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 3beta-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 (kcat) as that for cholesterol, but with a higher Km. Cholesterol methyl ether underwent side-chain cleavage to pregnenolone methyl ether with kcat and Km 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 Km values similar to those for cholesterol, but kcat 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 3beta-hydroxyl group is larger than previously believed.
Tuckey
R C
RC
Department of Biochemistry, The University of Western Australia, Nedlands. rtuckey@uniwa.uwa.edu.au.
Lawrence
J
J
Cameron
K J
KJ
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002788:Cholesterol Esters / Q000378:metabolism*; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000302:isolation & purification / Q000378:metabolism*; D005260:Female; D006801:Humans; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D013379:Substrate Specificity
D002788:Cholesterol Esters; D002786:Cholesterol Side-Chain Cleavage Enzyme