Enzyme-bound sterols of bovine adrenocortical cytochrome P-450scc

Bovine adrenocortical cytochrome P-450 capable of cleaving the side chain of cholesterol was purified to homogeneity by the method of Suhara et al. [Suhara, K., Gomi, T., Sato, H., Itagaki, E., Takemori, S., & Katagiri, M. (1978) Arch. Biochem Biophys. 190, 290]. The substrate-bound form of the enzyme preparation was shown to contain in addition to cholesterol (1.2-3.0 mol/mol of P-450) 0.4 mol of (22R)-22-hydroxycholesterol, 0.1 mol of (20R,22R)-20,22-dihydroxycholesterol, and a trace amount (0.005 mol) of (20S)-20-hydroxycholesterol per mol of P-450scc. This relatively large concentration of (22R)-22-hydroxycholesterol is in accord with the hypothesis that the major pathway leading to side-chain cleavage proceeds through initial hydroxylation at the 22 position. The presence of these sterols as native constituents of cytochrome P-450scc supports their role as enzyme-bound intermediates in the biosynthesis of pregnenolone from cholesterol.