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Comparative Studies on Structure and Function of Pyridoxal Dependent and Indepandent Amino Acid Racemases

Research Project

Project/Area Number 04680188
Research Category

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

Allocation TypeSingle-year Grants
Research Field 代謝生物化学
Research InstitutionKYOTO UNIVERSITY

Principal Investigator

ESAKI Nobuyoshi  Instiute for Chemical Research, Kyoto University, Associate Professor, 化学研究所, 助教授 (50135597)

Co-Investigator(Kenkyū-buntansha) HIRASAWA Toshiko  Instructor Institute for Chemical Research, Kyoto University, Technical Associat, 化学研究所, 教務職員 (80238352)
YOSHIMURA Tohru  Instructor Institute for Chemical Research, Kyoto Univirsity, 化学研究所, 助手 (70182821)
Project Period (FY) 1992 – 1993
Project Status Completed (Fiscal Year 1993)
Budget Amount *help
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1993: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1992: ¥1,100,000 (Direct Cost: ¥1,100,000)
KeywordsAlanine Racemase / Amino Acid Rasemase with Low Substrate Apecificity / Hydroxyproline Epimerase / Glutamate Racemase / Aspartate Racemase / Pyridoxal Phosphate
Research Abstract

Two distinct types of amino acid racemases are known : one requires pyridoxal phosphate as a coenzyme and is represented by alanine racemase, the other requires neither cofactors nor coenzymes and is represented by glutamate racemase. It is interesting and important to compare catalytic mechanisms of these two types of enzymes, both of which catalyze racemization of alph-amino acids. We aimed to study the structures and functions of both types of enzymes, thereby to clarify mechanisms of the amino acid racemization. As to pyridoxal phosphate dependent amino acid racemases, we have chosen thermostable alanine racemase from Bacillus stearothermophilus. We have constructed a mutant enzyme of it in which Lys39 binding the coenzyme pyridoxal phosphate is replaced by alanlne, purified it, and characterized its enzymological properties. In particular, we have studied spectrophotometric properties of the coenzyme complexes and reaction with beta-chloroalanine. Pyridoxal-dependent amino acid racemase with low substrate specificity from Pseudomonas putida has been studied as well. We have cloned its structural gene, purified the enzyme from the clone cells, characterized the enzyme, determined the stereochemistry of hydrogen transfer in the abortive transamination, and studied the reaction mechanism. On the other hand, as to pyridoxal phosphate-independent racemases, glutamate racemase and aspartate racemase, we studied the role of cysteine residues of both enzymes by site-directed mutagenesis and chemical modification. Glutamate racemase loses the ability to transfer alph-proton, when either Cys73 or Cys184 is replaced by Ala or modified. On the other hand, apsartate racemase is completely inactivated by modification or replacement of only one Cys73 per dimeric enzyme.
Therefore, we have proposed that asparate racemase probably has a composit active site.

Report

(3 results)
  • 1993 Annual Research Report   Final Research Report Summary
  • 1992 Annual Research Report
  • Research Products

    (31 results)

All Other

All Publications (31 results)

  • [Publications] Soo-Young Choi: "Reaction Mechanism of Glutamate Racemase,A Pyridoxal Phosphate-Independent Amino Acid Racemase" Journal of Biochemistry. 112. 139-142 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Jeong Weon Huh: "Synthesis of L-Proline from The Racemate by Coupling of Enzymatic Enantiospecific Oxidation and Chemical Non-Enantiospecific Reduction" Journal of Fermentation and Bioengineering. 74. 189-190 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Tohru Yoshimura: "Structure and Function of Alanine Racemase" Bulletin of the Institute for Chemical Research,Kyoto University. 70. 378-384 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Jeong Weon Huh: "Total Conversion of Racemic Pipecolic Acid into the L-Enantiomer by a Combination of Enantiospecific Oxidation with D-Amino Acid Oxidase and Reduction with Sodium Borohydride" Bioscience,Biotechnology,and Biochemistry. 56. 2081-2082 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Nobuyoshi Nakajima: "Purification and Characterization of Aldehyde Reductase from Leuconostoc dextranicum" Bioscience,Biotechnology,and Biochemistry. 57. 160-161 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Takashi Tamura: "Synthesis and Characterization of the Selenium Analog of Glutathione Disulfide" Analytical Biochemistry. 208. 151-154 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Kenji Soda: "Proceedings of the 1st International Congress on Vitamins and Biofactores in Life Science in Kobe,1991" Center for Academic Purificatons Japan, 606 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Nobuyoshi Esaki: "Proceedings of the 1st International Congress on Vitamins and Biofactores in Life Science in Kobe,1991" Center for Academic Purificatons Japan, 606 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] S.-Y.Choi, N.Esaki, T.Yoshimura, and K.Soda: "Reaction Mechanism of Glutamate Racemase, a Pyridoxal Phosphate-Independent Amino Acid Racemase." J.Biochem.112. 139-142 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] J.W.Huh, K.Yokoigawa, N.Eskai, and K.Soda: "Synthesis of L-Proline from the Racemase by Coupling of Enzymatic Enantiospecific Oxidation and Chemical Non-Enantiospecific Reduction." J.Ferment.Bioeng.74. 189-190 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] T.Yoshimura, N.Esaki, and K.Soda: "Structure and Function of Alanine Racemase." Bull.Inst.Chem.Res., Kyoto Univ.70. 378-384 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] J.W.Huh, K.Yokoigawa, N.Esaki, and K.Soda: "Total Conversion of Racemic Pipecolic Acid into the L-Enantiomer by a Combination of Enantiospecific Oxidation with D-Amino Acid Oxidase and Reduction with Sodium Borohydride" Biosci.Biotech.Biochem.56. 2081-2082 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] N.Nakajima, K.Nakamura, H.Sumi, A.Matsuyama, N.Esaki, and K.Soda: "Purification and Characterization of Aldehyde Raductase from Leuconostoc dextranicum" Biosci.Biotech.Biochem..57. 160-161 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] T.Tamura, T.Oikawa, A.Ohtaka, N.Fujii, N.Esaki, and K.Soda: "Synthesis and Characterization of the Selenium Analog of Glutathione Disulfide" Analytical Biochemistry. 208. 151-154 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] K.Soda, H.Toyama, and N.Esaki: ""Frontiers and New Horizons in Amino Acid Research"" (K.Takai ed.) Elsevier. 327-332 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Soo-Young Choi: "Reaction Mechanism of Glutamate Racemase,A Pyridoxal Phosphate-Independent Amino Acid Racemase" Journal of Biochemistry. 112. 139-142 (1992)

    • Related Report
      1993 Annual Research Report
  • [Publications] Jeong Weon Huh: "Synthesis of L-Proline from The Racemate by Coupling of Enzymatic Enantiospecific Oxidation and Chemical Non-Enantiospecific Reduction" Journal of Fermentation and Bioengneering. 74. 189-190 (1992)

    • Related Report
      1993 Annual Research Report
  • [Publications] Tohru Yoshimura: "Structure and Function of Alanine Racemase" Bulletin of the Institute for Chemical Research,Kyoto University. 70. 378-384 (1992)

    • Related Report
      1993 Annual Research Report
  • [Publications] Jeong Weon Huh: "Total Conversion of racemic Pipecolic Acid into the L-Enantiomer by a Combination of Enantiospecific Oxidation with D-Amino Acid Oxidase and Reduction with Sodium Borchydride" Bioscience,Biotechnology,and Biochemistry. 56. 2081-2082 (1992)

    • Related Report
      1993 Annual Research Report
  • [Publications] Nobuyoshi Nakajima: "Purification and Characterization of Aldehyde Reductase from Leuconostoc dextranicum" Bioscience,Biotechnology,and Biochemistry. 57. 160-161 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] Takashi Tamura: "Synthesis and Characterization of the Selenium Analog of Glutathione Disulfide" Analytical Biochemistry. 208. 151-154 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] Kenji Soda: "Proceedings of the 1st International Congress on Vitamins and Biofactores in Life Science in Kobe,1991" Center for Academic Purifications Japan, 606 (1992)

    • Related Report
      1993 Annual Research Report
  • [Publications] Nobuyoshi Esaki: "Proceedings of the 1st International Congress on Vitamins and Biofactores in Life Science in Kobe,1991" Center for Academic Purifications Japan, 606 (1992)

    • Related Report
      1993 Annual Research Report
  • [Publications] Soo-Young Choi: "Reaction Mechanism of Glutamate Racemase,A Pyridoxal Phosphate-Independent Amino Acid Racemase" Journal of Biochemistry. 112. 139-142 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Jerong Weon Huh: "Synthesis of L-Proline from The Racemate by Coupling of Enzymatic Enantiospecific Oxidation and Chemical Non-Enantiospecific Reduction" Journal of Fermentation and Bioengineering. 74. 189-190 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Tohru Yoshimura: "Structure and Function of Alanine Racemase" Bulletin of the Institute for Chemical Research,Kyoto University. 70. 378-384 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Jeong Weon Hush: "Total Conversion of Racemic Pipecolic Acid into the L-Enantiomer by a Combination of Enantiospecific Oxidation with D-Amino Acid Oxidase and Reduction with Sodium Borchydride" Bioscience,Biotechnology,and Biochemistry. 56. 2081-2082 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Nobuyoshi Nakajima: "Purification and Characterization of Aldehyde Reductase from Leuconostoc dextranicum" Bioscience,Biotechnology,and Biochemistry. 57. 160-161 (1993)

    • Related Report
      1992 Annual Research Report
  • [Publications] Takashi Tamura: "Synthesis and Characterization of the Selenium Analog of Glutathione Disulfide" Analytical Biochemistry. 208. 151-154 (1993)

    • Related Report
      1992 Annual Research Report
  • [Publications] Kenji Soda: "Proceedings of the 1st International Congress on Vitamins and Biofactores in Life Science in Kobe, 1991" Center for Academic Purificatons Japan, 606 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Nobuyoshi Esaki: "Proceedings of the 1st International Congress on Vitamins and Biofactores in Life Science in Kobe, 1991" Center for Academic Purificatons Japan, 606 (1992)

    • Related Report
      1992 Annual Research Report

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Published: 1992-04-01   Modified: 2016-04-21  

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