Active site structure and substrate specificity of cytochrome P450 4A1: Steric control of ligand approach perpendicular to heme plane

The active site of an engineered, expressed fusion protein containing the sequences of cytochrome P450 4A1 (lauric acid omega-hydroxylase) and NADPH-cytochrome P450 reductase, has been probed with olefinic, acetylenic, aromatic, and other analogs of its normal substrate. The fusion protein omega-hydroxylates lauric acid, epoxidizes 11-dodocenoic acid, oxidizes 11-dodecynoic acid to 1,12-dodecandioic acid, but does not oxidize 9-phenylnonanoic acid. Nevertheless, the latter is a tight-binding Type I ligand (K-s = 0.77 mu M) and a potent inhibitor of lauric acid hydroxylation. These and other observations are used to construct an active site model that accounts for its remarkable substrate and inhibitor specificity. (C) 1996 Academic Press, Inc.