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Proton-transfer mechanism of pyridoxal enzymes studied by 3D-free energy profile

Research Project

Project/Area Number 13680697
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Structural biochemistry
Research InstitutionOsaka Medical College

Principal Investigator

HAYASHI Hideyoshi  Osaka Medical College Faculty of Medicine Associate Professor, 医学部, 助教授 (00183913)

Project Period (FY) 2001 – 2002
Project Status Completed (Fiscal Year 2002)
Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2001: ¥2,200,000 (Direct Cost: ¥2,200,000)
Keywordsproton-transfer / pKa / catalytic-mechanism / aminotransferase / pyridoxal-phosphate / strain / free-energy-profile / energy-barrier / 自由エネルギー
Research Abstract

The detailed mechanism of proton transfer from the substrate to the coenzyme Schiff base in aspartate aminotransferase (AAT) and the structural basis for the driving force have been elucidated through 3D-free energy analysis. The use of V393F mutation, which perturbs the formation of the lid over the active site, clarified that the unfavorable interaction between Gly38 and the unprotonated Schiff base elevates the free energy level of the Michaelis complex, thereby increasing the k_<cat> value. Thus, the classical explanation for the increase in the Schiff base pK_a has been found to be nothing more than a reflection of the destabilization of the Michaelis complex.
Analysis of the reaction of AAT with C5 substrates revealed that the C5 substrate, unlike the C4 substrate such as aspartate, take an extended conformation in the open form of AAT. The decrease in the K_m value and unchanged k_<cat> value of the V39L mutant enzyme were assessed based on the 3D-free energy analysis in which the conformation of AAT was taken as the third axis of the 3D profile. The analysis showed that AAT takes a strategy to increase the k_<cat> value toward the C5 substrate at the expense of K_m by destabilizing the open form of the Michaelis complex.
The present study clearly showed the effectiveness of the 3D-free energy analysis. The method is considered to be applicable to many aspects of the enzyme catalysis, if an appropriate coordinate is taken as the third axis.

Report

(3 results)
  • 2002 Annual Research Report   Final Research Report Summary
  • 2001 Annual Research Report
  • Research Products

    (36 results)

All Other

All Publications (36 results)

  • [Publications] Hayashi, Hideyuki: "Conformational change in aspartate aminotransferase on substrate binding induces strain in the catalytic group and enhances catalysis"J.Biol.Chem.. 278(11). 9481-9488 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Goto, Masaru: "Crystal structures of branched-chain amino acid aminotransferase in the complex with glutamate and glutarate"Biochemistry. 42(印刷中). (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Omi, Rie: "Structure of imidazole glycerol phosphate synthase from Thermus thermophilus HB8"J.Biochem.. 132(5). 759-765 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Olmo, Maria.T.: "Spectroscopic analysis of recombinant rat histidine decarboxylase"J.Biochem.. 132(3). 433-439 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Watanabe, Akira: "Reaction mechanism of alanine racemase from Bacillus stearohermophilus x-ray crystallographic studies of the enzyme bound with N-(5'-phosphopyridoxyl)alanine"J.Biol.Chem.. 277(21). 19166-19172 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Okada, Kengo: "Structures of Escherichia coli branched-chain amino acid aminotransferase and its complexes with 4-methylvalerate and 2-methylleucine : Induced fit and substrate recognition of the enzyme"Biochemistry. 40(25). 7453-7463 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Nakayama, Toru: "Specificity analysis and mechanism of aurone synthesis catalyzed by aureusidin synthase, a polyphenol oxidase homolog responsible for flower coloration"FEBS Lett.. 499(1-2). 107-111 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Ikushiro, Hiroko: "A water-soluble homodimeric serine palmitoyltransferase from Sphingomonas paucimobilis EY2395T strain. purification, characterization, cloning, and overproduction"J.Biol.Chem.. 276(21). 18249-18256 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Haruyama, Kazuki: "Structures of Escherichia coli histidinol-phosphate aminotransferase and its complexes with histidinol-phosphate and N-(5'-phosphopyridoxyl)-L-glutamate"Biochemistry. 40(15). 4633-4644 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] 林 秀行: "酵素反応のエネルギー論"生化学. 74-1. 5-16 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Hayashi, Hideyuki: "Strain and catalysis in aspartate aminotransferase"Biochim.Biophys.Acta. (印刷中). (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Mizuguchi, Hiroyuki: "Characterization of histidinol phosphate aminotransferase from Escherichia coli"Biochim.Biophys.Acta. (印刷中). (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Ikushiro, Hiroko: "Bacterial serine palmitoyltransferase : a water-soluble homodimeric prototype of the eukarvotic enzyme"Biochim.Biophys.Acta. (印刷中). (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Hayashi, H., Mizuguchi, H., Miyahara, I., Nakajima, Y., Hirotsu, K., and Kagamiyama, H.: "Conformational change in aspartate aminotransferase on substrate binding induces strain in the catalytic group and enhances catalysis"J. Biol. Chem.. 278(11). 9481-9488 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Goto, M., Miyahara, I., Hayashi, H., Kagamiyama, H., and Hirotsu, K.: "Crystal structures of branched-chain amino acid aminotransferase in the complex with glutamate and glutarate: true reaction intermediate and double substrate recognition of the enzyme"Biochemistry. 42, in press. (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Omi, R., Mizuguchi, H., Goto, M., Miyahara, I., Hayashi, H., Kagamiyama, H., and Hirotsu, K.: "Structure of imidazole glycerol phosphate synthase from Thermus thermophilus HB8: Open-closed conformational change and ammonia tunneling"J. Biochem. 132(5). 759-765 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Olmo, M.T., Sanchez-Jimenez, F., Medina, M.A., and Hayashi, H.: "Spectroscopic analysis of recombinant rat histidine decarboxylase"J. Biochem. 132(3). 433-439 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Watanabe, A., Yoshimura, T., Mikami, B., Hayashi, H., Kagamiyama, H., and Esaki, N.: "Reaction mechanism of alanine racemase from Bacillus stearothermophilus: x-ray crystallographic studies of the enzyme bound with N-(5'-phosphopyridoxyl)alanine"J. Biol. Chem.. 277(21). 19166-19172 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Okada, K., Hirotsu, K., Hayashi, H., and Kagamiyama, H.: "Structures of Escherichia coli branched-chain amino acid aminotransferase and its complexes with 4-methylvalerate and 2-methylleucine: Induced fit and substrate recognition of the enzyme"Biochemistry. 40(25). 7453-7463 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Nakayama, T., Sato, T., Fukui, Y., Yonekura-Sakakibara, K., Hayashi, H., Tanaka, Y., Kusumi, T., and Nishino, T.: "Specificity analysis and mechanism of aurone synthesis catalyzed by aureusidin synthase, a polyphenol oxidase homolog responsible for flower coloration"FEBS Lett. 499(1-2). 107-111 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Ikushiro, H., Hayashi, H., and Kagamiyama, H.: "A water-soluble homodimeric serine palmitoyltransferase from Sphingomonas paucimobilis EY2395T strain, purification, characterization, cloning, and overexpression"J. Biol. Chem.. 276(21). 18249-18256 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Haruyama, K., Nakai, T., Miyahara, I., Hirotsu, K., Mizuguchi, H., Hayashi, H., and Kagamiyama, H.: "Structures of Escherichia coli histidinol-phosphate aminotransferase and its complexes with histidinol-phosphate and N-(5'-phosphopyridoxyl)-L-glutamate: Double substrate recognition of the enzyme"Biochemistry. 40(15). 4633-4644 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Hayashi, H., Mizuguchi, H., Miyahara, I., Islam, M.M., Ikushiro, H., Nakajima, Hirotsu, K., & Kagamiyama, H.: "Strain and catalysis in aspartate aminotransferase"Biochim. Biophys. Acta. in press. (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Mizuguchi, H., Hayashi, H., Miyahara, I., Hirotsu, K., and Kagamiyama, H.,: "Characterization of histidinol phosphate aminotransferase from Escherichia coli"Biochim. Biophys. Acta. in press. (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Ikushiro, H., Hayashi, H., and Kagamiyama, H.: "Bacterial serine palmitoyltransferase: a water-soluble homodimeric prototype of the eukaryotic enzyme"Biochim. Biophys. Acta. in press. (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Hayashi, Hideyuki: "Conformational change in aspartate aminotransferase on substrate binding induces strain in the catalytic group and enhances catalysis"J. Biol. Chem.. 278(11). 9481-9488 (2003)

    • Related Report
      2002 Annual Research Report
  • [Publications] Goto, Masaru: "Crystal structures of branched-chain amino acid aminotransferase in the complex with glutamate and glutarate"Biochemistry. 42(印刷中). (2003)

    • Related Report
      2002 Annual Research Report
  • [Publications] Olmo, Maria. T.: "Spectroscopic analysis of recombinant rat histidine decarboxylase"J. Biochem.. 132(3). 433-439 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Omi, Rie: "Structure of imidazole glycerol phosphate synthase from Thermus thermophilus HB8"J. Biochem.. 132(5). 759-765 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Watanabe, Akira: "Reaction mechanism of alanine racemase from Bacillus stearothermophilus x-ray crystallographic studies of the enzyme bound with N-5'-phosphopyridoxyl)alanine"J. Biol. Chem.. 277(21). 19166-19172 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Hayashi, Hideyuki: "Strain and catalysis in aspartate aminotransferase"Biochim. Biophys. Acta. (印刷中). (2003)

    • Related Report
      2002 Annual Research Report
  • [Publications] Haruyama, K.: "Structures of Escherichia coli histidinol-phosphate aminotransferase and its complexes with histidinol-phosphate and N-(5'-phosphopyridoxyl)-L-glutamate : Double substrate recognition of the enzyme"Biochemistry. 40・15. 4633-4644 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Ikushiro, H.: "A water-soluble homodimeric serine palmitoyltransferase from Sphingomohas paucimobilis EY2395T strain. purification, characterization, cloning, and overproduction"J. Biol. Chem.. 276・21. 18249-18256 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Okada, K.: "Structures of Excherichia coli branched-chain amino acid aminotransferase and its complexes with 4-methylvalerate and 2-methylleucine : Induced fit and substrate recognition of the enzyme"Biochemistry. 40・25. 7453-7463 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Watanabe, A.: "Reaction mechanism of alanine racemase from Bacillus stearothermophilus : X-ray crystallographic studies of the enzyme bound with N-(5'-phosphopyridoxyl)-alanine"J. Biol. Chem.. 277(in press). (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] 林 秀行: "酵素反応のエネルギー論"生化学. 74・1. 5-16 (2002)

    • Related Report
      2001 Annual Research Report

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Published: 2001-04-01   Modified: 2016-04-21  

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