Question

Why is it important that there is a “satisfactory linear portion” in the enzyme assay?

Answer 1

In enzyme assays, the data is plotted as Time versus product formed to get the enzyme reaction progress curve. The data obtained during initial stages of reaction is linear, while in later stages the line becomes non linear due to the depletion of the substraate and the results obtained from this non linear part will yield erronaeous results.

The velocity of the enzyme reaction can only determined from the satisfactory linear portion which is extra-polated linearly. The equation obtained from the linear line is in the form of y=mx + c whose slope and intercept can be used of calculation of velocity of the reaction, substrate concentration. When there is non linearity the regression (r2) of the line is low and hence cant be used for conclusive calculation of the experimental results

Alternatively Lineweaver Buke (double reciprocal plot) plot can also be used where, a graph is plotted between 1/[S] (on X axis) and I/V (on y axis) to give a straight line which can be conveniently used for calculation of Enzyme velocity and Micahelis menton constant. The slope of line is Km/Vmax and the intercept of the line 1/vmax. The results of this graph are more accurate when compared to a curvilinear plot as seen in the first case.