| Project/Area Number |
07458147
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Bioorganic chemistry
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| Research Institution | OKAZAKI NATIONAL RESEARCH INSTITUTES |
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Principal Investigator |
WATANABE Yoshihito OKAZAKI NATIONAL RESEARCH INSTITUTES,INSTITUTE FOR MOLECULAR SCIENCE,PROFESSOR, 分子科学研究所, 教授 (10201245)
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| Co-Investigator(Kenkyū-buntansha) |
SHIMADA Hideo KEIO UNIVERSITY,SCHOOL OF MEDICINE ASSOCIATE PROFESSOR, 医学部, 助教授 (80095611)
OZAKI Shin-ichi OKAZAKI NATIONAL RESEARCH INSTITUTES,INSTITUTE FOR MOLECULAR SCIENCE,RESEARCH AS, 分子科学研究所, 助手 (40280581)
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| Project Period (FY) |
1995 – 1996
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| Project Status |
Completed (Fiscal Year 1996)
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| Budget Amount *help |
¥5,800,000 (Direct Cost: ¥5,800,000)
Fiscal Year 1996: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1995: ¥4,400,000 (Direct Cost: ¥4,400,000)
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| Keywords | 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 |
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| Research Abstract |
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.
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