Isomerization, but not oxidation, is suppressed by a single point mutation, E361Q, in the reaction catalyzed by cholesterol oxidase
Journal Article
·
· Journal of the American Chemical Society
- State Univ. of New York, Stony Brook, NY (United States)
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}-hydroperoxy-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 H{sub 2}O{sub 2} 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. 42 refs., 5 figs., 2 tabs.
- OSTI ID:
- 460051
- Journal Information:
- Journal of the American Chemical Society, Journal Name: Journal of the American Chemical Society Journal Issue: 5 Vol. 119; ISSN JACSAT; ISSN 0002-7863
- Country of Publication:
- United States
- Language:
- English
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