ISOMERIZATION, BUT NOT OXIDATION, IS SUPPRESSED BY A SINGLE-POINT MUTATION, E361Q, IN THE REACTION CATALYZED BY CHOLESTEROL OXIDASE

The putative active site base of cholesterol oxidase from Streptomyces has been removed by site-directed mutagenesis and the mutant enzyme c haracterized. When glutamate-361 is mutated to a glutamine, the isomer ization chemistry catalyzed by cholesterol oxidase is suppressed and t he intermediate cholest-5-ene-3 one is isolated. The specific activity for oxidation is 20-fold slower than the wild-type reaction;though th e specific activity for isomerization is 10 000-fold slower. Furthermo re, incubation of cholest-5-ene-3-one with the E361Q cholesterol oxida se resulted in the production of cholest-4-ene-6 beta-hydroperoxy-3-on e (6%), cholest-4-ene-3,6-dione (32%), cholest-4-ene-6 beta-ol-3-one ( 36%), and cholest-4-ene-6 alpha-hydropesoxy-3-one/cholest-4-ene-6 alph a-ol-3-one (13%), in addition to cholest-4-ene-3-one (13%). Measuremen t of reaction Stoichiometry eliminated the possibility that H2O2 or th e C4a-hydroperoxy flavin was the oxygenation agent. It is proposed tha t cholest-4-ene-6-hydroperoxy-3-one is the product of radical chain au toxidation and that cholest-4-ene-3,6-dione and cholest-4-ene-6-ol-3-o ne are decomposition products of the hydroperoxy steroid radical. The characterization of the E361Q mutant chemistry has illuminated the imp ortance of intermediate sequestration in enzyme catalysis. The mutant enzyme will be used to obtain information about the structure of the e nzyme in the presence of the reaction intermediate. Moreover, the alte red activity of E361Q cholesterol oxidase will facilitate its applicat ion in studies of cell membranes.