THE HIGHLY STEREOSELECTIVE OXIDATION OF POLYUNSATURATED FATTY-ACIDS BY CYTOCHROME P450BM-3

Cytochrome P450BM-3 catalyzes NADPH-dependent metabolism of arachidoni c acid to nearly enantiomerically pure 18(R-hydroxyeicosatetraenoic ac id and 14(S),15(R)-epoxyeicosatrienoic acid (80 and 20% of total produ cts, respectively), P450BM-3 oxidizes arachidonic acid with a rate of 3.2 +/- 0.4 mu mol/min/nmol at 30 degrees C, the fastest ever reported for an NADPH dependent, P450-catalyzed reaction, Fatty acid, oxygen, and NADPH are utilized in an approximately 1:1:1 molar ratio, demonstr ating efficient coupling of electron transport to monooxygenation. Eic osapentaenoic and eicosatrienoic acids, two arachidonic acid analogs t hat differ in the properties of the C-15-C-18 carbons, are also active ly metabolized by P450BM-3 (1.4 +/- 0.2 and 2.9 +/- 0.1 mu mol/min/nmo l at 30 degrees C, respectively). While the 17,18-olefinic bond of eic osapentaenoic acid is epoxidized with nearly absolute regio- and stere ochemical selectivity to 17(S),18(R)-epoxyeicosatetraenoic acid (great er than or equal to 99% of total products, 97% optical purity), P450BM -3 is only moderately regioselective during hydroxylation of the eicos atrienoic acid omega-1, omega-2, and omega-3 sp(3) carbons, with 17-, 18-, and 19-hydroxyeicosatrienoic acid formed in a ratio of 2.4:2.2:1, respectively. Based on the above and on a model of arachidonic acid-b ound P450BM-3, we propose: 1) the formation by P450BM-3 of a single ox idant species capable of olefinic bond epoxidation and sp(3) carbon hy droxylation and 2) that product chemistry and, thus, catalytic outcome are critically dependent on active site spatial coordinates responsib le for substrate binding and productive orientation between heme-bound active oxygen and acceptor carbon bond(s).