Capdevila J H, Wei S, Helvig C, Falck J R, Belosludtsev Y, Truan G, Graham-Lorence S E, Peterson J A
Department of Biochemistry, Vanderbilt University Medical School, Nashville, Tennessee 37232, USA.
J Biol Chem. 1996 Sep 13;271(37):22663-71. doi: 10.1074/jbc.271.37.22663.
Cytochrome P450BM-3 catalyzes NADPH-dependent metabolism of arachidonic acid to nearly enantiomerically pure 18(R)-hydroxyeicosatetraenoic acid and 14(S), 15(R)-epoxyeicosatrienoic acid (80 and 20% of total products, respectively). P450BM-3 oxidizes arachidonic acid with a rate of 3.2 +/- 0.4 micromol/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, demonstrating efficient coupling of electron transport to monooxygenation. Eicosapentaenoic and eicosatrienoic acids, two arachidonic acid analogs that differ in the properties of the C-15-C-18 carbons, are also actively metabolized by P450BM-3 (1.4 +/- 0.2 and 2.9 +/- 0.1 micromol/min/nmol at 30 degrees C, respectively). While the 17,18-olefinic bond of eicosapentaenoic acid is epoxidized with nearly absolute regio- and stereochemical selectivity to 17(S),18(R)-epoxyeicosatetraenoic acid (>/=99% of total products, 97% optical purity), P450BM-3 is only moderately regioselective during hydroxylation of the eicosatrienoic acid omega-1, omega-2, and omega-3 sp3 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-bound P450BM-3, we propose: 1) the formation by P450BM-3 of a single oxidant species capable of olefinic bond epoxidation and sp3 carbon hydroxylation and 2) that product chemistry and, thus, catalytic outcome are critically dependent on active site spatial coordinates responsible for substrate binding and productive orientation between heme-bound active oxygen and acceptor carbon bond(s).
细胞色素P450BM-3催化花生四烯酸的NADPH依赖性代谢，生成几乎对映体纯的18(R)-羟基二十碳四烯酸和14(S),15(R)-环氧二十碳三烯酸（分别占总产物的80%和20%）。在30℃时，P450BM-3氧化花生四烯酸的速率为3.2±0.4微摩尔/分钟/纳摩尔，这是报道过的NADPH依赖性P450催化反应中最快的速率。脂肪酸、氧气和NADPH以大约1:1:1的摩尔比被利用，这表明电子传递与单加氧反应有效偶联。二十碳五烯酸和二十碳三烯酸这两种花生四烯酸类似物，在C-15 - C-18碳的性质上有所不同，它们也能被P450BM-3有效代谢（在30℃时分别为1.4±0.2和2.9±0.1微摩尔/分钟/纳摩尔）。虽然二十碳五烯酸的17,18-烯烃键被环氧化，生成几乎具有绝对区域和立体化学选择性的17(S),18(R)-环氧二十碳四烯酸（占总产物的≥99%，光学纯度为97%），但P450BM-3在二十碳三烯酸的ω-1、ω-2和ω-3 sp3碳羟基化过程中区域选择性适中，生成的17-、18-和19-羟基二十碳三烯酸的比例分别为2.4:2.2:1。基于上述内容以及花生四烯酸结合型P450BM-3的模型，我们提出：1）P450BM-3形成一种单一的氧化剂物种，能够进行烯烃键环氧化和sp3碳羟基化；2）产物化学性质以及催化结果关键取决于负责底物结合的活性位点空间坐标，以及血红素结合的活性氧与受体碳键之间的有效取向。
