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