title: Thr268 in substrate binding and catalysis in P450BM-3.
abstract: Members of the gene superfamily of proteins called "P450" catalyze monooxygenation reactions that require an input of two electrons and a molecule of oxygen per catalytic cycle. These proteins are widely distributed among living organisms, from bacteria to human. P450BM-3, a soluble protein isolated from Bacillus megaterium, is self-sufficient, containing P450 and reductase domains on the same polypeptide. P450BM-3 catalyzes the hydroxylation of various fatty acids at omega-1, omega-2, and omega-3 positions, as well as epoxidations of double bonds. We have constructed the active-site mutant, T268A, and analyzed the effect on arachidonic acid and palmitic acid oxidation. Data indicate that the mutation changes the coupling (ratio of NADPH consumed versus product formed) for both arachidonic acid and palmitic acid oxidation. We have also analyzed cumene hydroperoxide-driven reactions and shown that they are unaffected by this mutation. These data, as well as fatty acid binding studies, support the hypothesis of a role of the I-helix residue, T268, in maintaining fatty acid substrates in the correct position for productive hydroxylation during the catalytic cycle of this enzyme.
authors.affiliation: Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas 75235-9038, USA.
authors.forename: G
authors.initials: G
authors.lastname: Truan
authors.forename: J A
authors.initials: JA
authors.lastname: Peterson
mesh_terms: D016718:Arachidonic Acid / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009251:NADPH-Ferrihemoprotein Reductase; D010084:Oxidation-Reduction; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D016718:Arachidonic Acid; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases