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1990 Fiscal Year Final Research Report Summary

Alteration of Structure and Function of Protease Inhibitor SSI to Design a New Enzyme Inhibitor

Research Project

Project/Area Number 01470146
Research Category

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

Allocation TypeSingle-year Grants
Research Field 物質生物化学
Research InstitutionThe University of Tokyo

Principal Investigator

MIURA Kin-ichiro  Univ. of Fac. of Engin., Tokyo, Professor, 工学部, 教授 (30000227)

Co-Investigator(Kenkyū-buntansha) MITSUI Yukio  Nagaoka Uni. of Technology, Fac. of Engin. Professor, 工学部, 教授 (40012637)
KUMAGAI Izumi  Univ. of Tokyo, Fac. of Engin., Ass. Professor, 工学部, 助教授 (10161689)
Project Period (FY) 1989 – 1990
KeywordsProtein engineering / Inhibitor / Protease inhibitor / Site-directed mutagenesis / Streptomyces / Gene engineering / Active site of protein / Protein structure
Research Abstract

In order to design new inhibitors for enzymes, we have investigated the structure-function relationship of the well-characterized protease inhibitor, Streptomyces subtilisin inhibitor (SSI), by the method of gene engineering technique. First, replacement of the amino acid at P1 site, the center of reactive site, of SSI by Lys or Arg resulted in attainment of inhibitory activity for trypsin, and by Tyr, Phe, or Trp resulted in that for chymotrypsin. These mutants inhibited subtilisin as strongly as wild type, whereas inhibitory activity of the mutants whose P1 site residue was Glu, Ile, or Pro was decreased. These observations demonstrated that inhibitory activities of P1 site mutants of protease inhibitors are related to the substrate specificity of protease. Substitution of the P4 site residue (Met70) of the mutant SSI with Lys at P1 site by Gly, and the mutant SSI with Gly at P1 site by Phe resulted in increase of inhibitory activity toward trypsin and subtilisin, respectively. These results showed that stronger inhibitors could be produced by replacements of amino acid residues around the reactive site of inhibitors, so that they were complementary to the subsite structure of protease. Furthermore, removal of disulfide dridge near the reactive site of SSI by replacements of Cys to Ser resulted in conversion of SSI into temporary inhibitor, in which inhibitory activity for the protease decreased with increasing incubation time after mixing with subtilisin. This finding suggested that conformational rigidity around the reactive site was required for the inhibitory action of SSI.

  • Research Products

    (22 results)

All Other

All Publications (22 results)

  • [Publications] S.Taguchi,I.Kumagai,K.Miura: "Comparison of secretory expression in Escherichia coli and Streptomyces of streptomyces subtilisin inhibitor(SSI)gene." Biochim.Biophys.Acta. 1049. 278-285 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] S.Kojima,I.Kumagai,K.Miura: "Effect on inhibitory activity of mutation at reaction site P4 of the streptomyces subtilisin inhibitor,SSI." Protein Engineering. 3. 527-530 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] I.Kumagai,S.Takeda,T.Hidino,K.Miura: "Expression of goat αーlactalbumin in Escherichia coli and its refolding to biologically active protein." Protein Engineering. 3. 449-452 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] S.Kojima,S.Obata,I.Kumagai,K.Miura: "Alteration of the specificity of the streptomyces subtilisin inhibitor by gene engineering." BIO/TECHNOLOGY. 8. 449-452 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] S.Taguchi,I.Kumagai,J.NaKayama,A.Suzuki,K.Miura: "Efficient extracellular expression of a foreign protein in Streptomyces using secretory protease inhibitor(SSI)gene fusions." BIO/TECHNOLOGY. 7. 1063-1066 (1989)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] S.Taguchi,K.Nishiyama,I.Kumagai,K.Miura: "Analysis of transctiptional control regions in the Streptomyces subtilisinーinhibitorーencoding gene." Gene. 84. 279-286 (1989)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] I.Kumagai,K.Miura: "Enchanced bacteriolytic activity of hen eggーwhite lysozyme due to conversion of trp62 to other aromatic amino acid residues." J.Biochem.105. 946-948 (1989)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] S.Obata,S.Furukubo,I.Kumagai,H.Takahashi,K.Miura: "Highーsevel expression in Streptomyces lividans 66 of a gene encoding Streptomyces subtilisin inhibitor from Streptomyces albogriseolus Sー3253" J.Biochem.105. 372-376 (1989)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] S.Obata,S.Taguchi,I.Kumagai,K.Miura: "Molecular cloning and nucleotide sequence setermination of gene encoding Streptomyces subtilisin inhibitor(SSI)." J.Biochem.105. 367-371 (1989)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] H.Kaji,I.Kumagai,A.Takeda,K.Miura T.Samejima: "Studies on Chemical synthesis of human cystatin A gene and its expression in Escherichia coli." J.Biochem.105. 143-147 (1989)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] K.Miura,I.Kumagai,S.Obata,S.Kojima S.Taguchi: "Partial alteration of a protein Streptomyces subtilisin inhibitor by siteーdirected mutagenesis." Proc.Japan Acad.64. (1988)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Miura, K., Kumagai. I., Obata, S., Kojima, S., Taguchi, S. ;: "Partial Alteration of a Protein Streptomyces Subtilisin Inhibitor by Site-Directed Mutagenesis." Proc. Jpn. Acad.,. 64B,. 147-149 (1988)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kumagai, I., Miura. K. :"Enhanced Bacteriolytic Activity of Hen Egg-White Lysozyme Due to Conversion of Trp62 to Other Aromatic Amino Acid Residues." J. Biochem.,. 105,. 946-948 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kaji, H., Kumagai, I., Takeda, A., Miura, K., Samejima, T. ;: "Studies on Chemical Synthesis of Human Cystatin A and Its Expression in Escherichiacoli." J. Biochem.,. 105,. 143-147 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Obata, S., Taguchi, S., Kumagai, I., Miura, K. ;: "Molecular Cloning and Nucleotide Sequence Determination of Gene Encoding Streptomyces Subtilisin Inhibitor (SSI)." J. Biochem.,. 105,. 367-371 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Obata, S., Furukubo, S., Kumagai, I., Takahashi, H., Miura, K. ;: "High-Level in Expression Streptomyces lividans 66 of a Gene Encoding Streptomyces Subtilisin Inhibitor from Streptomyces albogriseolus S-3253." J. Biochem.,. 105,. 372-376 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Taguchi, S., Kumagai, I., Nakayama, J., Suzuki, A., Miura, K. ;: "Efficient Extracellular Expression of a Foreign Protein in Streptomyces Using Secretory Protease Inhibitor (SSI) Gene Fusion." Bio/Technology,. 7,. 1063-1066 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Taguchi, S., Nishiyama, K., Kumagai, I., Miura, K. ;: "Analysis of Transcriptional Control Region in the Streptomyces Subtilisin-Inhibitor-Encoding Gene." Gene,. 84,. 279-286 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kojima, S., Obata, S., Kumagai, I., Miura, K. ;: "Alteration of the Specifity of the Streptomyces Subtilisin Inhibitor by Gene Engineering." Bio/Technology,. 8,. 449-452 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kojima, S., Kumagai, I., Miura, K. ;: "Effect on Inhibitory Activity of Mutation at Reaction Site P4 of the Streptomyces Subtilisin Inhibitor, SSI." Protein Engineering,. 3,. 527-530 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Taguchi, S., Kumagai, I., Miura, K. ;: "Comparison of Secretory Expression in Escherichia coli and Streptomyces of Streptomyces Subtilisin Inhibitor (SSI) Gene." Biochim. Biophys. Acta,. 1049,. 278-285 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kumagai, I., Takeda, S., Hibino, T., Miura, K.: "Expression of goat alpha-Lactalbumin in Escherichea coli and Its Refolding to Biologically Active Protein." Protein Engineering,. 3,. 449-452 (1990)

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

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Published: 1993-08-12  

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