CHARACTERIZATION OF UNSTABLE INTERMEDIATES OF HEME ENZYMES BY UTILIZING ENZYMES AND THEIR HYBRIDES

利用酶及其混合物表征血红素酶的不稳定中间体

基本信息

批准号：
07458147
负责人：
WATANABE Yoshihito
金额：
$ 3.71万
依托单位：
OKAZAKI NATIONAL RESEARCH INSTITUTES
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-07458147/
关键词：
PEROXIDASE COMPOUND I PEG-HRP MYOGLOBIN HEME ヘム蛋白質ミューテーションハイブリッド

项目摘要

Polyethylene glycolated horseradish peroxidase (PEG-HRP) can catalyze one- and two-electron oxidation reactions in organic solvents as well as in aqueous buffer. Even though the oxidation of guaiacol in benzene and chlorobenzene is five order of magnitude slower than in phosphate buffer, compound I and II are also involved in the catalytic cycle in organic media. Factor analysis and global fittings of rapid scan data set reveal that the formation of compound I of PEG-HRP in organic media consists of two steps : the first fast and the second slow process, and suggest the involvement of a H2O2-HRP complex in the catalytic cycle. The labile precursor of compound I is stabilized when PEG-HRP reacts with hydrogen peroxide in chlorobenzene at -20ﾟC.The absorption spectrum of the precursor does not exhibit the features of hyperporphyrin spectrum but has a normal Soret as previously observed in R38L HRP.More importantly, compound I of PEG-HRP can be maintained for more than an hour at -20ﾟC in chlorobenzene.Comparison of the X-ray crystal structures of sperm whale myoglobin (Mb) and cytochrome c peroxidase (CcP) allows us to design Mb mutants to mimic the active site of peroxidase. We have found that relocating the distal histidine of Mb from position 64 to 43 increases peroxygenase activities with respect to those of peroxidase as well as wild type Mb. More importantly, a ferryl radical cation like species has been identified as the catalytic intermediate of the Mb mutant for the first time. The relevance of the alignment of distal histidine to the functions of hemoproteins is discussed.

聚乙烯糖化的辣根过氧化物酶（PEG-HRP）可以在有机溶液以及水缓冲液中催化一氧化物和两电子氧化物反应。即使在苯和氯苯中的鸟醇的氧化速度比磷酸盐缓冲液慢五个数量级，但化合物I和II也参与了有机培养基中的催化循环。因子分析和快速扫描数据集的全球配件表明，有机培养基中PEG-HRP的化合物I的形成由两个步骤组成：第一个快速和第二个慢速过程，并建议在催化循环中涉及H2O2-HRP复合物。当PEG -HRP与-20 ﾟC中的氯化氢反应时，化合物I的不稳定前体被稳定。氯苯氯苯氯苯甲酸盐的X射线晶体结构的比较，使鲸鱼肌红蛋白（MB）和细胞色素C过氧化物酶（CCP）使我们能够设计MB突变体以模仿过氧化物酶的活性位点。我们发现，将MB的远端组氨酸从位置64转移到43位会增加过氧化物酶活性相对于过氧化物酶和野生型MB的活性。更重要的是，渡轮自由基阳离子类似物种首次被确定为MB突变体的催化中间体。讨论了远端组氨酸对齐与血蛋白功能的相关性。