Wild-type enzyme as a reporter of inhibitor binding by catalytically impaired mutant enzymes.
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 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-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.
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DuPont Agricultural Products, Stine-Haskell Research Center, 1094 Elkton Road, Newark, Delaware, 19714-0030, USA.
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D019943:Amino Acid Substitution; D001578:Benzopyrans / Q000494:pharmacology*; D002384:Catalysis; D020134:Catalytic Domain; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006836:Hydro-Lyases / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry
C111394:2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one; D001578:Benzopyrans; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D006836:Hydro-Lyases; C082741:scytalone dehydratase