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Cytochrome P450BM-3 preferentially oxidized fatty acids with terminal double or triple bonds to the omega-2 hydroxylated fatty acids rather than, respectively, to the epoxide or diacid metabolites. The enzyme is inactivated during catalytic turnover of long, terminally unsaturated fatty acids but not by the analogous medium-length fatty acids. Enzyme inactivation by 17-octadecynoic acid and 16-hydroxy-17-octadecynoic acid is due to alkylation of the prosthetic heme group to given an adduct tentatively identified as N-(2-oxo-3-hydroxy-17-carboxyheptadecyl)protoporphyrin IX by its chromatographic and spectroscopic properties. Catalytic turnover of 17-octadecenoic acid also results in heme modification. Fatty diacid monoethyl thioesters are introduced as a new class of irreversible inhibitors that exploit the omega-2 oxidation specificity of cytochrome P450BM-3. Catalytic oxidation of the monoethyl thioesters of dodecanedioic and hexadecanedioic acids results in enzyme inactivation and formation of the parent diacids as metabolites. Limited tryptic digestion of the enzyme after incubation with the monoethyl thioester of [14C]hexadecanedioic acid shows that the inactivating agent binds covalently to both the heme and flavin domains. This finding, and the observation that glutathione prevents inactivation of the enzyme by the monoethyl thioesters, indicate that a diffusible metabolite, probably the sulfoxide, is responsible for enzyme inactivation. The strong preference for omega-2 allylic or propargylic hydroxylation over terminal pi-bond oxidation is opposite to the usual cytochrome P450 pattern and requires that the enzyme actively suppress terminal pi-bond oxidation. The inference that the enzyme binds and sequesters the terminal carbon in a lipophilic pocket is consistent with the crystal structure of the hemoprotein domain of cytochrome P450BM-3.

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