Competitive inhibitors

If an inhibitor binds reversibly to the same site as the substrate, the inhibition will be competitive. Competitive inhibitors are common in nature.

One way to measure the effect of an inhibitor is to measure enzyme velocity at a variety of substrate concentrations in the presence and absence of an inhibitor. As the graph below shows, the inhibitor substantially reduces enzyme velocity at low concentrations of substrate, but doesn't alter velocity very much at very high concentrations of substrate.

As the graph above shows, the inhibitor does not alter Vmax, but increases the observed KM (concentration of substrate that produces half-maximal velocity, in the presence of a competitive inhibitor). The observed KM is defined by the following equation, where Ki is the dissociation constant for inhibitor binding (in the same concentration units as [Inhibitor]):

If you have determined the Km plus and minus a single concentration of inhibitor, you can rearrange that equation to determine the Ki.

You'll get a more reliable determination of Ki if you determine the observed Km at a variety of concentrations of inhibitor. Fit each curve to determine the observed Km. Enter the results onto a new table, where X is the concentration of inhibitor, and Y is the observed Km. If the inhibitor is competitive, the graph will be linear. Use linear regression to determine the X- and Y-intercepts. The Y-axis intercept equals the KM and the X-axis intercept equals the negative Ki.

Another experimental design is to measure enzyme velocity at a single concentration of substrate with varying concentrations of a competitive inhibitor. The results will look like this.

The concentration of competitor that reduces enzyme velocity by half is called the EC50 or IC50. Its value is determined by three factors:

	•	The dissociation constant for binding of inhibitor to enzyme, the Ki. If the Ki is low (the affinity is high), the EC50 will be low. The subscript i is used to indicate that the competitor inhibited enzyme activity. It is the concentration of the competitor that will bind to half the enzyme sites at equilibrium in the absence of substrate or other competitors.

	•	The concentration of the substrate. If you choose to use a higher concentration of substrate, it will take a larger concentration of inhibitor to compete for 50% of the activity.

	•	The KM. It takes more inhibitor to compete for a substrate with a low KM than for a substrate with a high KM.

To determine the Ki with Prism (from data collected with a single concentration of substrate):

1. Enter data with X equal to logarithm of inhibitor concentration and Y equal to velocity.

2. Press Analyze, and choose built-in analyses. From the curves section, choose nonlinear regression.

3. Choose the one-site competitive binding equation.

4. If you want to fix the top and bottom plateaus to constant values, click the Constants button and enter the values. Most often, you will fix the bottom plateau to zero, assuming that there is no enzyme velocity at maximum concentrations of inhibitor.

5. On the nonlinear regression dialog, choose the option "Ki from IC50". This option is usually used for radioligand binding studies, so enter the KM where it asks for the Kd, and enter substrate concentration where it asks for radioligand concentration. Enter both in mM (or any concentration units; only the ratio matters). Enter concentrations, not the logarithm of concentrations.

Inhibitors that are not competitive

If an inhibitor binds to a site on the enzyme distinct from the site that binds substrate, the inhibition cannot be overcome by increasing the concentration of substrate. The inhibition is not competitive, and the inhibitor decreases the observed Vmax (and may also increase the observed KM). Consult an advanced text on enzyme kinetics for information about non-competitive, uncompetitive, and mixed inhibition.

Competitive and non-competitive inhibitors bind reversibly. An inhibitor that binds covalently to irreversibly inactivate the enzyme is called an irreversible inhibitor or inactivator.