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

• 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

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.

聚乙二醇化辣根过氧化物酶（PEG-HRP）在有机溶剂和水溶液中均能催化单电子和双电子氧化反应。尽管愈创木酚在苯和氯苯中的氧化比在磷酸盐缓冲液中慢五个数量级，但化合物I和II也参与有机介质中的催化循环。因子分析和全局拟合表明，PEG-HRP复合物I在有机介质中的形成过程包括两个步骤：第一个快速过程和第二个缓慢过程，表明H_2O_2-HRP复合物参与了催化循环。化合物Ⅰ的前体在-20 ℃氯苯中与过氧化氢反应后稳定，其吸收光谱不具有超卟啉光谱的特征，但具有R38 L HRP的Soret吸收峰，更重要的是，PEG-HRP复合物Ⅰ在氯苯中于-20 ℃可保持1小时以上。抹香鲸肌红蛋白（Mb）的X射线晶体结构比较细胞色素c过氧化物酶（CcP）允许我们设计Mb突变体来模拟过氧化物酶的活性位点。我们已经发现，从位置64到43的Mb的远端组氨酸的重新定位增加过氧化物酶活性相对于那些过氧化物酶以及野生型Mb。更重要的是，铁基阳离子自由基样物种已被确定为Mb突变体的催化中间体的第一次。远端组氨酸的对齐的血红素蛋白的功能的相关性进行了讨论。

项目成果

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