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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
PROPERTIES OF ENZYMES

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.
PROPERTIES OF ENZYMES

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.
PROPERTIES OF ENZYMES

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.
PROPERTIES OF ENZYMES

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.
PROPERTIES OF ENZYMES

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.
PROPERTIES OF ENZYMES

TEMPERATURE SENSITIVITY
PROPERTIES OF ENZYMES

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.
PROPERTIES OF ENZYMES

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
FACTORS AFFECTING ENZYME ACTIVITY

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.
FACTORS AFFECTING ENZYME ACTIVITY

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.
FACTORS AFFECTING ENZYME ACTIVITY

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.
FACTORS AFFECTING ENZYME ACTIVITY

SUBSTRATE CONCENTRATION
FACTORS AFFECTING ENZYME ACTIVITY

SUBSTRATE CONCENTRATION
Michaelis-Menten Equation
• Michaelis-Menten equation is a rate equation for
reaction kinetics in enzyme catalysed reaction
• Written as
Vmax(S)
V = ---------------
Km + S
FACTORS AFFECTING ENZYME ACTIVITY

SUBSTRATE CONCENTRATION
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.
FACTORS AFFECTING ENZYME ACTIVITY

SUBSTRATE CONCENTRATION
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.
FACTORS AFFECTING ENZYME ACTIVITY

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)
FACTORS AFFECTING ENZYME ACTIVITY

SUBSTRATE CONCENTRATION
Enzyme kinetics & Km value

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
FACTORS AFFECTING ENZYME ACTIVITY

SUBSTRATE CONCENTRATION
Enzyme kinetics & Km value
• 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
FACTORS AFFECTING ENZYME ACTIVITY

SUBSTRATE CONCENTRATION
Enzyme kinetics & Km value
V = V max (S)
Km + (S)

Measured velocity (V) is equal to ½ Vmax.


So,
V max (S)
½ V max = Km + (S)
FACTORS AFFECTING ENZYME ACTIVITY

SUBSTRATE CONCENTRATION
Enzyme kinetics & Km value

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


V max

Km + (S) = 2 (S)

Km = (S)

K stands for constant & M stands for Michaelis


FACTORS AFFECTING ENZYME ACTIVITY

SUBSTRATE CONCENTRATION
Effect of enzyme concentration on Km
FACTORS AFFECTING ENZYME ACTIVITY

SUBSTRATE CONCENTRATION
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
FACTORS AFFECTING ENZYME ACTIVITY

SUBSTRATE CONCENTRATION
LINEWEAVER-BURK PLOT
FACTORS AFFECTING ENZYME ACTIVITY

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.
FACTORS AFFECTING ENZYME ACTIVITY

EFFECT OF TEMPERATURE
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.


FACTORS AFFECTING ENZYME ACTIVITY
EFFECT OF TEMPERATURE
FACTORS AFFECTING ENZYME ACTIVITY

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.
FACTORS AFFECTING ENZYME ACTIVITY

EFFECT OF pH
• Pepsin (optimum pH 1-2); Alkaline phosphatase (optimum pH 9-
10) & Acid phosphatase (4-5)
FACTORS AFFECTING ENZYME ACTIVITY

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
FACTORS AFFECTING ENZYME ACTIVITY

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.
FACTORS AFFECTING ENZYME ACTIVITY

EFFECT OF ACTIVATORS
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+)
FACTORS AFFECTING ENZYME ACTIVITY

EFFECT OF ACTIVATORS
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)
FACTORS AFFECTING ENZYME ACTIVITY

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.
FACTORS AFFECTING ENZYME ACTIVITY

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.