THR268 IN SUBSTRATE-BINDING AND CATALYSIS IN P450BM-3

Members of the gene superfamily of proteins called ''P450'' catalyze m onooxygenation reactions that require an input of two electrons and a molecule of oxygen per catalytic cycle. These proteins are widely dist ributed among living organisms, from bacteria to human. P450BM-3, a so luble protein isolated from Bacillus megaterium, is self-sufficient, c ontaining 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 effec t on arachidonic acid and palmitic acid oxidation. Data indicate that the mutation changes the coupling (ratio of NADPH consumed versus prod uct formed) for both arachidonic acid and palmitic acid oxidation. We have also analyzed cumene hydroperoxide-driven reactions and shown tha t they are unaffected by this mutation. These data, as well as fatty a cid binding studies, support the hypothesis of a role of the I-helix r esidue, T268, in maintaining fatty acid substrates in the correct posi tion for productive hydroxylation during the catalytic cycle of this e nzyme. (C) 1998 Academic Press.