Wild-Type Enzyme as a Reporter of Inhibitor Binding by Catalytically Impaired Mutant Enzymes

Abstract

A method for the determination of inhibition constants for catalytically-debilitated mutant enzymes is described. The inhibitor is partitioned between the mutant and wild-type enzymes. Catalytic rates of the wild-type enzyme are used as the signal of inhibitor binding to the mutant enzyme. The method is validated with scytalone dehydratase, the Y50F mutant, and a potent inhibitor. The Ki value for Y50F determined by this method is 0.49 ± 0.10 nM. The Ki value determined using the Y50F catalytic report for inhibitor binding in the absence of wild-type enzyme is 0.20 ± 0.030 nM. The wild-type enzyme binds the inhibitor ten-fold less tightly, thus indicating that the hydrogen-bonding interaction between the Y50 hydroxyl group and the inhibitor (suggested by X-ray crystallography) is weak. The method is most useful when the catalytic activity of the wild-type enzyme is the most sensitive report of inhibitor binding and the mutant enzyme is greatly crippled in catalytic activity.