title: Effects of steric bulk and conformational rigidity on fatty acid omega hydroxylation by a cytochrome P450 4A1 fusion protein.
abstract: The action of cytochrome P450 4A1 (CYP4A1) on fatty acid substrates is characterized by a pronounced regioselectivity for omega-hydroxylation. To elucidate the chemical basis of this specificity we probed the active site of a CYP4A1 fusion protein (f4A1) with bulky and/ or rigid analogs of lauric acid, the optimum natural substrate for f4A1 and CYP4A1. f4A1 efficiently omega-hydroxylates lauric acid, epoxidizes 11-dodecenoic acid, and oxidizes 11-dodecynoic acid to 1,12-dodecanedioic acid. Medium length fatty acids having omega-terminal groups as large as t-butyl or m-tolyloxy bind tightly to f4A1 as Type I ligands and are efficiently hydroxylated on their methyl termini. omega-Phenylnonanoic acid also induces a Type I binding spectrum (Ks = 0.77 microM) but fails to undergo hydroxylation and strongly inhibits lauric acid hydroxylation by f4A1. Slightly shorter acids such as omega-phenyloctanoic acid, naproxen, and ibuprofen also strongly inhibit lauric acid hydroxylation but do not induce a Type I spectrum and do not undergo hydroxylation. Like 10-methoxydecanoic acid, the rigid and rod-like 4'-methoxy-4-biphenylcarboxylic acid is O-demethylated by f4A1, which also omega-hydroxylates m- and p-heptyloxybenzoic acids but not m- or p-amyloxyhydrocinnamic acids. The histamine antagonist cimetidine and the peroxisome proliferator perfluorodecanoic acid are both potent inhibitors of f4A1. Thus the active site of f4A1 is quite tolerant of steric bulk and rigidity around the heme region and the polar group recognition site, but perhaps less so in the midchain region. Although CYP4A enzymes are not usually regarded as "drug metabolizing P450s," the fact that commonly used therapeutic agents strongly inhibit lauric acid omega-hydroxylation by f4A1 as well as liver microsomes from clofibrate-induced rats suggests these and related agents could potentially interfere with the contribution of CYP4A enzymes to the metabolism of endogenous lipids.
authors.affiliation: Department of Medicinal Chemistry, University of Kansas, Lawrence 66045-2506, USA.
authors.forename: R B
authors.initials: RB
authors.lastname: Bambal
authors.forename: R P
authors.initials: RP
authors.lastname: Hanzlik
mesh_terms: D000495:Allosteric Site; D000818:Animals; D001665:Binding Sites; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D006900:Hydroxylation; D066298:In Vitro Techniques; D007850:Lauric Acids / Q000737:chemistry / Q000378:metabolism*; D050356:Lipid Metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D007850:Lauric Acids; D011993:Recombinant Fusion Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D042926:Cytochrome P-450 CYP4A