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The novel substrate recognition mechanism utilized by thermophilic aspartate aminotransferase

Research Project

Project/Area Number 09680619
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Functional biochemistry
Research InstitutionOsaka University

Principal Investigator

KURAMITSU Seiki  Osaka University, Graduate School of Science Professor, 大学院・理学研究科, 教授 (60153368)

Project Period (FY) 1997 – 1998
Project Status Completed (Fiscal Year 1998)
Budget Amount *help
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 1998: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1997: ¥2,600,000 (Direct Cost: ¥2,600,000)
KeywordsThermus thermophilus / aminotransferase / transferase / X-ray crystallography / lysine / substrate recognition mechanism / arginine / 蛋白質工学 / 酸性基質 / 変異型酵素 / カルボキシル基 / 基質結合部位 / X線結晶解析 / 疎水性基質
Research Abstract

It has been believed that an enzyme is quite specific for its substrate.But aminotransferases are quite unique enzymes which can bind quite different binds of substrate.
These enzymes have two different types of binding pockets for acidic and hydrophobic substrates. (Catalytic residue is identical for each substrates.) Acidic substrate binds rigid region (Kawaguchi et al.(1997) J.Biochem.122, 55-63) and hydrophobic substrate binds flexible region (Kawaguchi and Kuramitsu, (1998) J.Biol. Chem. 273, 18353-18364).
The three-dimensional structure of aspartate aminotransferase from extreme thermophile, Thermus thermophilus HB8 have been determined by X-ray crystallography.In view of the X-ray crystallographic structure of T.thermophilus AspAT, K1O9V mutant was prepared.Replacing K109 with Val resulted in loss of activity toward acidic substrates, but increased that toward the neutral substrate, alanine, considerably.These results indicate that K109 is a major determinant of the acidic substrate specificity of T.thermophilus AspATs. Kinetic analysis of the interactions with neutral substrates indicated that T.thermophilus AspAT is subject to less steric hindrance and its substrate-binding pocket has a more flexible conformation than E.coli AspAT.A flexible active site in the rigid T.thermophilus AspAT molecule may explain its high activity even at room temperature.

Report

(3 results)
  • 1998 Annual Research Report   Final Research Report Summary
  • 1997 Annual Research Report
  • Research Products

    (20 results)

All Other

All Publications (20 results)

  • [Publications] Nakai,T.: "Structure of Thermus thermophilus HB8 Aspartate Aminotransferase and Its Complex with Maleate" Biochemistry. 38・8. 2413-2424 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Nobe,Y.: "The Novel Substrate Recognition Mechanism Utilized in Aspartate Aminotransferase of Thermus thermophilus HB8" J.Biol.Chem.273・45. 29554-29564 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Kawaguchi,S.: "Thermodynamics and Molecular Simulation Analysis of Hydrophobic Substrate Recognition by Aminotransferases" J.Biol.Chem.273・29. 18353-18364 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Kawaguchi,S.: "Enzyme Flexibility : New Concept in Recognition of Hydrophobic Substrates" J.Biochem.55・1. 55-63 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Kawaguchi, S., Nobe, Y., Yasuoka, J., Wakamiya, T., Kusumoto, S., and Kuramitsu, S.: "Enzyme Flexibility : New Concept in Recognition of Hydrophobic Substrates" J.Biochem.122(No.1). 55-63 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Kawaguchi, S.and Kuramitsu, S.: "Thermodynamics and Molecular Simulation Analysis of Hydrophobic Substrate Recognition by Aminotransferases" J.Biol.Chem.273(No, 29). 18353-18364 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Nobe, Y., Kawaguchi, S., Ura, H., Nakai, T., Hirotsu, K., Kato, R., and Kuramitsu, S.: "The Novel Substrate Recognition Mechanism Utilized in Aspartate Aminotransferase of Extreme Thermophile, Thermus thermophilus HB8" J.Biol.Chem.273(No, 45). 29554-29564 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Nakai, T., Okada, K., Akutsu, S., Miyahara, I., Kawaguchi, S., Kato, R., Kuramitsu, S., and Hirotsu, K.: "Structure of Thermus thermophilus HB8 Aspartate Aminotransferase and Its Complex with Maleate" Biochemistry. 38(No.8). 2413-2424 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Kawaguchi,S.: "Thermodynamics and Molecular Simulation Analysis of Hydrophobic Substrate Recognition by Aminotransferases" J.Biol.Chem.273. 18353-18364 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Nobe,Y.: "The Novel Substrate Recognition Mechanism Utilized in Aspartate Aminotransferase of Thermus thermophilus HB8" J.Biol.Chem.273. 29554-29564 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Nakai,T.: "Crystallization and Preliminary X-Ray Characterization of Aspartate Aminotransferase from Thermus thermophilus HB8" Acta Cryst.D54. 1032-1034 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] 倉光成紀: "「高度好熱菌丸ごと一匹プロジェクト-基本的生命現象の系統的解析-」へ向けてのボランティア" 生産と技術. 50. 70-72 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] 増井良治: "好熱菌全ゲノムの配列決定とその意義" 蛋白質核酸酵素. 44. 165-170 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] 河口真一: "朝倉書店,東京" 「シリーズ分子生物学 6.構造生物学」, 11 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Kawaguchi,S.: "Enzyme Flexibility : New Concept in Recognition of Hydrophobic Substrates" J.Biochem.55. 55-63 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Putukhov,M.: "Insights into Thermal Resistance of Proteins from Intrinsic Stability of Their α-Helices" Proteins : Structure,Function,and Genetics. 29. 309-320 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Kawaguchi,S.: "New Concept of the Enzymatic Recognition of Hydrophobic Substrates" Protein Science. 6. 66-66 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Nakai,T.: "Structural Study of Extremely Thermophilic Bacterium Aminotransferase" Protein Science. 6. 94-94 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] 倉光成紀: "「高度好熱菌丸ごと一匹プロジェクト -基本的生命現象の系統的解析-」へ向けてのボランティア" 生産と技術. (印刷中). (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] 倉光成紀: "アミノ基転移酵素" 朝倉書店,東京, 11 (1997)

    • Related Report
      1997 Annual Research Report

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Published: 1997-04-01   Modified: 2016-04-21  

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