Pivotal role of water in the mechanism of P450BM-3
Pivotal role of water in the mechanism of P450BM-3
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DOI：
10.1021/bi011197q
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发表时间：
2001-11-13
期刊：
影响因子：
2.9
通讯作者：
Peterson, JA
中科院分区：
文献类型：
--
作者：
Haines, DC;Tomchick, DR;Peterson, JA
细胞色素P450是一个超家族的酶，可以催化大量结构和化学结构不同的疏水底物的氧化。在这里，我们描述了细菌P450BM-3与新底物N-棕榈酰甘氨酸之间的配合物的晶体结构，分辨率为1.65埃，揭示了底物结合时活性部位重组的以前无法识别的特征。N-棕榈酰甘氨酸结合的亲和力比任何其他已知底物都高，反应的周转数比棕榈酸高，但沿着脂肪酸部分具有不变的区域特异性。底物结合诱导酶的不同区域的构象变化，包括与活性部位相邻的部分I-螺旋。这些变化导致连接血红素铁的关键水分子位移约1埃，导致铁从低自旋到高自旋的转化。水分子被困在靠近血红素基团的地方，这使得它可以在铁和新的结合位置之间进行分配。这种分配解释了底物结合后高自旋-低自旋平衡的存在。水分子与血红素铁的接近表明它也可能参与了导致P450BM-3中氧的异解键断裂的质子转移级联。
Cytochrome P450s constitute a superfamily of enzymes that catalyze the oxidation of a vast number of structurally and chemically diverse hydrophobic substrates. Herein, we describe the crystal structure of a complex between the bacterial P450BM-3 and the novel substrate N-palmitoylglycine at a resolution of 1.65 Angstrom, which reveals previously unrecognizable features of active site reorganization upon substrate binding. N-Palmitoylglycine binds with higher affinity than any other known substrate and reacts with a higher turnover number than palmitic acid but with unaltered regiospecificity along the fatty acid moiety. Substrate binding induces conformational changes in distinct regions of the enzyme including part of the I-helix adjacent to the active site. These changes cause the displacement by about 1 Angstrom of the pivotal water molecule that ligands the heme iron, resulting in the low-spin to high-spin conversion of the iron. The water molecule is trapped close to the heme group, which allows it to partition between the iron and the new binding site. This partitioning explains the existence of a high-spin-low-spin equilibrium after substrate binding. The close proximity of the water molecule to the heme iron indicates that it may also participate in the proton-transfer cascade that leads to heterolytic bond scission of oxygen in P450BM-3.