Biosynthesis of Pregnenolone from Cholesterol by Mitochondrial Enzymes of Bovine Adrenal Cortex: The Question of the Participation of the 20(22)‐Olefins and 20,22‐Epoxides of Cholesterol

The two isomeric 20(22)‐olefins and the four isomeric 20,22‐epoxides of cholesterol have been examined as possible intermediates in the conversion of cholesterol to pregnenolone both by crude extracts of acetone‐dried bovine adrenal cortex mitochondria, and by a partially purified cytochrome P‐450 specific for the side chain cleavage (P‐450_SCC). The quantities of pregnenolone (measured by radioimmunoassay) formed from these putative intermediates and from cholesterol, (20S)‐20‐hydroxycholesterol, (22R)‐22‐hydroxycholesterol, and (20R,22R)‐20,22‐dihydroxycholesterol have been compared. In both crude and purified systems, the conversion of the monohydroxycholesterols is somewhat more rapid than that of cholesterol. However, the dihydroxycholesterol is a very much more efficient precursor of pregnenolone. Whereas small amounts of pregnenolone were formed from the four 20,22‐epoxycholesterols, and slightly larger amounts from the two 20(22)‐didehydrocholesterols in the crude enzyme systems, almost no conversion of these six compounds was observed with the purified system containing cytochrome P‐450_SCC, adrenoxin, adrenodoxin reductase, and an NADPH‐generating system. It is concluded that the olefins and epoxides are not obligatory intermediates in the conversion of cholesterol to pregnenolone, and the conversions of cholesterol olefins and epoxides observed by us and by others may reflect the presence of extraneous enzymes such as double bond reductases and epoxide hydratases which are unrelated to pregnenolone biosynthesis. The relative effectiveness of the cholesterol olefins and epoxides, as well as 20‐isocholesterol in inhibiting the formation of pregnenolone from [4‐¹⁴C]cholesterol has been determined. The compounds which have the same configuration as cholesterol at C‐20 are more potent inhibitors of this conversion than the olefins in which the C‐21, C‐20, C‐22, C‐23 carbon system is planar, or than those compounds which have the opposite configuration to cholesterol at C‐20 (i.e. 20‐isocholesterol, (20S,22S)‐20,22‐epoxycholesterol and (20S,22R)‐20,22‐epoxycholesterol). The enzyme appears to be more fastidious in its steric requirement at C‐20 than at C‐22. Since the olefins and epoxides are stereochemically rigidified around the C‐20 to C‐22 bond, these inhibitor studies suggest that the natural conformation of cholesterol on the side chain cleavage enzyme system is such that the side chain is fully extended and thus closely resembles the conformation of cholesterol in the crystal state.