ACTIVE-SITE TOPOLOGY OF BOVINE CHOLESTEROL SIDE-CHAIN CLEAVAGE CYTOCHROME-P450 (P450SCC) AND EVIDENCE FOR INTERACTION OF TYROSINE-94 WITH THE SIDE-CHAIN OF CHOLESTEROL

Combining site-directed mutagenesis with analysis of the active-site t opology of bovine cholesterol sidechain cleavage cytochrome P450scc (P 450scc), we have investigated the roles of tyrosine residues 93 and 94 on substrate binding. Four single mutants (Y93A, Y93S, Y94A, and Y94S ) and one double mutant (Y93S/Y94S) were examined. The largest increas e in K-s was observed for binding of cholesterol and 25-hydroxycholest erol to the Y94S mutant (similar to 5.5-fold), with a smaller increase (<2.5-fold) for binding of 22-hydroxycholesterol. Mutation of Y94 thu s appears to influence the interaction with cholesterol, 25-hydroxycho lesterol, and possibly 22-hydroxycholesterol. Y93 is not involved in b inding of 22- and 25-hydroxycholesterol but may interact with choleste rol. The active-site topologies of P450scc and its mutants were probed by reaction with three arylhydrazines. The N-arylprotoporphyrin IX re gioisomer patterns obtained with phenyl- and 2-naphthylhydrazine indic ate that the active site is primarily open above pyrrole ring A and su ggest that a region some distance above pyrrole ring D is also open. T he single mutations Y93S, Y93A, Y94A, and Y94S do not detectably alter the regioisomer patterns obtained with the phenyl- and 2-naphthyl pro bes, but a small, reproducible change is observed with the 2-naphthyl probe for the Y93S/Y94S double mutant, The conformational alteration i mplied by this change could not be detected by titration with 22- and 25-hydroxycholesterol but is detectable by titration with cholesterol. The results indicate that cholesterol binds over pyrrole ring D of th e heme in bovine P450scc, strongly suggest that Y94 interacts with the side chain of cholesterol, and provide evidence that the side chains of 22- and 25-hydroxycholesterol bind to a different region of the act ive site than the side chain of cholesterol. (C) 1995 Academic Press I nc.