SIDE-CHAIN SPECIFICITIES OF HUMAN AND BOVINE CYTOCHROMES-P-450SCC

Cytochrome P-450scc catalyses the conversion of cholesterol to pregnen olone by the sequential hydroxylation of the side chain of cholesterol . This occurs at a single active site and produces 22R-hydroxycholeste rol and 22R-20alpha-dihydroxycholesterol as intermediates. To further define the active site of human and bovine cytochromes P-450scc, we ha ve examined the kinetics of the conversion of structural analogues of cholesterol with modified side chains, to pregnenolone. Analysis of th e side-chain cleavage of analogues of cholesterol modified at C22 conf irmed the high degree of structural specificity for the 22R position b y cytochrome P-450scc, the major effect being on the turnover number ( k(cat)) rather than on binding. The analogues of cholesterol that had a polar group at C24, C25 or C26 had much lower K(m) values and genera lly lower k(cat) values than the non-polar analogues which were tested . K(m) values of the polar analogues were 3-25-times lower than the K( m) for cholesterol and k(cat) values were also much lower than the k(c at) values for cholesterol, particularly for the human enzyme. The dat a suggest that the tight binding of the analogues with a hydroxyl or k etone group at C24, C25 or C26 places C20 and C22 in a poor orientatio n relative to the heme group for hydroxylation to occur. Many of the p olar analogues which were tested are postulated regulators of cellular cholesterol metabolism. Several of these analogues are good substrate s for bovine and human cytochromes P-450scc at low substrate concentra tion, as determined from their k(cat)/K(m) values. This study also ind icates that the active site of cytochrome P-450scc is well conserved b etween bovine and human cytochromes. However, small species difference s are evident since lower k(cat), values relative to the k(cat) of cho lesterol are observed for some polar side-chain analogues of cholester ol with the human enzyme.