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MECHANISM FOR OXYGEN ACTIVATION BY CYTOCHROME P450cam

CYTOCHROME P450cam 的氧气激活机制

基本信息

批准号：
07458163
负责人：
ISHIMURA Yuzuru
金额：
$ 4.1万
依托单位：
SCHOOL OF MEDICINE,KEIO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07458163/
关键词：
Cytochome P450 P450cam Activation of molecular oxygen Acid-base catalyst Site-directed mutagenesis Artificial amino acids Dioxygen-bond cleavage Threonine Hydrogen bonding

项目摘要

Cytochrome P450cam is the terminal mono-oxygenase in the d-camphor hydroxylating system of Pseudomonas putida. Recently the x-ray crystal structure of this enzyme has been solved by Poulos and his associates enabling us to study its exact structure-function. relationship. The enzyme catalyzes the reaction indicated below.d-Camphor+NADH+H^++O_2*5-exo-hydroxycamphor+NAD^++H_2OOur previous study with conventional site-directed mutagenesis of the enzyme revealed that a hydroxy (OH) group of Thr252 at the oxygen pocket of the cytochrome plays an important role in the mono-oxygenase reaction : In the absence of the OH group, oxygen consumption and the hydroxylation of d-camphor was uncoupled (Proc.Natl.Acad.Sci.U.S.A.86,7823-7827). Based on this and other evidence, the role of Thr252 has been proposed by us to form a hydrogen bonding net work which is formed this threonine and carboxyl group of Asp251. Such a hydrogen-bonding net work is required for the activation of heme-bound dioxygen (O_ … More 2), i.e.reductive cleavage of the O-O bond in O_2, in the above reaction.to investigate further the role of the OH group of Thr252 in cytochrome P450cam, artificial amino acids with oxygen-, sulfur-, or nitrogen-containing groups were introduced to the 252 position (in place of OH group of Thr) by the aid of in vitro translation system. Measurrements of the enzyme activity of such mutants showed that only those containing oxygen atoms at that position (-OCH_3, COOCH_3) in place of the OH group retained a large portion of the mono-oxygenase activity (>75% formation of hydroxylated product per O_2 consumed). On the other hand, mutants containing a residue with sulfur or nitrogen (-SCH_3, -NH_3^+) had lost most of the activity (<10% formation of hydroxylated product per O_2 consumed). Thus oxygen atom at the residue of 252 position is important for the mono-oxygenase activity.On the basis of these results, we propose that the property of the oxygen atom in Thr252 as an acceptor of a hydrogen bond is essential for the formation of a hydrogen-bonding network for proton transfer necessary for the mono-oxygenase reaction described above. Less

细胞色素P450cam是恶臭假单胞菌d-樟脑羟化系统中的末端单加氧酶。最近，Poulos和他的同事们已经解开了这种酶的X射线晶体结构，使我们能够研究它的确切结构-功能。两性关系。该酶催化的反应表明below.d-Camphor+NADH+H^++O_2*5-exo-hydroxycamphor+NAD^++H_2OOur以前用常规定点突变的研究表明，Thr252在细胞色素氧囊的羟基(OH)在单加氧酶反应中起着重要作用：在没有羟基的情况下，氧的消耗和d-樟脑的羟基化是解偶联的(Proc.Natl.Academy.Suc.U.S.A.86,7823-7827)。基于这一点和其他证据，我们提出Thr252的作用是形成一个氢键网络，该网络形成了Asp251的这个苏氨酸和羧基。这种氢键网络是激活血红素结合的氧气(O_…)所必需的为了进一步研究Thr252的羟基在细胞色素P450cam中的作用，在体外翻译系统的帮助下，在252位引入了含氧、含硫或含氮的人工氨基酸。对这些突变体的酶活性测定表明，只有那些在该位置上含有氧原子的突变体(-OCH3，CoOCH3)才保留了很大一部分单加氧酶活性(&gt；每消耗O_2有75%的羟化产物形成)。另一方面，含有硫或氮残基的突变体(-SCH_3，-NH_3^+)失去了大部分活性(每消耗O_2产生&lt；10%的羟化产物)。在这些结果的基础上，我们认为Thr252中氧原子作为氢键受体的性质对于形成上述单加氧酶反应所需的质子转移氢键网络是必不可少的。较少