Graham-Lorence S, Truan G, Peterson J A, Falck J R, Wei S, Helvig C, Capdevila J H
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235, USA.
J Biol Chem. 1997 Jan 10;272(2):1127-35. doi: 10.1074/jbc.272.2.1127.
Cytochrome P450 BM-3 catalyzes the high turnover regio- and stereoselective metabolism of arachidonic and eicosapentaenoic acids. To map structural determinants of productive active site fatty acid binding, we mutated two amino acid residues, arginine 47 and phenylalanine 87, which flank the surface and heme ends of the enzyme's substrate access channel, respectively. Replacement of arginine 47 with glutamic acid resulted in a catalytically inactive mutant. Replacement of arginine 47 with alanine yielded a protein with reduced substrate binding affinity and arachidonate sp3 carbon hydroxylation activity (72% of control wild type). On the other hand, arachidonic and eicosapentaenoic acid epoxidation was significantly enhanced (154 and 137%, of control wild type, respectively). As with wild type, the alanine 47 mutant generated (18R)-hydroxyeicosatetraenoic, (14S,15R)-epoxyeicosatrienoic, and (17S,18R)-epoxyeicosatetraenoic acids nearly enantiomerically pure. Replacement of phenylalanine 87 with valine converted cytochrome P450 BM-3 into a regio- and stereoselective arachidonic acid epoxygenase ((14S,15R)-epoxyeicosatrienoic acid, 99% of total products). Conversely, metabolism of eicosapentaenoic acid by the valine 87 mutant yielded a mixture of (14S,15R)- and (17S,18R)-epoxyeicosatetraenoic acids (26 and 69% of total, 94 and 96% optical purity, respectively). Finally, replacement of phenylalanine 87 with tyrosine yielded an inactive protein. We propose that: (a) fatty acid oxidation by P450 BM-3 is incompatible with the presence of residues with negatively charged side chains at the surface opening of the substrate access channel or a polar aromatic side chain in the vicinity of the heme iron; (b) the high turnover regio- and stereoselective metabolism of arachidonic and eicosapentaenoic acids involves charge-dependent anchoring of the fatty acids at the mouth of the access channel by arginine 47, as well as steric gating of the heme-bound oxidant by phenylalanine 87; and (c) substrate binding coordinates, as opposed to oxygen chemistries, are the determining factors responsible for reaction rates, product chemistry, and, thus, catalytic outcome.
细胞色素P450 BM-3催化花生四烯酸和二十碳五烯酸的高效区域和立体选择性代谢。为了确定活性位点脂肪酸有效结合的结构决定因素，我们对两个氨基酸残基进行了突变，分别是精氨酸47和苯丙氨酸87，它们分别位于酶底物通道的表面和血红素末端两侧。用谷氨酸取代精氨酸47导致产生一个催化无活性的突变体。用丙氨酸取代精氨酸47产生一种蛋白质，其底物结合亲和力和花生四烯酸sp3碳羟基化活性降低（为对照野生型的72%）。另一方面，花生四烯酸和二十碳五烯酸的环氧化作用显著增强（分别为对照野生型的154%和137%）。与野生型一样，丙氨酸47突变体产生的（18R）-羟基二十碳四烯酸、（14S,15R）-环氧二十碳三烯酸和（17S,18R）-环氧二十碳四烯酸几乎是对映体纯的。用缬氨酸取代苯丙氨酸87将细胞色素P450 BM-3转化为一种区域和立体选择性花生四烯酸环氧化酶（（14S,15R）-环氧二十碳三烯酸，占总产物的99%）。相反，缬氨酸87突变体对二十碳五烯酸的代谢产生了（14S,15R）-和（17S,18R）-环氧二十碳四烯酸的混合物（分别占总量的26%和69%，光学纯度分别为94%和96%）。最后，用酪氨酸取代苯丙氨酸87产生一种无活性的蛋白质。我们提出：（a）P450 BM-3对脂肪酸的氧化与底物通道表面开口处带有负电荷侧链的残基或血红素铁附近的极性芳香侧链的存在不相容；（b）花生四烯酸和二十碳五烯酸的高效区域和立体选择性代谢涉及精氨酸47使脂肪酸在通道口处进行电荷依赖性锚定，以及苯丙氨酸87对血红素结合氧化剂的空间门控作用；（c）与氧化学性质相反，底物结合坐标是决定反应速率、产物化学性质以及催化结果的因素。
