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.