Identification of the binding site on cytochrome P450 2B4 for cytochrome b5 and cytochrome P450 reductase
Identification of the binding site on cytochrome P450 2B4 for cytochrome b5 and cytochrome P450 reductase
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DOI：
10.1074/jbc.273.27.17036
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发表时间：
1998-07-03
影响因子：
4.8
通讯作者：
Waskell, L
中科院分区：
文献类型：
--
作者：
Bridges, A;Gruenke, L;Waskell, L
细胞色素P450 2B 4的模型，其通过用细胞色素P450的四种已知晶体结构的同源性建模而构建（Chang，T. T.，Stiffelman，O. B.，瓦克瑟岛A，Loew，G. H、Bridges，A和Waskell，L.（1997）Protein Eng.10，119-129）用于选择通过计算机对接研究和许多先前的生物化学和定点诱变研究预测的参与结合细胞色素B的血红素结构域的氨基酸（5）。选择位于分子远端和近端表面的24个氨基酸残基进行诱变。这24种突变蛋白在大肠杆菌中表达，纯化，并就其结合细胞色素B（5）和支持底物氧化的能力进行表征。7个突变体，R122 A、R126 A、R133 A、F135 A、M137 A、K139 A和K433 A，均位于细胞色素P450 2B 4的近端表面，靠近血红素配体，被鉴定为表现出结合细胞色素B的能力降低（5）。除K433 A外，所有突变体均位于C或C*：螺旋或其末端。此外，这7个突变体和细胞色素P450近端表面上的另外两个突变体R422 A和R443 A显示出与细胞色素P450还原酶的结合降低。这些研究表明，细胞色素B（5）和细胞色素P450还原酶的结合位点如预测的那样位于细胞色素P450 2B 4的近端表面，并且部分重叠但不相同。
A model of cytochrome P450 2B4, which was constructed by homology modeling with the four known crystal structures of the cytochromes P450 (Chang, T.-T., Stiffelman, O. B., Vakser, I. A, Loew, G. H., Bridges, A, and Waskell, L. (1997) Protein Eng. 10, 119-129), was used to select amino acids predicted, by computer docking studies and numerous previous biochemical and site-directed mutagenesis studies, to be involved in binding the heme domain of cytochrome b(5),. Twenty-four amino acid residues located on both the distal and the proximal surface of the molecule were chosen for mutagenesis. These 24 mutant proteins were expressed in Escherichia coli, purified, and characterized with respect to their ability to bind cytochrome b(5), and support substrate oxidation. Seven mutants, R122A, R126A, R133A, F135A, M137A, K139A, and K433A, all on the proximal surface of cytochrome P450 2B4 near the heme ligand, were identified that exhibited decreased ability to bind cytochrome b(5),. All of the mutants except K433A are located in either the C or C*: helices or their termini. In addition, these seven mutants and two additional mutants on the proximal surface of cytochrome P450, R422A and R443A, were shown to exhibit decreased binding to cytochrome P450 reductase. These studies indicate that the binding sites for cytochrome b(5), and cytochrome P450 reductase are, as predicted, located on the proximal surface of cytochrome P450 2B4 and are partially overlapping but not identical.