GraphPad Curve Fitting Guide

Equation: Substrate inhibition

Equation: Substrate inhibition

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Equation: Substrate inhibition

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At high concentrations, some substrates also inhibit the enzyme activity. Substrate inhibition occurs with about 20% of all known enzymes. It happens when two molecules of substrate can bind to the enzyme, and thus block activity.

Step by step

Create an XY data table. Enter substrate concentration into the X column, and enzyme activity into the Y columns. If you have several experimental conditions, place the first into column A, the second into column B, etc.

After entering data, click Analyze, choose nonlinear regression, choose the panel of enzyme kinetics equations, and choose Substrate inhibition.


Y=Vmax*X/(Km + X*(1+X/Ki))


Vmax is the maximum enzyme velocity, if the substrate didn't also inhibit enzyme activity, expressed in the same units as Y.

Km is the Michaelis-Menten constant, expressed in the same units as X. It describes the interaction of substrate and enzyme in the absence of inhibitor.

Ki is the dissociation constant for substrate binding in such a way that two substrates can bind to an enzyme. It is expressed in the same units as X.

Note: Prism 7 uses a different (improved) set of rules for initial values than did Prism 6.


Equation 5.44, in RA Copeland, Enzymes, 2nd edition, Wiley, 2000.