Stereospecific, mechanism-based, suicide inhibition of a cytochrome P 450 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-P 450, 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-α-ol) of the ecdysteroids and has been thought to involve the initial formation of α-5,6-epoxy-7-dehydrocholesterol (αepo7dC). Pharmacological studies indicated that conversion of C to 7dC in prothoracic gland homogenates was strongly and equally inhibited by the isomeric cholesterol substrate analogues α- and β-5,6-epoxycholesterol (α- and βepoC) and α- and β-5,6-iminocholesterol (α- and βiminoC). With respect to the conversion of C to ecdysteroids by disrupted glands, however, the two α-isomeric substrates were 10-fold more inhibitory than were their β-analogues. Indeed, αiminoC was as active as the non-specific pyrimidyl cytochrome-P 450, monooxy-genase inhibitor fenarimol that shows moderate toxicity in many insect species. All four cholesterol analogues competitively inhibited cholesterol 7,8-dehydrogenation, but only αepoC and possibly αiminoC were desaturated to Δ 7-products. Although the K m (and K i s) for all the substrates were similar (1.7-6.0 × 10 −5 M), the V max for αepoC 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 αepoC and αiminoC by prothoracic glands would produce the potentially reactive intermediates, αepo7dC and αimino7dC, 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 αepoC and probably αiminoC 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.