STEREOSPECIFIC, MECHANISM-BASED, SUICIDE INHIBITION OF A CYTOCHROME-P-450 INVOLVED IN ECDYSTEROID BIOSYNTHESIS IN THE PROTHORACIC GLANDS OFMANDUCA-SEXTA

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 mono oxygenase specific to the larval prothoracic gland. A subsequent serie s of unknown ''black-box'' oxidations of 7dC result in the unusual rin g geometry (cis-A/B) and functionality (6-keto-7-ene-14-alpha-ol) of t he ecdysteroids and has been thought to involve the initial formation of alpha-5,6-epoxy-7-dehydrocholesterol (alpha epo7dC). Pharmacologica l studies indicated that conversion of C to 7dC in prothoracic gland h omogenates was strongly and equally inhibited by the isomeric choleste rol substrate analogues alpha- and beta-5,6-epoxycholesterol (alpha- a nd beta epoC) and alpha- and beta-5,6-iminocholesterol (alpha- and bet a iminoC). With respect to the conversion of C to ecdysteroids by disr upted glands, however, the two alpha-isomeric substrates were 10-fold more inhibitory than were their beta-analogues. Indeed, alpha iminoC w as as active as the non-specific pyrimidyl cytochrome-P-450 monooxygen ase inhibitor fenarimol that shows moderate toxicity in many insect sp ecies. All four cholesterol analogues competitively inhibited choleste rol 7,8-dehydrogenation, but only alpha epoC and possibly alpha iminoC were desaturated to Delta(7)-products. Although the K(m)s (and K(i)s) for all the substrates were similar (1.7-6.0 x 10(-5) M), the V-max f or alpha epoC dehydrogenation was eight-fold higher than that of C, ma king it a superior substrate for following this reaction in ecdysteroi dogenic tissues rich in endogenous C. The 7,8-dehydrogenation of alpha epoC and alpha iminoC by prothoracic glands would produce the potenti ally reactive intermediates, alpha epo7dC and alpha imino7dC, respecti vely. They, in turn, could then undergo facile, acid-catalyzed ring-op ening to the allylic-stabilized carbo-cation electrophiles. These very reactive, transient species, if formed in the active site of the mono oxygenase, 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 i minoC are mechanism-based suicide inhibitors of the enzyme catalyzing cholesterol 7,8-dehydrogenation and may be the prototypes of a new cla ss of selective insect control agents.