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1991 Fiscal Year Final Research Report Summary

Studies on Active Site of Transaminase by Site-Directed Mutagenesis

Research Project

Project/Area Number 01480524
Research Category

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

Allocation TypeSingle-year Grants
Research Field 物質生物化学
Research InstitutionOsaka Medical College

Principal Investigator

KAGAMIYAMA Hiroyuki  Osaka Medical College, Medical School, Professor, 医学部, 教授 (80028555)

Project Period (FY) 1989 – 1991
KeywordsMutagenesis / Transaminase / Aspartate Aminotransferase / Enzyme Mechanism / Pyridoxal Phosphate / Active Site / Protein Engineering
Research Abstract

We have examined the functional role of some active site residues of E. coli aspartate aminotransferase by using kinetic analysis on mutant enzymes produced by the site-directed mutagenesis. Aspartate aminotransferase has been the most extensively studied representative of many transaminases ; X-ray analysis has been done. Our findings are as follows :
1. Lys258 is essential for catalysis, acting as a catalytic base to withdraw an a -proton from the amino acid substrate, which is a prerequisite for the catalysis.
2. A negative charge at position 222, and a hydrogen bond between the hydroxyl group of Tyr225 and the unprotonated hydroxyl group of the coenzyme probably help in lowering the electron density of the coenzyme to facilitate the a- proton removal.
3. Arg292 and Arg386 are essential for the recognition of the dicarboxylic substrates.
4. Substitution of Arg292 to uncharged residues greatly enhanced the catalytic efficiency of the transamination of neutral amino acid without. any effect on the binding.
5. The endole ring of Trpl4O not only regulates the rotational movement of the coenzyme ring during the catalysis, but it may be also involved in the binding of the carboxyl side chain of the dicarboxylic acid substrates.
6. The phenol group of Tyr7O is essential for the stabilization of the transition states with all substrates. The presence of the benzen ring at position 70 is necessary to recognize the glutanate-2-oxoglutarate substrate pair.

  • Research Products

    (42 results)

All Other

All Publications (42 results)

  • [Publications] Nagashima,F.: "cDNA Cloning and Expression of Pig Cytosolic Aspartate Aminotransferase in Escherichia coli:Aminoterminal Heterogeneity of Expressed Products and Lack of Its Correlation with Enzyme Function." Biochemistry. 28. 1153-1160 (1989)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Tanizawa,K.: "The Primary Structure of Thermostable D-Amino Acid Aminotransferase from A Thermophilic Bacillus Species,and Its Correlation with L-Amino Acid Aminotransferase." J.Biol.Chem.264. 2450-2454 (1989)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Inoue,Y.: "Substitution of A Lysyl Residue for Arginine386 of Escherichia coli Aspartate Aminotransferase." J.Biol.Chem.9673-9681 (1989)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Hayashi,H.: "〔Arg292--Val〕or 〔Arg292--Leu〕 Mutation Enhances The Reactivity of Escherichia coli Aspartate Aminotransferase with Aromatic Amino Acids." Biochem.Biophys.REs.Commun.159. 337-342 (1989)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] kamitori,S.: "Crystallization and Preliminary X-Ray Characterization of Branched-Chain Amino acid Aminotransferase From Escherichia coli." J.Biochem.105. 671-672 (1989)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 倉光 成紀: "Siteーdirected mutagenesisによるタンパク質機能の解析" 臨床科学. 25. 249-259 (1989)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 林 秀行: "アミノ酸転移酵素研究の新展開(蛋白質化学から蛋白質工学へ)" 蛋白質 核酸 酵素. 34. 1893-1903 (1989)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Hayashi,H.: "Effect of Replacement of Tryptophan-140 by Phenylalanine or Glycine on The Function of Escherichia coli Aspartate Aminotransferase." Biochem.,Biophys.Rse.Commun.167. 407-412 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Sung,M-H.: "Purification and Characterization of Thermostable Aspartate Aminotransferase From A Thermophilic Bacillus Species." J.Bacteriol.172. 1345-1351 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kuramitsu,S.: "Pre-Steady State Kinetics of Escherichia coli Aspartate Aminotransferase Catalyzed Reactions and Thermodynamic Aspects of Its Sustrate Specificty." Biochemistry. 29. 5469-5476 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Morino,Y.: "Mammalian Aspartate Aminotransferase Isozymes:From DNA to Protein." Annals of The New York Academy of Sciences. 585. 32-47 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kamitori,S.: "Three-Dimensional Structures of Aspartate Aminotransferase Form Escherichia coli and Its Mutant Enzyme at 2.5A Resolution." J.Biochem.108. 175-184 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 倉光 成紀: "アスパラギン酸トランスアミナ-ゼ" プロテインエンジニアリング(現代化学). 増刊19. 64-73 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Sung,M-H.: "Thermostable Aspartate Aminotransferase From A Thermophilic Bacillus species:Gene Cloning,Sequence Determination,and preliminary X-Ray Characterization." J.Biol.Chem.266. 2567-2572 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Inoue,K.: "Tyr225 in Aspartate Aminotransferase:Contribution of The Hydrogen Bond Between Tyr225 and Coenzyme to The Catalytic Reaction." J.Biochem.109. 570-576 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Hayashi,H.: "Replacement of An Interdomain Residue Va139 Escherichia coli Aspartate aminotransferase Affects The Catalytic Copetence Without Altering the Substrate Specificity of The Enzyme." J.Biochem.109. 699-704 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Yano,T.: "The Rolr of His143 in The Catalytic Mechanism of Escherichia coli Aspartate Aminotransferase." J.Biol.Chem.266. 6079-6085 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Inoue,K.: "Site-Directed Mutagenesis of Escherichia coli Aspartate Aminotransferase:Role of Tyr70 in The Catalytic Process." Biochemistry. 30. 7796-7801 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Sung,M-H.: "Molecular Cloning and Sequence Determination of Thermostable aspartate Aminotransferase From Thermophilic Bacillus Species." Enzymes Dependent on Pyridoxal Phosphate and Other Carbonyl Compounds as Cofactors.(Fukui,T.et al.eds.). 55-57 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Okamota,A.: "Three-Dimensional Structure of Aspartate Aminotransferase Form Escherichia coli." Enzymes Dependent on Pyridoxal Phosphate and Other Carbonyl Compounds as Cofactors.(Fukui,T.et al eds.). 107-109 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kuramitsu,S.: "Asp222,His143,and Tyr70 in Escherichia coli Aspartate Aminotransferase." Enzymes Dependent on Pyridoxal Phosphate and Other Carbonyl Compounds as Cofactors.(Fukui,T.et al eds.). 179-181 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Hayashi,H.: "Escherichi coli Aspartate Aminotransferase:Effect of Site-Specific Mutations on Substrate Binding and Catalysis." Enzymes Dependent on Pyridoxal Phosphate and Other Carbonyl Compounds as Cofactors.(Fukui,T.et al eds.). 183-186 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 倉光 成紀: "ピリドキサル酵素の蛋白工学" 蛋白工学シリ-ズ蛋白質立体構造の新しい視点(森川耿右編). 152-173 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Nagashima, F.: "cDNA Cloning and Expression of Pig Cytosolic Aspartate Amino-transferease in Escherichia coli : Aminoterminal Heterogeneity of Expressed Products and Lack of Its Correlation with Enzyme Function." Biochemistry. 28. 1153-1160 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Tanizawa, K.: "The Primary Structure of Thermostable D-Amino Acid Aminotransferase from A Thermophilic Bacillus Species, and Its Correlation with L-Amino Acid Aminotransferase." J. Biol. Chem.264. 2450-2454 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Inoue, Y.: "Substitution of A Lysyl Residue for Arginine386 of Escherichia coli Aspartate Aminotransferase." J. Biol. Chem.9673-9681 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Hayashi, H.: "[Arg292--Val] or [Arg292--Leu] Mutation Enhances The Reactivity of Escherichia coli Aspartate Aminotransferase with Aromatic Amino Acids." Biochem. Biophys. Res. Commun.159. 337-342 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kamitori, S.: "Crystallization and Preliminary X-Ray Characterization of Branched-Chain Amino acid Aminotransferase From Escherichia coli." J. Biochem.105. 671-672 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Hayashi, H.: "Effect of Replacement of Tryptophan-140 by Phenylalanine or Glycine on The Function of Escherichia coli Aspartate Aminotrans-ferase." Biochem., Biophys. Rse. Commun.167. 407-412 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Sung, M-H.: "Purification and Characterization of Thermostable Aspartata Aminotransferase From A Thermophilec Bacillus Species." J. Bacteriol.172. 1345-1351 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kuramitsu, S.: "Pre-Steady State Kinetics of Escherichia coli Aspartate Amino-transferase Catalyzed Reactions and Thermodynamic aspects of Its Sustrate Specificty." Biochmeistry. 29. 5469-5476 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Morino, Y.: "Mammalian Aspartate Aminotransferase Isozymes : From DNA to Protein." Annals of The Nwe York Academy of Sciences. 585. 32-47 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kamitori, S.: "Three-Dimensional Structures of Aspartate Aminotransferase From Escherichia coli and Its Mutant Enzyme at 2.5A Resolution." J. Biochem.108. 175-184 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Sung, M-H.: "Thermostable Aspartate Aminotransferase From A Thermophilic Bacillus Species : Gene Cloning, Sequence Determination, and Preliminary X-Ray Characterization." J. Biol. Chem.266. 2567-2572 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Inoue, K.: "Tyr225 in Aspartate Aminotransferase : Contribution of The Hydrogen Bond Between Tyr225 and Coenzyme to The Catalytic Reaction." J. Biochem.109. 570-576 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Hayashi, H.: "Replacement of An Interdomain Residue Va139 of Escherichia coli Aspartate aminotransferase Affects The Catalytic Competence With-out Altering the Substrate Specificity of The Enzyme." J. Biochem.109. 699-704 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Yano, T.: "The Rolr of His143 in The Catalytic Mechanism of Escherichia coli Aspartate Aminotransferase." J. Biol. Chem.266. 6079-6085 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Inoue, K.: "Site-Directed Mutagenesis of Escherichia coli Aspartate Aminotransferase : Role of Tyr70 in The Catalytic Process." Biochemistry. 30. 7796-7801 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Sung, M-H.: "Molecular Cloning and Sequence Determination of Thermostable aspartate Aminotransferase From Thermophilic Bacillus Species." Enzymes Dependent on Pyridoxal Phosphate and Other Carbonyl Compounds as Cofactors.(Fukui, T. et al. eds.). 55-57 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Okamota, A.: "Three-Dimensional Structure of Aspartate Aminotransferase From Escherichia coli." Enzymes Dependent on Pyridoxal Phosphate and Other Carbonyl Compounds as Cofactors.(Fukui, T. et al. eds.). 107-109 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kuramitsu, S.: "Asp222, His143, and Tyr70 in Escherichia coli Aspartate Amino-transferase." Enzymes Dependent on Pyridoxal Phosphate and Other Carbonyl Compounds as Cofactors.(Fukui, T. et al. eds.). 179-181 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Hayashi, H.: "Escherichi coli Aspartate Aminotransferase : Effect of Site-Specific Mutations on Substrate Binding and Catalysis." Enzymes Dependent on Pyridoxal Phosphate and Other Carbonyl Compounds as Cofactors.(Fukui, T. et al. eds.). 183-186 (1991)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 1993-03-16  

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