1989 Fiscal Year Final Research Report Summary
Studies on structure-function relationship of enzymes catalyzing asymmetric syntheses of Michael addition type.
| Project/Area Number |
63470136
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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 |
TOKUSHIGE Masanobu Kyoto University, Faculty of Science, Professor, 理学部, 教授 (20025266)
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| Co-Investigator(Kenkyū-buntansha) |
YUNOTO Noboru Kyoto University, Faculty of Science, Instructor, 理学部, 助手 (30200877)
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| Project Period (FY) |
1988 – 1989
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| Keywords | Michael addition / Aspartase / Fumarase / Asymmetric synthesis / Sequence homology / Hybridization / Site-directed modification / Fe-S cluster |
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| Research Abstract |
In order to elucidate the structure-function characteristics of aspartases and fumarases, which catalyze asymmetric synthetic reactions of Michael addition type, enzymatic and genetic studies were carried out, and the following results were obtained.
1) Aspartase of Escherichiac coli, composed of 4-identical subunits was inactivated by chemical-modification with N-ethylmaleimide (NEM) and denatured in 4 M guanidine-HCl (GuHC1) in the presence of various ratio of the active enzyme. The denatured mixture was then renatured by dilution with dilute phosphate buffer, pH 7.4. The hybridized enzyxne exhibited the aspartase activity in proportion to the content of the unmodified subunit, indicating that as long as the quaternary structure is proper, the enzyme activity can be exhibited, even if some subunits are inactive.
2) Cysteind 430 of aspartase, which participates in the activation of the catalytic activity was genetically converted to tryptophan using synthetic oligonucleotides. Trp-430-containing aspartase exhibited 4 times higher enzyme activity at acidic pH, and the requirement for divalent metal ions was also increased.
3) Available-evidence suggests that E. colicells contain three fumarases, FUMA, FUMB, dnd FUMC. We purified a fumarase which required divalent ferrous ions for its activity. The purified enzyme was found to contain iron-S cluster like aconitase and was identified as a product of fumA. FUMC, which did not require ferrous ions was also purified to homogeneety.
4) Freviously we utilized aspartic beta-semialdehyde (ASA) as a kind of suicide substrate of aspartase. In order to extend this study, we established a rapid purification procedure for homoserine dehydrogenase of yeast, by which a large amount of ASA supply became possible.
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