SIDE-CHAIN CLEAVAGE OF CHOLESTEROL ESTERS BY HUMAN CYTOCHROME P-450(SCC)

In order to define the substrate binding site of human cytochrome P-45 0(scc) in the vicinity of the 3 beta-hydroxyl group of cholesterol, we have tested the ability of the cytochrome to cleave the side chain of a range of cholesterol esters and cholesterol methyl ether. Using a T ween-20 detergent reconstituted system we found that cholesterol sulph ate could undergo side-chain cleavage with the same turnover number (k (cat)) as that for cholesterol, but with a higher K-m. Cholesterol met hyl ether underwent side-chain cleavage to pregnenolone methyl ether w ith k(cat) and K-m values 30% of those for cholesterol. Cholesterol fa tty acid esters with acyl chain lengths of up to four carbons were abl e to undergo side-chain cleavage with K-m values similar to those for cholesterol, but k(cat) values only 12-23% of those for cholesterol. T urnover numbers decreased as the acyl group length increased beyond fo ur carbons, although some activity was still detected with cholesterol palmitate as substrate. Analysis of bovine cytochrome P-450(scc) reve aled that it could also cleave the side chain of acyl and sulphate est ers of cholesterol. This study indicates that the substrate binding si te of cytochrome P-450(scc) in the vicinity of the 3 beta-hydroxyl gro up is larger than previously believed. Copyright (C) 1996 Elsevier Sci ence Ltd.