CRYSTAL-STRUCTURE OF SUBSTRATE-FREE PSEUDOMONAS-PUTIDA CYTOCHROME-P-450
CRYSTAL-STRUCTURE OF SUBSTRATE-FREE PSEUDOMONAS-PUTIDA CYTOCHROME-P-450
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DOI：
10.1021/bi00366a049
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发表时间：
1986-09-09
期刊：
影响因子：
2.9
通讯作者：
HOWARD, AJ
中科院分区：
文献类型：
--
作者：
POULOS, TL;FINZEL, BC;HOWARD, AJ
无底物形式的恶臭假单胞菌细胞色素P-450 cam的晶体结构已在2.20埃下精制。分辨率，并与酶的底物结合形式进行比较。在不存在底物樟脑的情况下，P-450 cam血红素铁原子与Cys-357的硫原子六配位，提供一个轴向血红素配体，并且水分子或氢氧根离子提供另一个轴向配体。除了铁连接的水配体之外，氢键键合的溶剂分子的网络占据基底口袋。当樟脑分子结合时，包括水配体的活性部位沃茨被置换，导致五配位高自旋血红素铁原子。Fno樟脑- Fcamphor差异傅立叶分析和两种精制结构的定量比较表明，除了接触樟脑分子的苯丙氨酸侧链的小的重新定位之外，没有可检测到的构象变化来自樟脑结合。然而，樟脑结合导致以Tyr-96，Thr-185和Asp-251为中心的蛋白质的三个独立片段的平均温度因子大幅下降。这表明樟脑的结合降低了P-450 cam分子这三个区域的灵活性，而没有改变所涉及原子的平均位置。
The crystal structure of Pseudomonas putida cytochrome P-450cam in the substrate-free form has been refined at 2.20-.ANG. resolution and compared to the substrate-bound form of the enzyme. In the absence of the substrate camphor, the P-450cam heme iron atom is hexacoordinate with the sulfur atom of Cys-357 providing one axial heme ligand and a water molecule or hydroxide ion providing the other axial ligand. A network of hydrogen-bonded solvent molecules occupies the substrate pocket in addition to the iron-linked aqua ligand. When a camphor molecule binds, the active site waters including the aqua ligand are displaced, resulting in a pentacoordinate high-spin heme iron atom. Analysis of the Fno camphor - Fcamphor difference Fourier and a quantitative comparison of the two refined structures reveal that no detectable conformational change results from camphor binding other than a small repositioning of a phenylalanine side chain that contacts the camphor molecule. However, large decrease in the mean temperature factors of three separate segments of the protein centered on Tyr-96, Thr-185, and Asp-251 result from camphor binding. This indicates that camphor binding decreases the flexibility in these three regions of the P-450cam molecule without altering the mean position of the atoms involved.