Side-chain cleavage of cholesterol esters by human cytochrome P-450(scc)

In order to define the substrate binding site of human cytochrome P-450(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 Tween-20 detergent reconstituted system we found that cholesterol sulphate could undergo side-chain cleavage with the same turnover number (k(cat)) as that for cholesterol, but with a higher K-m. Cholesterol methyl ether underwent side-chain cleavage to pregnenolone methyl ether with k(cat) and K-m values 30% of those for cholesterol. Cholesterol fatty acid esters with acyl chain lengths of up to four carbons were able 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. Turnover numbers decreased as the acyl group length increased beyond four carbons, although some activity was still detected with cholesterol palmitate as substrate. Analysis of bovine cytochrome P-450(scc) revealed that it could also cleave the side chain of acyl and sulphate esters of cholesterol. This study indicates that the substrate binding site of cytochrome P-450(scc) in the vicinity of the 3 beta-hydroxyl group is larger than previously believed. Copyright (C) 1996 Elsevier Science Ltd.