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1995 Fiscal Year Final Research Report Summary

Molecular Basis of Lysine Specificity of Lysylendopeptidase

Research Project

Project/Area Number 06680585
Research Category

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

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

Principal Investigator

NORIOKA Shigemi  Institute for Protein Research, Associate Professor, たんぱく質研究所, 助教授 (70198638)

Project Period (FY) 1994 – 1995
KeywordsLysylendopeptidase / Substrate Specificity / Site-directed Mutagenesis / Protein Engineering / Hydrogen Bond
Research Abstract

Achromobacter protease I (API) is a lysine-specific serine protease. Crystal structural analyzes of API and API-TLCK complex revealed that the carboxyl oxygen of Asp225 formed several hydrogen bonds with its surrounding amino acid residues and a water molecule (W420) in S1-pocket. Odelta2 of Asp225 formed hydrogen bonds with Ogamma1 of Thr189, Ogamma of Ser214, and Nepsilon1 of Trp182. W420 also formed hydrogen bonds with Odelta1 of Asp225, CO of Thr189, CO of Ser214 and Ogamma of Ser214. From these observations, it was thought that besides Asp225, Thr189, Ser214 and Trp182 might also contribute to the strict specificity for lysine and the high proteolytic activity of API.
In order to investigate role of the hydrogen bonds, Trp182, Thr189 and Ser214 were substituted for other amino acids. The kcat/Km of T189V,S214A,and T189VS214A were decreased to 1/4,1/3 and 1/46 of that of native API,respectively. The decreased activities were mainly due to the increase of Km value, suggesting that the partial loss of the hydrogen bond network and a local minute structural change in S1-pocket decreased substrate binding ability of these mutants. On the other hand, the similarity of enzymatic properties between W182F and native API suggested that the hydrogen bond between Odelta2 of Asp225 and Nepsilon1 of Trp182 was not directly related to the substrate binding.
The strict specificity and high proteolytic activity of API were primarily based on the electrostatic interaction between Asp225 and lysine substrate. But this interaction was not sufficient for strong binding of the lysine substrate to API and the hydrogen bonding system in the S1-pocket assisted the binding of a substrate to API.

  • Research Products

    (6 results)

All Other

All Publications (6 results)

  • [Publications] Shigemi Norioka: "Identification of three catalytic triad constituents and Asp-225 essential for function of lysine-specific serine protease,Achromobacter protease I." J. Biol. Chem.269. 17025-17029 (1994)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Shigemi Norioka: "Site-directed mutagenic study of the substrate specificity of Streptomyces erythraeus trypsin-like protease." Protein Engineering. 7. 1153-1154 (1994)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Yuko Nagamine-Natsuka: "Molecular cloning,nucleotide sequence,and expression of the gene encoding a trypsin-like protease from Streptomyces erythraeus." J. Biochem.118. 1007-1013 (1995)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Shigemi Norioka, Shigeki Ohta, Takeshi Ohara, Scong-Il Lim, and Fumio Sakiyama: "Identification of three catalytic triad constituents and Asp-225 essential for function of lysine-specific serine protease, Achromobacter protease I." J.Biol.Chem.269. 17025-17029 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Shigemi Norioka, Yuko Nagamine, and Fumio Sakiyama: "Site-directed mutagenic study of the substrate specificity of Streptomyces erythraeus trypsin-like protease." Protein Engineering. 7. 1153-1154 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Yuko Nagamine-Natsuka, Shigemi Norioka, and Fumio Sakiyama: "Mocecular cloning, nucleotide sequence, and expression of the gene encoding a trypsin-like protease from Streptomyces erythraeus." J.Biochem.118. 338-346 (1995)

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

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Published: 1997-03-04  

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