ( i.c km = [S] when, V =1/2 Vmax )
We know the Michaelis-Menten
Vmax × [S]
V =
Km + [S]

Vmax × [S]
=> 1/2 Vmax = [V = 1/2 Vmax ]
Km + [S]
=> 1/2 Vmax (Km + [S]) = Vmax × [S]
=>1/2 (Km + [S]) = [S]
=> Km + [S] = 2[S]
=> Km = 2[S] - [S]
=> Km = [S]

So, it is proved that Km is equal to substrate concentration when the initial

velocity is half of the maximum velocity.
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 COFACTORS
Definition:
A large number of enzymes require the presence of an
additional component (non protein) in order to carry
out their catalytic functions and are combination with
the active site of the enzyme. These additional
components are called cofactor.

Cofactors may be divided into three groups

1. Coenzyme
2. Prosthetic group
3. Metals ions/ metal activators

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Coenzyme:
When the molecule is loosely bound to the enzyme and
dialyzable is called coenzyme. Coenzymes are vitamin in
nature.
e.g. NAD+,TPP
Prosthetic group:
When the additional group is tightly bind by covalent
linkage to the enzyme is called prosthetic group.
e.g FAD
Metals ions/ Metal activators:
Any metal ions are sometime bound with the enzyme
molecules is called metal activators.
e.g Cu++ divalent = tightly bound, Na+ monovalent =
loosely
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Difference between enzyme and coenzyme:

Sl.no. Enzyme Coenzyme

01. Enzymes are protein Coenzymes are vitamin in

in nature. nature.
02. Enzymes are Coenzymes are
macromolecules. micromolecules.
03. Not dialyzable Dialyzable
04. They are not thermo They are thermo (heat)
(heat) stable. stable.
05. They are really They are really catalyst of
catalyst. catalyst.
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Holoenzyme:
The enzyme and cofactor (Non protein) part is called
holoenzyme

Apoenzyme:
If the cofactor is removed, the enzyme loss the catalytic
activity, such inactive protein part is called apoenzyme.
.

Substrate:
It is a substance on which the
enzyme act is called substrate.
e.g Maltose is substrate for maltase
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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. The active
site has a unique geometric shape that is
complementary to the geometric shape of a substrate
molecule. similar to the fit of puzzle pieces. This
means that enzymes specifically react with only one or
a very few similar compounds

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 Mechanism of Enzyme Action

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❖ The basic mechanism by which enzymes catalyze chemical
reactions begins with the binding of the substrate (or
substrates) to the active site on the enzyme.

❖ The binding of the substrate to the enzyme causes changes

in the distribution of electrons in the chemical bonds of the
substrate and ultimately causes the reactions that lead to
the formation of products.

❖ The products are released from the enzyme surface to

regenerate the enzyme for another reaction cycle.
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 Fischer’s Lock and Key Theory

Enzymes are very specific and it was suggested

by Fischer in 1890.

The lock and key model assumes that the active

site of the enzyme and the substrate are equal
shaped. It supposes that the substrate fits perfectly
into the active site of the enzyme.

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•According to this model, the shape of the active site of
the enzyme is complementary to the shape of substrate
molecules. I.e. the substrate is like a key whose shape is
complementary to the enzyme which is supposed to be
locked and they fit perfectly.

•Enzymes catalyze only those substrates which fit

perfectly on the active site of that enzyme.

•Once the product is formed, they no longer fit into the

active site and escape into the surrounding medium.

•According to the lock and key model, enzymes behave as

rigid molecules. However, most enzymes are globular and
are flexible with varying shape.
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 Induced fit model
•In 1959, Koshland suggested a modification to the ‘Lock
and Key’ hypothesis which is known as ‘Induced
fit’ hypothesis.

This model suggests that an enzyme, when binding

with its substrate, optimizes the interface through
physical interactions to form the final complex structure.

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•The amino acids which make up the active site are
molded into precise shapes which enable the enzyme to
perform its catalytic function most efficiently.

•For instance, a suitable analogy to describe the Induced

fit model would be that of a hand changing the shape of
the glove as the individual put on the glove. Therefore, in
this case, the glove is the active site of an enzyme and
the hand is the substrate.

•However, in some cases, the substrate molecules change

slightly as it enters the active site before binding.

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