Experiment : Effect of substrate concentration - Michealis – Menten kinetics

Principle

Enzyme kinetics is the study of rates of reactions catalyzed by enzymes. This

information will help in understanding the mechanism of action of an enzyme, how its
activity is controlled in the cell and how the activity can be increased or decreased. Michealis
– Menten kinetics is applicable for many enzymes, but it is valid only when the enzyme
concentration is less than the substrate concentration and when the enzyme is not allosteric.
The Michealis – Menten equation shows how the rate of the reaction,V varies with the
substrate concentration.

V = Vmax S / Ks + S

Here Vmax is the maximum rate of the reaction or maximum velocity and Ks is
the saturation constant and is given by the value of S at half the Vmax value

To estimate the Vmax and Ks values, researches have developed linearizations such as
Lineweaver – Burk plot and Eadiee- Hoftsee plot.The Lineweaver – Burk plot or double
reciprocal plot is a common way of estimating the above given constants. This is produced by
taking the reciprocal of both sides of the Michaelis- Menten equation. This produces a
straight line in the form of the equation - y = mx + C, where m is the slope given by Ks / Vmax
and the intercept being 1 / Vmax
 Fig. Lineweaver Burk plot

Procedure

1. A range of volumes( 0 – 2ml, 0.1,0.3,0.5,0.7,1.0,1.2) of the standard starch (4

%) solution is pippetted out in test tubes.
2. Volume is made upto 2 ml with ph 6 sodium phosphate buffer.
3. 0.5 ml of the given Amylase solution is added to each of the test tubes and
incubated for 15 min at room temperature.
4. The reaction is stopped by adding DNS and the amount of glucose present in the
test tubes is measured by using DNS method
5. V, the rate of the reaction is given by dividing the amount of glucose produced
by incubation time or by estimating Activity of Amylase.
6. The plot of V vs S and 1/V vs 1/S is drawn and the Michealis – Menten
parameters are estimated.
Result :