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Studies on structure-function relationship of enzymes catalyzing asymmetric syntheses of Michael addition type.

Research Project

Project/Area Number 63470136
Research Category

Grant-in-Aid for General Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field 物質生物化学
Research InstitutionKYOTO UNIVERSITY

Principal Investigator

TOKUSHIGE Masanobu  Kyoto University, Faculty of Science, Professor, 理学部, 教授 (20025266)

Co-Investigator(Kenkyū-buntansha) YUNOTO Noboru  Kyoto University, Faculty of Science, Instructor, 理学部, 助手 (30200877)
Project Period (FY) 1988 – 1989
Project Status Completed (Fiscal Year 1989)
Budget Amount *help
¥7,500,000 (Direct Cost: ¥7,500,000)
Fiscal Year 1989: ¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 1988: ¥4,700,000 (Direct Cost: ¥4,700,000)
KeywordsMichael addition / Aspartase / Fumarase / Asymmetric synthesis / Sequence homology / Hybridization / Site-directed modification / Fe-S cluster / フラマ-ゼ / 不斉合成反応 / 相同性 / 部位指向遺伝子変異 / 鉄-硫黄クラスタ- / マイケル付加反応 / アスパルターゼ / フマラーゼ / オリゴマー酵素 / 遺伝子操作 / 化学修飾 / 構造ー機能相関
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.

Report

(3 results)
  • 1989 Annual Research Report   Final Research Report Summary
  • 1988 Annual Research Report
  • Research Products

    (27 results)

All Other

All Publications (27 results)

  • [Publications] Yumoto,N.&Tokushige,M.: "Characterization of multiple fumarase proteins in Escherichia coli." Biochem.Biophys.Res.Commun.,. 153. 1236-1243 (1988)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Imaishi,H.,Yumoto,N.&Tokushige,M.: "Preparation of active hybrid enzymes composed of the native and chemically inactivated aspartase subunits from Escherichia coli" Biotechnol.Appl.Biochem.,in press. (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Imaishi,H.,Yumoto,N.&Tokushige,M.: "Characterization of intermediate species during the molecular assembly of aspartase." Physiol.Chem.Phys.Med.NMR,in press.(1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Yumoto,N.,Kawata,Y.,Noda,S.&Tokushige,M.: "Rapid purification and characterization of homoserine dehydrogenase from Saccharomyces cerevisiae.,submitted" Eur.J.Biochem.

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Ueda,Y.,Yumoto,N.&Tokushige,M.: "Purification and Characterization of two types of fumarase from Escherichia coli.,submitted" J.Biol. Chem.

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] 徳重正信: "UPバイオロジ-NO.70酵素のはたらき" 東京大学出版会, (1988)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] 徳重正信: "生命の化学最前線" さんえい出版, (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Yumoto,N. & Tokushige,M.: "Characterization of multiple fumarase proteins in Escherichia coli." Biochem. Biophys. Res. Commun., 153, 1236-1243, 1988.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Imaishi,H., Yumoto,N., & Tokushige,M.: "Preparation of active hybrid enzymes composed of the native and chemically inactivated aspartase subunits from Escherichia coli." Biotechnol. Appl. Biochem. 1990.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Imaishi,H., Yumoto,N., & Tokushige,M.: "Characterization of intermediate species during the molecular assembly of aspartase." Physiol. Chem. Phys. Med. NMR, 1990.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Yumoto,N., Kawata,Y., Noda,S., & Tokushige,M.: "Rapid purification and characterization of homoserine dehydrogenase from Saccharomyces cerevisiae." Eur. J. Biochem.,.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Ueda,Y., Yumoto,N., & Tokushige,M.: "Purification and characterization of two types of fumarase from Escherichia coli." J. Biol. Chem.,.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Hiromasa Imaishi: "Preparation of active hybrid enzymes composed of the native and chemically insctivated aspartase subunits from Escherichio coli" Biotrchnology and Applied Biochem. 12. (1990)

    • Related Report
      1989 Annual Research Report
  • [Publications] Hiromasa Imaishi: "Charactorizction of intermediate species during the moleculor assemly of aspartase" Physiol Chem.Phys.Med.NMR.(1990)

    • Related Report
      1989 Annual Research Report
  • [Publications] Noboru Yumoto: "Rapid punification and characterization of homoserine dehydrogenase from Saccharomyces cerevisiae" Eur J.Biochem. (1990)

    • Related Report
      1989 Annual Research Report
  • [Publications] Noboru Yumoto: "Characterization of multiple fumarase proteins in Escheri chio coli" Biochem.Biophys.Res.Commun. 153. 1236-1243 (1988)

    • Related Report
      1989 Annual Research Report
  • [Publications] Yuji Ueda: "Punfication and characterization of two types of fumarase from Escherichia coli" J.Biol.Chem. (1990)

    • Related Report
      1989 Annual Research Report
  • [Publications] Noboru Yumoto: "Site-directed mutagenesis of Cys-430 to Trp-430 of aspartase" (1990)

    • Related Report
      1989 Annual Research Report
  • [Publications] 徳重正信: "酵素のはたらき" 東大出版会, 123 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] 徳重正信偏著: "(株)さんえい出版" 生命の化学最前線, 165 (1990)

    • Related Report
      1989 Annual Research Report
  • [Publications] Yumoto,N.: Biochem.Biophys.Res.Commun. 153. 1236-1243 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] Higashi,Y: Biochem.Int.17. 103-109 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] Higashi,Y: Biochem.Int.16. 449-452 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] Imaishi,H.: Biochim.Biophys.Acta. (1989)

    • Related Report
      1988 Annual Research Report
  • [Publications] Oda,T.: J.Biochem.104. 178-183 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] Tokushige,M.: Biochimie. (1989)

    • Related Report
      1988 Annual Research Report
  • [Publications] 徳重正信: "酵素のはたらき" 東京大学出版会, 1-123 (1988)

    • Related Report
      1988 Annual Research Report

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Published: 1988-04-01   Modified: 2016-04-21  

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