| Project/Area Number |
05454631
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Functional biochemistry
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| Research Institution | Keio University |
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Principal Investigator |
ISHIMURA Yuzuru School of Medicine Keio University, Professor, 医学部, 教授 (40025599)
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| Co-Investigator(Kenkyū-buntansha) |
EGAWA Tsuyoshi School of Medicine Keio University, Assistant, 医学部, 助手 (10232935)
MIKI Hideho School of Medicine Keio University, Assistant, 医学部, 助手 (90229667)
SHIMADA Hideo School of Medicine Keio University, Assist.Prof., 医学部, 講師 (80095611)
HIROSE Tadaaki School of Medicine Keio University, Assist.Prof., 医学部, 講師 (60051405)
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| Project Period (FY) |
1993 – 1994
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| Project Status |
Completed (Fiscal Year 1994)
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| Budget Amount *help |
¥6,900,000 (Direct Cost: ¥6,900,000)
Fiscal Year 1994: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1993: ¥5,400,000 (Direct Cost: ¥5,400,000)
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| Keywords | Cytochrome P450 / Oxygen metabolism / Activated forms of oxygen / Oxygenases / Monooxygenases / Site-directed mutagenesis / NADPH-oxidase / Oxgen Activation mechanism / NADPHオキシダーゼ |
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| Research Abstract |
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.).
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