PROCHIRAL SULFIDES AS IN-VITRO PROBES FOR MULTIPLE FORMS OF THE FLAVIN-CONTAINING MONOOXYGENASE

A homologous series of alkyl-substituted p-tolyl sulfides have been sy nthesized and evaluated as in vitro, isozyme-selective substrate probe s for the microsomal flavin containing monooxygenases. Straight-chain and branched-chain alkyl homologs were metabolized to the correspondin g (R)- and (S)-sulfoxides which were analyzed by chiral phase high-per formance liquid chromatography. Initial studies demonstrated that the stereochemical composition of alkyl p-tolyl sulfoxides generated by FM O2, purified from rabbit lung, was a function of the degree of steric crowding about the prochiral center, In contrast, purified rabbit live r FMO1 formed the (R)-sulfoxide from the n-alkyl series of substrates in a highly stereoselective manner (>90%), Similar results were obtain ed with these two rabbit cDNAs expressed in E. coli. In contrast to ra bbit FMO1 and FMO2, a characteristic feature of catalysis by cDNA-expr essed rabbit FMO3 was the lack of stereoselectivity observed for forma tion of methyl p-tolyl sulfoxide. Collectively, these data demonstrate that the stereochemical composition of sulfoxides generated from the n-alkyl series of sulfides is isozyme-dependent. Metabolism of : methy l p-tolyl sulfide by detergent-solubilized hepatic microsomes from a w ide variety of experimental animals yielded predominantly (R)- methyl p-tolyl sulfoxide, which, at least in rabbit liver, is indicative of c atalysis dominated by FMO1, However, solubilized human and macaque liv er preparations catalyzed this reaction in a relatively non-stereosele ctive manner, Macaque liver FMO was purified and the metabolite profil e generated from the n-alkyl p-tolyl sulfides was found to be most sim ilar to rabbit FMO3. Moreover, antibodies directed against macaque liv er FMO selectively reacted with rabbit FMO3 and a microsomal protein e xpressed in adult human, but not fetal human liver, adult human kidney or adult human lung. Therefore, an FMO isoform expressed selectively in adult primate liver has catalytic and immunochemical properties con sistent with its classification in the FMO3 family.