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Protein Engineering of Enzymes

Research Project

Project/Area Number 06454654
Research Category

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

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

Principal Investigator

KURAMITU Seiki  Osaka Univ., Fac.Sci., Professor, 理学部, 教授 (60153368)

Project Period (FY) 1994 – 1995
Project Status Completed (Fiscal Year 1995)
Budget Amount *help
¥7,000,000 (Direct Cost: ¥7,000,000)
Fiscal Year 1995: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1994: ¥5,600,000 (Direct Cost: ¥5,600,000)
KeywordsEnzymatic Mechanism / One Enzyme-Two Substrates / Hydrophobic Substrate / Hydrophobic Interaction / Dynamics / Fluctuation / Protein Engineering / Gene Engineering / 一酵素二基質 / 多機能酵素 / 触媒反応機構 / 基質認識機構 / 部位特異的変異法 / キメラ / X線結晶解析 / アミノ基転移酵素
Research Abstract

Many enzymes have reactivities for only one kind of substrate and their substrate specificities are very narrow. It was shown that the hydrophobic substrate specificity of aminotransferase is surprisingly broad. Aminotransferase catalyzes reversible transamination between amino acid and keto acid substratevia the ping-pong bi-bi mechanism. A series of aliphatic amino acids and keto acids with straight and terminal-branched side chains were used to study the hydrophobic substrate specificity. These substrates were useful for studying the effect of one additional substrate methyl group. The activity of aminotransferase generally increased with an increase in the chain length of the substrate. In particular, when the number of carbons of the substrate was between 4 and 7, the additional effect of one methyl group for stabilization of the transition state was constant. This suggests that the hydrophobic substrate pocket has a uniform hydrophobic environment and can equally recognize the methyl group in the substrate-binding pocket. It is suggested that the determinant of the hydrophobic substrate specificity is a spatial distribution of residues with plasticity in the substrate-binding pocket. The recognition of manifold hydrophobic substrates with different shapes requires precise re-packing by the enzyme and might, therefore, require flexibility of the binding pocket and/or the whole enzyme molecule. In other words, the strict substrate specificity is in contrast to the plasticity of the enzyme.This concept will be applicable to many enzymes that recognize hydrophobic substrates and will be useful for constructing a hydrophobic ligand binding protein.

Report

(3 results)
  • 1995 Annual Research Report   Final Research Report Summary
  • 1994 Annual Research Report
  • Research Products

    (44 results)

All Other

All Publications (44 results)

  • [Publications] Matsushima, Y.: "Replacement of Active Site Lysine-239 of Thermostable Aspartate Amino-transferase by S-(2-Aminoethyl) Cysteine: Properties of the Mutant Enzyme" J. Biochem.115. 108-112 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Miyazawa, K.: "Construction of Aminotransferase Chimeras and Analysis of Their Substrate Specificity" J. Biochem.115. 568-577 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Okamoto, A.: "X-Ray Crystallographic Study of Pyridoxal 5′-Phosphate-Type Aspartate Aminotransferase from Escherichia coli in Open and Closed Forms" J. Biochem.116. 95-107 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Kawaguchi, S.: "Homologus Ligation" Trends Genet.10. 420-420 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Metzler, D. E.: "NMR Studies of ^1H Resonances in the 10-18-ppm Range for Aspartate Aminotransferase from Escherichia coli" J. Biol. Chem.269. 28027-28033 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Kawaguchi, S.: "Construction of Aminotransferase Chimeras and Analysis of Their Substrate Specificity" Protein Science. Vol. 3,. 87-87 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Kuramitsu, S.: "Enzymes with Two Substrate Binding Pockets" Protein Science. Vol. 3,. 129-129 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Kuramitsu, S.: "Enzymes that Recognize Two Different Kinds of Substrate" Protein Eng.7. 1152-1153 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 河口真一: "酵素反応のダイナミックス" 蛋白質核酸酵素. 39. 1083-1090 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Kawaguchi, S.: "Separation of Heat-Stable Proteins from Thermus thermophilus HB8 by Two-Dimensional Electrophoresis" Electophoresis. 16. 1060-1066 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Kawaguchi, S.: "Homologous Ligation" Protein Science. Vol. 4,. 162-162 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Kuramitsu, S.: "Database of Heat-Stable Proteins from Thermus thermophilus HB8" Protein Eng.8. 964-964 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Kawaguchi, S.: "Homologous Ligation" Protein Eng.8. 965-965 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Okamoto, A.: "An Aspartate Aminotransferase from an Extremely Thermophilic Bacterium, thermus thermophilus HB8" J. Biochem.119. 135-144 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Metzler, D. E.: "Biochemistry of Vitamin B6 and PQQ" Birkhaeuser Verlag, 5 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 倉光成紀: "分子生物学の展開,第6巻「アミノ基転移酵素」" 朝倉書店, ((印刷中))

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Matsushima, Y.: "Replacement of Active Site Lysine-239 of Thermostable Aspartate Aminotransferase by S- (2-Aminoethyl) Cysteine : Properties of the Mutant Enzyme" J.Biochem.115 (No.1). 108-112 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Miyazawa, K.: "Construction on Aminotransferase Chimeras and Analysis of Their Substrate Specificity" J.Biochem.115 (No.3). 568-577 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Okamoto, A.: "X-Ray Crystallographic Study of Pyridoxal 5'-Phosphate-Type Aspartate Aminotransferase from Escherichia coliin Open and Closed Forms" J.Biochem.116 (No.1). 95-107 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Kawaguchi, S.: "Homologus Ligation" Trends Genet.10 (No.12). 420 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Metzler, D.E.: "NMR Studies of ^1H Resonances in the 10-18-ppm Range for Aspartate Aminotransferase from Escherichia coli" J.Biol.Chem.269 (No.45). 28027-28033 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Kawaguchi, S.: "Construction on Aminotransferase Chimeras and Analysis of Their Substrate Specificity" Protein Science. 3 (Suppl.1). 87-87 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Kuramitsu, S.: "Enzymes with Two Substrate Binding Pockets" Protein Science. 3 (Suppl.1). 129-129 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Kuramitsu, S.: "Enzymes that Recognize Two Different Kinds of Substrate" Protein Eng.7 (No.9). 1152-1153 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Metzler, D.E.: "Using ^1H-and ^<15>N-NMR Spectroscopy to Observe Active Sites of Porcine Cytosolic and Escherichia coli Aspartate Aminotransferase" "Biochemistry of Vitamin B6 and PQQ" (Marino, G., Sannia, G., and Bossa, F., eds.) Birkhaeuser Verlag, Basel. 55-59 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Kawaguchi, S.: "Separation of Heat-Stable Proteins from Thermus thermophilus HB8 by Two-Dimensional Electrophoresis" Electophoresis. 16. 1060-1066 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Kawaguchi, S.: "Homologous Ligation" Protein Science. 4 (Suppl.1). 162-162 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Kuramitsu, S.: "Database of Heat-Stable Proteins from Thermus thermophilus HB8" Protein Eng.8 (No.9). 964-964 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Kawaguchi, S.: "Homologous Ligation" Protein Eng.8 (No.9). 965-965 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Okamoto, A.: "An Aspartate Aminotransferase from an Extremely Thermophilic Bacterium, Thermus thermophilus HB8" J.Biochem.119 (No.1). 135-144 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Kawaguchi,S.: "Separation of Heat-Stable Proteins from Thermus thermophilus HB8 by Two-Dimensional Electrophoresis" Electophoresis. 16. 1060-1066 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Kuramitsu,S.: "Database of Heat-Stable Proteins from Thermus thermophilus HB8" Protein Eng.8. 964- (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Kawaguchi,S.: "Homologous Ligation" Protein Eng.8. 965- (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Okamoto,A.: "An Aspartate Aminotransferase from an Extremely Thermophilic Bacterium,Thermus thermophilus HB8" J.Biochem.119. 135-144 (1996)

    • Related Report
      1995 Annual Research Report
  • [Publications] Takamatsu,S.: "Mismatch DNA Recognition Protein from an Extremely Thermophilic Bacterium,Thermus thermophilus HB8" Nucleic Acids Res.24. 640-648 (1996)

    • Related Report
      1995 Annual Research Report
  • [Publications] Kato,R.: "ATPase Activity of UvrB Protein from Thermus thermophilus HB8,and Its Interaction with DNA" J.Biol.Chem.(in press). (1996)

    • Related Report
      1995 Annual Research Report
  • [Publications] 倉光成紀: "分子生物学の展開,第6巻「アミノ基転移酵素」" 朝倉書店,東京(印刷中),

    • Related Report
      1995 Annual Research Report
  • [Publications] Kuramitsu,S.: "Replacement of Active Site Lysine-239 of Thermostable Aspartate Aminotransferase by S-(2-Aminoetyl)Cysteine:Properties of the Mutant Enzyme" J.Biochem.115. 108-112 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Kuramitsu,S.: "X-Ray Crystallographic Study of Pyridoxal 5'-Phosphate-Type Aspartate Aminotransferase from Escherichia coli in Open and Closed Forms" J.Biochem.116. 95-107 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Kuramitsu,S.: "Homologus Ligation" Trends Genet.10. 420 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Kuramitsu,S.: "NMR Studies of H Resonances in the 10-18-ppm Range for Aspartate Aminotransferase From Escherichia Coli" J.Biol.Chem.269. 28027-28033 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Kuramitsu,S.: "Separation of Heat-Stable Proteins from Thermus thermophilus HB8 by Two-Dimensional Electrophoresis" Electophoresis. (in press).

    • Related Report
      1994 Annual Research Report
  • [Publications] 倉光成紀: "酵素反応のダイナミックス" 蛋白質核酸酵素. 39. 1083-1090 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] 倉光成紀: "新生化学実験講座 第1巻 VII.タンパク質工学 天然タンパク質の安定性の分子設計" 東京化学同人,

    • Related Report
      1994 Annual Research Report

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Published: 1994-04-01   Modified: 2016-04-21  

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