Enzymes

CONTENTS
• Definition of Enzyme
• Properties of Enzymes
• Classification of Enzymes
• Factors affecting the Enzyme
• Enzyme unit
DEFINITION :
Enzymes are macromolecular biological catalysts.
Enzymes are known to catalyse more than 5,000

biochemical reaction types.
Most enzymes are proteins, although a few

are catalytic RNA molecules.
PROPERTIES OF ENZYMES
• Catalytic Activity
• Colloidal Nature
• Specificity
• Temperature Sensitivity
• pH Sensitivity
CATALYTIC ACTIVITY
• Enzymes work by weakening bonds which lowers

activation energy.
• The reduction of activation energy (Ea) increases

the amount of reactant molecules that achieve a
sufficient level of energy, such that they reach the
activation energy and form the product.
COLLOIDAL NATURE
• They are colloidal in

nature due to which they
present a large surface
area for reactions to take
place.
• The more enzyme exposed

to substrate or the more
substrate exposed
to enzyme.
SPECIFICITY
• Enzymes are highly specific in nature i.e., a particular
enzyme can catalyse only a particular type of reaction
e.g., the enzyme malic dehydrogenase removes
hydrogen atom from malic acid and not from other keto
acids.
• The enzymes possess active sites which are highly

specific centres composed of varying number and
sequence of amino acids.
SPECIFICITY
• The active site possess a

particular binding site
which complexes only
with specific substrate.
• Thus a suitable substrate

fulfils the requirements
of active site and closely
fixes with it.
TEMPERATURE SENSITIVITY
• Enzymes are inactivated or destroyed at temperatures
considerably below the boiling point of water. At 50°C most
enzymes in a liquid medium are inactivated.
• Slow inactivation takes place even at low temperatures.
Some enzymes can endure temperatures of 100°C for short
periods.
• But dried enzyme extracts can endure temperature of 100°C
to 120°C or even higher. Thus enzymes are thermolabile.
TEMPERATURE SENSITIVITY
pH SENSITIVITY
• pH can have an effect of the state of ionization of acidic or

basic amino acids.
• If the state of ionization of amino acids in a protein is altered
then the ionic bonds that help to determine the 3-D shape of
the protein can be altered.
• This can lead to altered protein recognition or an enzyme
might become inactive.
pH SENSITIVITY
• Changes in pH may not only affect the shape of an enzyme
but it may also change the shape or charge properties of the
substrate so that either the substrate cannot bind to the active
site or it cannot undergo catalysis.
• In general enzymes have a pH optimum .However the
optimum is not the same for each enzyme.
CLASSIFICATION OF ENZYMES
Enzymes are classified according to the type of
reaction they catalyze:
Class Reactions catalyzed
• Oxidoreductases Oxidation-reduction.
• Transferases Transfer groups of atoms.
• Hydrolases Hydrolysis.
• Lyases Add atoms/remove atoms
to/from a double bond.
• Isomerases Rearrange atoms .
• Ligases Use ATP to combine
molecules.
CLASSIFICATION OF ENZYMES
Oxidoreductases
oxidases - oxidize ,reductases – reduce
Transferases
transaminases – transfer amino groups
kinases – transfer phosphate groups
Hydrolases
proteases - hydrolyze peptide bonds
lipases – hydrolyze lipid ester bonds
Lyases
carboxylases – add CO2
hydrolases – add H2O
FACTORS AFFECTING ENZYME ACTIVITY
The contact between the enzyme and substrate is the

most essential pre-requisite for enzyme activity.
1. Enzyme concentration
2. Substrate concentration
3. Temperature
4. Hydrogen ion concentration (pH)
5. Product concentration
6. Presence of activators
7. Time
8. Light & radiation
ENZYME CONCENTRATION
• Rate of a reaction or velocity (V) is directly proportional
to the enzyme concentration, when sufficient substrate is
present.
• Velocity of reaction is increased proportionately with the
concentration of enzyme, provided substrate concentration
is unlimited.
ENZYME CONCENTRATION
Vmax = Kcat (e)
E = enzyme concentration
Kcat = catalytic rate constant.
Kcat (catalytic rate constant) – defined
as the number of substrates molecules
formed by each enzyme molecule in unit
time.
Expressed as moles produced/mol
enzyme/time.
SUBSTRATE CONCENTRATION
• Increase in the substrate concentration gradually
increases the velocity of enzyme reaction within the
limited range of substrate levels.
• A rectangular hyperbola is obtained when velocity is
plotted against the substrate concentration
• Three distinct phases of the reaction are observed in the

graph (A-linear; B-curve; C-almost unchanged.
Michaelis-Menten Equation
• Michaelis-Menten equation is a rate equation for
reaction kinetics in enzyme catalysed reaction
• Written as
Vmax(S)
V = ---------------
Km + S
Michaelis-Menten Plot
• The velocity of enzyme catalysed reactions is altered as the

substrate concentration is increased.
• First order reaction:
At low substrate concentration, velocity increases proportionally as
the concentration of the substrate is increased.
Michaelis-Menten Plot
• Mixed order reaction:
When the concentration of the substrate is further increased (at mid
substrate concentration), the velocity increases, but not
proportionally to substrate concentration.
• Zero order reaction:

At high substrate concentration, the velocity is maximum & is
independent of substrate concentration.
Enzyme kinetics & Km value
The enzyme (E) reacts with substrate (S) to form unstable enzyme-
substrate (ES) complex.
The ES complex is either converted to product (P) or can dissociate
back to enzyme (E) & substrate (S).
Substrate (S) + Enzyme (E)  Enzyme substrate (ES)  Product (P) + Enzyme (E)
K1 K3
E+S ES E+P
K2
K1,K2 & K3 are velocity constants.

Km, Michaelis-menten constant is given by the formula…
K2 + K3
Km =
K1
• Michaelis-menten set up mathematical expressions for the rate of all the three
reactions in the equation.
• V as the initial rate of reaction (velocity).

• S as the initial concentration of the substrate.
• V max as the maximum velocity attained with high substrate concentration
when all the enzyme molecules are occupied.
• Km as Michaelis-menten constant
V = V max (S)
Km + (S)
Measured velocity (V) is equal to ½ Vmax.

So,
V max (S)
½ V max = Km + (S)
Km + (S) = 2V max (S)

V max
Km + (S) = 2 (S)
Km = (S)
K stands for constant & M stands for Michaelis

Effect of enzyme concentration on Km
DOUBLE RECIPROCAL PLOT
• Sometimes it is impractical to achieve high substrate concentrations to
reach the maximal velocity conditions.
• So, ½Vmax or Km may be difficult to determine.
• The experimental data at lower concentrations is plotted as reciprocals.
• The straight line thus obtained is extrapolated to get the reciprocal of
Km.
• Called as Lineweaver-Burk Plot or Double Reciprocal Plot which can
be derived from the Michaelis-Menten equation
LINEWEAVER-BURK PLOT
EFFECT OF TEMPERATURE
• The velocity of enzyme reaction increases when temperature of
the medium is increased; reaches a maximum and then falls (Bell
shaped curve).
• The temperature at which maximum amount of the substrate is
converted to the product per unit time is called the optimum
temperature.
• Temperature is increased, more molecules get activation energy, or
molecules are at increased rate of motion.
• Their collision probabilities are increased and so the reaction
velocity is enhanced.
Temperature Coefficient Q10
• The temperature coefficient (Q10) is the factor by which the rate of
catalysis is increased by a rise in 10°C.
• Generally, the rate of reaction of most enzymes will double by a rise in
10°C.
• When temperature is more than 50°C, heat denaturation and consequent
loss of tertiary structure of protein occurs.
• Activity of the enzyme is decreased.

EFFECT OF pH
• Each enzyme has an optimum pH (usually pH between 6 and 8).

• On both sides of which the velocity will be drastically reduced.
• The graph will show a bell shaped curve
• The pH decides the charge on the amino acid residues at the active
site.
• The net charge on the enzyme protein would influence substrate
binding and catalytic activity.
• Optimum pH may vary depending on the temperature,
concentration of substrate, presence of ions etc.
EFFECT OF pH
• Pepsin (optimum pH 1-2); Alkaline phosphatase (optimum pH 9-
10) & Acid phosphatase (4-5)
EFFECT OF PRODUCT CONCENTRATION
• The accumulation of reaction products generally decreases

the enzyme velocity.
• For certain enzymes, the products combine with the active
site of enzyme and form a loose complex and, thus, inhibit
the enzyme activity.
• In the living system, this type of inhibition is generally
prevented by a quick removal of products formed
EFFECT OF ACTIVATORS
• Some of the enzymes require certain inorganic metallic cations
like Mg2+, Mn2+, Zn2+, Ca2+, Co2+, Cu2+, Na+, K+, for their
optimum activity
• Anions are also needed for enzyme activity e.g. chloride ion for
amylase
• Metals function as activators of enzyme velocity through various
mechanisms combining with the substrate, formation of ES-
metal complex, direct participation in the reaction and bringing a
conformational change in the enzyme.
Two categories of enzymes requiring metals for their activity
 Metal-activated enzymes
 Metalloenzyme
Metal-activated enzymes : The metal is not tightly held by the
enzyme and can be exchanged easily with other ions.
e.g. ATPase (Mg2+ and Ca2+) & Enolase (Mg2+)
Metalloenzyme : These enzymes hold the metals rather
tightly which are not readily exchanged.
e.g. Alcohol dehydrogenase, carbonic anhydrase, alkaline
phosphatase, carboxypeptidase and aldolase contain zinc.
Phenol oxidase (copper)
Pyruvate oxidase (manganese)
Xanthine oxidase (molybdenum)
Cytochrome oxidase (iron and copper)
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 LIGHT AND RADIATION

Exposure of enzymes to ultraviolet,
beta, gamma & X-rays inactivates
certain enzymes due to the
formation of peroxides. e.g. UV
rays inhibit salivary amylase
activity.
ENZYME UNIT
• The enzyme unit (U) is a unit for the amount of a

particular enzyme.
• One U is defined as the amount of the enzyme that produces

a certain amount of enzymatic activity, that is, the amount
that catalyses the conversion of 1 micro mole of substrate
per minute.
• The conditions also have to be specified: one usually takes a
temperature of 25°C and the pH value and substrate
concentration that yield the maximal substrate conversion
rate.
ENZYME UNIT
• The enzyme unit was adopted by the International Union of

Biochemistry in 1964. Since the minute is not an SI unit, the
enzyme unit is discouraged in favour of the katal, the unit
recommended by the General Conference on Weights and
Measures in 1978 and officially adopted in 1999.
• One katal is the amount of enzyme that converts 1 mole of

substrate per second, so
1 U = 1/60 micro katal = 16.67 nano katal.
The enzyme unit should not be confused with the International
Unit (IU), an unrelated measure of biologically active substances.
BIBLIOGRAPHY
www.google.co.in
www.wikipedia.org
www.slideshare.net
www.publishyourarticles.com
www.academic.brooklyn.edu
www.worthington-biochem.com
ACKNOWLEDGEMENT
I owe a great many thanks to a great many people who guided and
supported me during this project. My deepest thanks to my teacher
Mrs. Swathi, for giving me an opportunity to work on this topic.
I thank my institution members and also extend my heartfelt thanks
to my family and well wishers.

Enzymes

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Enzymes

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CONTENTS

Enzymes are macromolecular biological catalysts.

Enzymes are known to catalyse more than 5,000

Most enzymes are proteins, although a few

• Enzymes work by weakening bonds which lowers

• The reduction of activation energy (Ea) increases

• They are colloidal in

• The more enzyme exposed

• The enzymes possess active sites which are highly

• The active site possess a

• Thus a suitable substrate

• pH can have an effect of the state of ionization of acidic or

The contact between the enzyme and substrate is the

• Three distinct phases of the reaction are observed in the

• The velocity of enzyme catalysed reactions is altered as the

• Zero order reaction:

K1,K2 & K3 are velocity constants.

• V as the initial rate of reaction (velocity).

Measured velocity (V) is equal to ½ Vmax.

Km + (S) = 2V max (S)

K stands for constant & M stands for Michaelis

• Activity of the enzyme is decreased.

• Each enzyme has an optimum pH (usually pH between 6 and 8).

EFFECT OF PRODUCT CONCENTRATION

• The accumulation of reaction products generally decreases

• Variations in the time of the reaction are

EFFECT OF LIGHT AND RADIATION

• The enzyme unit (U) is a unit for the amount of a

• One U is defined as the amount of the enzyme that produces

• The enzyme unit was adopted by the International Union of

• One katal is the amount of enzyme that converts 1 mole of

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