Stereospecific, mechanism-based, suicide inhibition of a cytochrome P450 involved in ecdysteroid biosynthesis in the prothoracic glands of Manduca sexta

Abstract

The first required step in ecdysteroid (molting hormone) biosynthesis, dietary cholesterol (C) conversion to 7-dehydrocholesterol (7dC) via 7,8-dehydrogenation, is mediated by a microsomal cytochrome-P450 monooxygenase specific to the larval prothoracic gland. A subsequent series of unknown "black-box" oxidations of 7dC result in the unusual ring geometry (cis-A/B) and functionality (6-keto-7-ene-14-alpha-ol) of the ecdysteroids and has been thought to involve the initial formation of alpha-5,6-epoxy-7-dehydrocholesterol (alpha epo7dC). Pharmacological studies indicated that conversion of C to 7dC in prothoracic gland homogenates was strongly and equally inhibited by the isomeric cholesterol substrate analogues alpha- and beta-5,6-epoxycholesterol (alpha- and beta epoC) and alpha- and beta-5,6-iminocholesterol (alpha- and beta iminoC). With respect to the conversion of C to ecdysteroids by disrupted glands, however, the two alpha-isomeric substrates were 10-fold more inhibitory than were their beta-analogues. Indeed, alpha amino C was as active as the non-specific pyrimidyl cytochrome-P450 monooxygenase inhibitor fenarimol that shows moderate toxicity in many insect species. All four cholesterol analogues competitively inhibited cholesterol 7,8-dehydrogenation, but only alpha epoC and possibly alpha iminoC were desaturated to delta 7-products. Although the KmS (and KiS) for all the substrates were similar (1.7-6.0 x 10(-5) M), the Vmax for alpha epoC dehydrogenation was eight-fold higher than that of C, making it a superior substrate for following this reaction in ecdysteroidogenic tissues rich in endogenous C. The 7,8-dehydrogenation of alpha epoC and alpha iminoC by prothoracic glands would produce the potentially reactive intermediates, alpha epo7dC and alpha imino7dC, respectively. They, in turn, could then undergo facile, acid-catalyzed ring-opening to the allylic-stabilized carbo-cation electrophiles. These very reactive, transient species, if formed in the active site of the monooxygenase, would then alkylate either the heme group or the apoprotein of the cytochrome or both, leading to the irreversible inhibition of the enzyme. The present data show that alpha epoC and probably alpha iminoC are mechanism-based suicide inhibitors of the enzyme catalyzing cholesterol 7,8-dehydrogenation and may be the prototypes of a new class of selective insect control agents.