| Project/Area Number |
61480460
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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 |
物質生物化学
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| Research Institution | Kyoto University |
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Principal Investigator |
TOKUSHIQE Masanobu Kyoto University,Faculty of Science Professor, 理学部, 教授 (20025266)
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| Co-Investigator(Kenkyū-buntansha) |
NISHINO Tokuzo Kyoto University, Faculty of Science Instructor, 理学部, 助手 (90005827)
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| Project Period (FY) |
1986 – 1987
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| Project Status |
Completed (Fiscal Year 1987)
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| Budget Amount *help |
¥6,000,000 (Direct Cost: ¥6,000,000)
Fiscal Year 1987: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1986: ¥4,000,000 (Direct Cost: ¥4,000,000)
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| Keywords | Aspartase / fumarase / tryptophanase / Site-directed mutagenesis / Schiff base-dependent deamination / Schiff base-independent deamination / マイケル付加反応 / 酵素機能発現機構 / 酵素の機能変換 / 酵素の一次構造と活性部位構造 / シッフ塩基 / 脱アミノ酵素 |
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| Research Abstract |
Comparative studies were carride out on the molecular structure and reaction mechanisms of tryptophanase, a Schiff-base-dependent enzyme, and aspartase, a Schiff base-nondependent enzyme, for amino group elimination and the following results were obtained.
I. Studies on tryptophanase:
1) Some monovalent cations such as K^+ and NH^+_ were found to convert the inactive holoenzyme into the active species and also to stabilize the latter species.
2) The Cys-298 was found to be essential for the above effects of the cations by means of the analysis with fluorescent probes.
3) In addition to the sulfhydryl groups, the methionyl residues were found to be also essential for the enzyme activity by chemical modification with chloramine-T.
II. Studies on aspartases.
1) Aspartase gene in Pseudomonas fluorescens cell was cell was cloned and the amino acid sequence was deduced from the base sequence.
2) Extensive homology was found in aspartases and fumarases by computer analysis.
3) Fumalaldehydic acid, a structural analog of the reaction intermediate, irreversibly inactivated aspartase form E. coli.
4) S-2, 3-Dicarboxyazairidine, an antibacterial agent isolated from the soil, competitively inhibited the activity of aspartase as well as fumarase.
5) Cys-430 in the E. coli aspartase was changed to tryptophan by the site-directed mutagenesis and the latter enzyme was characterized in comparison with the former enzyme.
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