You can get by with about 5 or 6 substrate concentrations. Ideally, if you plan to use Lineweaver-Burk analysis, the substrate concentrations should be spaced evenly along the 1/[S] axis, and they should also be grouped both above and below the Km.

Dear Adam B Shapiro,

1) I should use a concentration serious substrate with constant enzyme concentration.

2) Then should find the km Km value of substrate.

Is this correct?

I hope to use 3 inhibitor concentrations of an inhibitor to perform the Lineweaver Burk plot. Moreover, I have 3 inhibitors and limited substrate. Thus, I need to use minimum substrate to optimize the substrate concentration. Hope you can help me.

You should measure the Km of the substrate first, which will help you choose the substrate concentration for the inhibition experiment. Usually, people use a fixed enzyme concentration, but this is not absolutely necessary - you can use different enzyme concentrations if necessary, then divide the rate by the enzyme concentration to account for it. The important points are to measure the initial rate of the reaction, and to make sure the enzyme concentration is much lower than the substrate concentration.

You can measure a Km with a relatively small number of substrate concentrations (5 or 6) as long as they are well chosen to cover the range both below and above the Km. This may take some experimentation if you don't have any idea of the right range to work in. I recommend using 2-fold serial dilutions, meaning that each concentration is half of the next higher one and twice the next lower one.