| Project/Area Number |
05454636
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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 |
Biophysics
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| Research Institution | KEIO UNIVERSITY |
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Principal Investigator |
SHIMADA Hideo KEIO UNIVERSITY BIOCHEMISTRY ASSISTANT PROFESSOR, 医学部, 講師 (80095611)
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| Co-Investigator(Kenkyū-buntansha) |
EGAWA Tuyoshi KEIO UNIVERSITY BIOCHEMISTRY INSTRUCTOR, 医学部, 助手 (10232935)
MUKAI Kuniaki KEIO UNIVERSITY BIOCHEMISTRY INSTRUCTOR, 医学部, 助手 (80229913)
KIMATA Yoko KEIO UNIVERSITY BIOCHEMISTRY INSTRUCTOR, 医学部, 助手 (60255429)
HIROSE Tadaaki KEIO UNIVERSITY PHARMACEUTICAL ASSISTANT PROFESSOR, 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 |
¥6,600,000 (Direct Cost: ¥6,600,000)
Fiscal Year 1994: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1993: ¥5,000,000 (Direct Cost: ¥5,000,000)
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| Keywords | CYTOCHROME P450 / CAMPHOR / OXYGENATEDFORM / ACID CATALYST / ARTIFICIAL ENZYME / O-O BOND CLEAVAGE / ARTIFICIAL AMINO ACID / ELECTRON TRANSFER / プロトン / 非天然型アミノ酸 |
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| Research Abstract |
Cytochrome P450cam (P450cam) is a heme-containing monooxygenase that catalyzes the reaction : d-camphor + NADH + H^+ + O_2 * 5-exo-hydroxycamphor + H_2O + NAD^+. In this reaction, 2 electrons from NADH are deliveredto P450cam via putidaredoxinreductaseand putidaredoxin (Pdx). P450cam is reduced by Pdx at the step where it is in the ferric and subsequent oxy-ferrous states.
During two years of this project, we have found that a surface residue, Arg112 in P450cam is crucial for the first and second reduction steps. Kinetic studies suggests that : 1) Arg112 forms the binding site for Pdx. 2) Arg112 is an important residue for intramolecular electron transfer (Pdx-P450cam) and also for controlling redox potentials of the P450cam heme-moiety. Another important results of this project is that we have successfully incorporated site-specifically unnatural amino acid, 0-methyl-threonine to the 112 position of P450cam. Catalytic activity measurement of this mutant shows that the mutant enzyme incorporates all the oxygen atom of molecular dioxygen consumed to 5-exo-position of d-camphor, although oxygen consuming activity is one third of that of the wild-type enzyme. This results indicate that the hydroxygroup of threonine is not indispensable for the monooxygenation reaction.
We previously showed that monooxygnation of d-camphor was only accomplished efficiently when the hydroxygroup is at 112 position of P450cam. Based on these and other results, we proposed a proton donor and/or acid catalysis as a role of Thr252. In order to validate this proposed role of Thr, we have planned this project and showed successfully the importance of the site-specific incorporation of unnatural amino acid to elucidate the catalytic mechanism of the enzyme.
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