The putative active site base of cholesterol oxidase from Streptomyces has been removed by site-directed mutagenesis and the mutant enzyme characterized. When glutamate-361 is mutated to a glutamine, the isomerization chemistry catalyzed by cholesterol oxidase is suppressed and the intermediate cholest-5-ene-3 one is isolated. The specific activity for oxidation is 20-fold slower than the wild-type reaction;though the specific activity for isomerization is 10 000-fold slower. Furthermore, incubation of cholest-5-ene-3-one with the E361Q cholesterol oxidase resulted in the production of cholest-4-ene-6 beta-hydroperoxy-3-one (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 alpha-ol-3-one (13%), in addition to cholest-4-ene-3-one (13%). Measurement of reaction Stoichiometry eliminated the possibility that H2O2 or the C4a-hydroperoxy flavin was the oxygenation agent. It is proposed that cholest-4-ene-6-hydroperoxy-3-one is the product of radical chain autoxidation and that cholest-4-ene-3,6-dione and cholest-4-ene-6-ol-3-one are decomposition products of the hydroperoxy steroid radical. The characterization of the E361Q mutant chemistry has illuminated the importance of intermediate sequestration in enzyme catalysis. The mutant enzyme will be used to obtain information about the structure of the enzyme in the presence of the reaction intermediate. Moreover, the altered activity of E361Q cholesterol oxidase will facilitate its application in studies of cell membranes.