Question

How do you find Km values?

Answer 1

Km and Vmax are determined by incubating the enzyme with varying concentrations of substrate; the results can be plotted as a graph of rate of reaction (v) against concentration of substrate ([S], and will normally yield a hyperbolic curve, as shown in the graphs above.
The relationship is defined by the Michaelis-Menten equation:

v = Vmax / (1 + (Km/[S]))

It is difficult to fit the best hyperbola through the experimental points, and difficult to determine Vmax with any precision by estimating the limit of the hyperbola at infinite substrate concentration. A number of ways of re-arranging the Michaelis-Menten equation have been devised to obtain linear relationships which permit more precise fitting to the experimental points, and estimation of the values of Km and Vmax