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Oxygen activation mechanisms in cytochrome P450-and NADPH oxidase-catalyzed reactions

细胞色素 P450 和 NADPH 氧化酶催化反应中的氧活化机制

基本信息

批准号：
05454631
负责人：
ISHIMURA Yuzuru
金额：
$ 4.42万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

To elucidate oxygen activation mechanisms in cytochrome P450 (P450) -and NADPH oxidase-catalyzed reactions, we prepared both P450cam and porcine neutrophil NADPH oxidase in large quantities and examined their catalytic properties.The enzymes prepared include site-directed mutants of P450cam and multiple components of the oxidase.P450cam catalyzes the hydroxylation of d-camphor to 5-exo-hydroxycamphor.The NADPH oxidase convertss molecular oxygen (O_2) to superoxide anion (O_2). Among the results, we believe the folowings are most important.(1) Thr252 in the active center of P450cam has been shown to be essential for the enzyme to be a monooxygenase. It was therefore considered as the proton donor to the oxygenated intermediate of the enzyme. In this study we were able to prepare O-CH_3-Thr252 mutant in which the OH group of Thr was no more dissociable and hence can not suply proton nor act as an acid catalyst.(2) On the basis of above and previously reported facts, we postulated a new scheme for the proton transfer network necessary for the oxygen activation(see Shimada et al.). The net work consists of Thr252, H_2O,Asp251 and Lys178/Arg186(Kimata et al.).(3) Compound I of P450cam was detected during its reaction with m-chloroperbenzoate serving as the first successful demonstration of Fe(IV)=O type intermediate in cytochrome P450 type of reactions(Egawa et al.).(4) We studied also an NADPH oxidase in B lymphocytes. The results suggested that the reaction center for the generation of O_2- was exposed to the outside of the cell-surface in the plasma membrane(Kanegasaki et al.).

为了阐明氧在细胞色素P450 （P450）和NADPH氧化酶催化反应中的激活机制，我们大量制备了P450cam和猪中性粒细胞NADPH氧化酶，并检测了它们的催化性能。所制备的酶包括P450cam的定点突变体和氧化酶的多种组分。P450cam催化d-樟脑羟基化生成5-外羟基樟脑。NADPH氧化酶将分子氧（O_2）转化为超氧阴离子（O_2）。在这些结果中，我们认为以下几点最为重要。(1) P450cam活性中心的Thr252已被证明是该酶成为单加氧酶所必需的。因此，它被认为是酶的氧合中间体的质子供体。在这项研究中，我们能够制备O-CH_3-Thr252突变体，其中Thr的OH基团不再可解，因此不能提供质子，也不能作为酸催化剂。(2)基于上述和先前报道的事实，我们假设了氧活化所需的质子转移网络的新方案（见Shimada等人）。网络由Thr252、H_2O、Asp251和Lys178/Arg186组成（Kimata et al.）。(3) P450cam的化合物I在与间氯过苯甲酸酯反应时被检测到，这是Fe(IV)=O型中间体在细胞色素P450型反应中的首次成功证明（Egawa等人）。(4)我们还研究了B淋巴细胞中的NADPH氧化酶。结果表明，生成O_2-的反应中心暴露在细胞质膜外的细胞表面（Kanegasaki et al.）。