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Studies on the entry site of protons necessary for the monooxygenation reaction catalyzed by cytochrome P450cam

细胞色素P450cam催化单氧化反应所需质子进入位点的研究

基本信息

批准号：
13680750
负责人：
SHIMADA Hideo
金额：
$ 2.3万
依托单位：
KEIO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

The monooxygenation reaction catalyzed by cytochrome P450 requires two equivalents of proton and electron to cleave dioxygen. This dioxygen scission generates a molecule of water and an oxygen atom that reacts with the substrate, yielding the monooxygenated product. The protons are transferred from the solvent to the active site of the enzyme located interior of the protein. However, how proton is conducted to the active site is still controversial. The objective of this research is to study on the mechanism and pathway of the proton transfer in the d-camphor monooxygenase, cytochrome P450cam, focusing on the entry site of the protonsWe have previously proposed the hydrogen-bonding network composed of Thr252-water-Asp251, which extends from the active site to near the protein surface and also proposed this network functions as the proton transfer pathway. The end of the network, Asp251 is within a hydrogen-bonding distance from the surface residue Asp182. This leads us to hypothesize t … More hat Asp182 is the proton entry site.The mutation of Asp182 to Asn slowed the proton-coupled electron transfer from reduced Pdx to oxy-ferrous P450cam (2nd ET) to 7% of the wild type rate. The same mutation, however, did not alter the electron transfer from reduced Pdx to ferric P450cam (1st ET), suggesting that slow proton transfer reduces the rate for 2nd ET. Mutation to Leu reduced the rates for 2nd and 1st ETs to 56 and 77% of the wild type rates, respectively. This small effect of the mutation on the proton transfer contradicts to that of Asn. X-ray structures of the wild type and Asn- and Leu-mutants enzymes demonstrated that the Leu side-chain showed high temperature factors in contrast to those for the side chains of Asn and Asp, both of which formed hydrogen bonds with Arg178 and Arg186. The high temperature factor is deduced to be caused by flexible or mobile side chain, possibly allowing a transient access of water to the protein interior. This possibility was supported by molecular surface analysis of the Leu-mutant with the side chain conformation different from those of Asn and Asp. Therefore, the flexible or mobile side chain explains the fast proton transfer in the Leu-mutant. These results indicate that Asp182 mediates proton transfer from the solvent interface to the active site of P450cam through the previously proposed hydrogen bonded network. Less

由细胞色素P450催化的单加氧反应需要两当量的质子和电子来裂解分子氧。该分子氧断裂产生水分子和与底物反应的氧原子，产生单氧化产物。质子从溶剂转移到位于蛋白质内部的酶的活性位点。然而，质子是如何传导到活性中心的，目前仍存在争议。本研究的目的是研究d-樟脑单加氧酶-细胞色素P450 cam中质子转移的机制和途径，重点是质子的进入位点，我们以前提出了由Thr 252-水-Asp 251组成的氢键网络，该网络从活性位点延伸到蛋白质表面附近，并提出该网络作为质子转移途径。网络的末端Asp 251与表面残基Asp 182在氢键距离内。这使我们假设t ...更多信息 Asp 182突变为Asn使还原型Pdx向氧合亚铁P450 cam（第二ET）的质子偶联电子转移速率降低到野生型的7%。然而，相同的突变并没有改变从还原型Pdx到铁P450 cam（第一ET）的电子转移，这表明缓慢的质子转移降低了第二ET的速率。突变为Leu使第二和第一次突变的比率分别降低至野生型比率的56%和77%。突变对质子转移的这种小的影响与Asn的相反。X射线结构的野生型和天冬氨酸和亮氨酸突变体酶表明，亮氨酸侧链显示高温因素，相反，为天冬氨酸和天冬氨酸，这两者形成氢键与Arg 178和Arg 186的侧链。高温因子被推断为由柔性或移动的侧链引起，可能允许水瞬时进入蛋白质内部。这种可能性得到了支持的分子表面分析的Leu突变体的侧链构象不同的天冬酰胺和天冬氨酸。因此，柔性或移动的侧链解释了Leu突变体中的快速质子转移。这些结果表明，Asp 182介导的质子转移从溶剂界面的P450 cam的活性位点，通过先前提出的氢键网络。少