How to determine Vmax and KM

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Title of page! How to determine Vmax and KM

To determine Vmax and KM with Prism:

1. Enter substrate concentrations into the X column and velocity into the Y column (entering replicates if you have them).
2. Click Analyze and choose built-in analyses. Pick nonlinear regression from the list of curves and regressions.
3. Choose more equations. Enter this equation as a new equation, or choose from the enzyme kinetics equation library.

Y = (Vmax * X)/(Km + X)

Variable	Comment
X	Substrate concentration. Usually expressed in mM or mM.
Y	Enzyme velocity in units of concentration of product per time. It is sometimes normalized to enzyme concentration, so concentration of product per time per concentration of enzyme.
Vmax	The maximum enzyme velocity. A reasonable rule for choosing an initial value might be that Vmax equals 1.0 times YMAX. Vmax is expressed in the same units as the Y values.
Km	The Michaelis-Menten constant. A reasonable rule for choosing an initial value might be 0.2*XMAX

Checklist for enzyme kinetics

When evaluating results of enzyme kinetics, ask yourself these questions:

Question

Comment

Was only a small fraction of the substrate converted to product?

The analysis assumes that the free concentration of substrate is almost identical to the concentration you added during the time course of the assay. You can test this by comparing the lowest concentration of substrate used in the assay with the concentration of product created at that concentration.

Is the production of product linear with time?

Check the concentration of product at several times to test this assumption.

Did you use high enough concentrations of substrate?

Calculate the ratio of the highest substrate concentration you used divided by the best-fit value of KM (both in the same concentration units). Ideally, the highest concentration should be at least 10 times the KM.

Are the standard errors too large? Are the confidence intervals too wide?

Divide the SE of the Vmax by the Vmax, and divide the SE of the KM by the KM. If either ratio is much larger than about 20%, look further to try to find out why.

Is product produced in the absence of enzyme?

The analysis assumes that all product formation is due to the enzyme. If some product is produced spontaneously, you'll need to do a fancier analysis to account for this.

Did you pick a time point at which enzyme velocity is constant.

Measure product formation at several time points straddling the time used for the assay. The graph of product concentration vs. time should be linear.

Is there any evidence of cooperativity?

The standard analysis assumes no cooperativity. This means that binding of substrate to one binding site does not alter binding of substrate to another binding pocket. Since many enzymes are multimeric, this assumption is often not true. If the graph of V vs. [S] looks sigmoidal, see Allosteric enzymes.

Comparison of enzyme kinetics with radioligand binding