Enzyme Technology

Introduction
Biological catalyst
protein, higher MW
specific

LOCK AND KEY MODEL

3D structure of Protein

Properties of enzymes
accelerate rate of reaction
does not alter reaction equilibrium
high catalytic power
work over moderate range of
temp,pH,press.
specificity
regulation of enzyme activity by
small ions or molecules

General features

folded polypeptide
one
or
more
subunit
with non protein
group
called
cofactor
different molecular
forms but catalyze
same
reaction
called isoenzyme

Enzyme catalysis (proximity

and orientation effect)
Chemical reactions go faster when the reactants are in
proximity, that is, near each other. In solution or in the gas
phase, this means that increasing the concentrations of
reacting molecules, which raises the number of collisions,
causes higher rates of reaction. Enzymes, which have
specific binding sites for particular reacting molecules,
essentially take the reactants out of dilute solution and
hold them close to each other. This proximity of reactants
is said to raise the effective concentration over that of
the substrates in solution, and leads to an increased
reaction rate.
Enzymes not only bring substrates and catalytic groups
close together, they orient them in a manner suitable for
catalysis as well

Comparison of calytsed and

uncatalysed reaction

ENZYME KINETICS

Developed by V.C.R. Henri in 1902 and

by L.Michalis and M.L.Menten in 1913
also known as Michalis Menten
kinetics or saturation kinetics
An enzyme solution has a fixed
number of active sites to which
substrate can bind. At high substrate
concentrations, all these sites may be
occupied by substrates or enzyme is
saturated.

The ES complex is established rather rapidly and the

rate of the reverse reaction of the second step is
negligible.
The assumption of an irreversible second reaction
often holds only when product accumulation is
negligible at the beginning of the reaction.
Two major approaches
1. rapid equilibrium
2. quasi-steady state

Michaelies-Menten/rapid equilibrium
Approach(1913)

The balance of equilibrium between free

components and enzymesubstrate was
assumed to occur quickly, compared
with the formation of product ,so that
k2 can be neglected and ES depends on
k1 & k-1.

Integral form of
Michaelies-Menten

Steady state
Approximation(1925)

the total enzyme concentration and

the concentration of the intermediate
complex do not change over time.
the quasi-steady-state assumption (or
pseudo-steady-state
hypothesis),
namely that the concentration of the
substrate-bound
enzyme
([ES])
changes much more slowly than those
of the product ([P]) and substrate
([S]).

Michaelis constant
The Michaelis constant is an approximation
of the affinity of the enzyme for the
substrate based on the rate constants
within the reaction, and it is numerically
equivalent to the substrate concentration
at which the rate of conversion is half of
vmax. A small KM indicates high affinity, and a
substrate with a smaller KM will approach
vmax more quickly. Very high [S] values are
required to approach vmax, which is reached
only when [S] is high enough to saturate
the enzyme.
KM is expressed in units of concentration,
usually in Molar units.

Determination of Rate parameters

Experimental data are obtained

from initial rate experiments.
The batch reactor is charged
with known amount of substrate
and enzyme
The product or substrate conc.
is plotted against time
The initial slope of the curve is
estimated.
Rate depends on the value of Eo
and So.
Many experiments can be used
to generate the plot as shown.

Evaluation of Kinetic parameters

Linear Burk Method

Eadie-hofstee

Hanes Woolf Method