Wild-type enzyme as a reporter of inhibitor binding by catalytically impaired mutant enzymes

A method for the determination of inhibition constants for catalytically-de bilitated 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 metho d is validated with scytalone dehydratase, the Y50F mutant, and a potent in hibitor. The K-i value for Y50F determined by this method is 0.49 +/- 0.10 nM. The K-i 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-t ype enzyme binds the inhibitor ten-fold less tightly, thus indicating that the hydrogen-bonding interaction between the Y50 hydroxyl group and the inh ibitor (suggested by X-ray crystallography) is weak. The method is most use ful when the catalytic activity of the wild-type enzyme is the most sensiti ve report of inhibitor binding and the mutant enzyme is greatly crippled in catalytic activity. (C) 1999 Academic Press.