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1995 Fiscal Year Final Research Report Summary

Researches on the mechanism of enzymatic action and base recognition of base non-specific ribonucleases

Research Project

Project/Area Number 05454574
Research Category

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

Allocation TypeSingle-year Grants
Research Field Biological pharmacy
Research InstitutionHoshi College of Pharmacy

Principal Investigator

IRIE Masachika  Hoshi College of Pharmacy, Professor, 薬学部, 教授 (70061265)

Co-Investigator(Kenkyū-buntansha) NAKAMURA Kazuo T.  Showa University, Pharmacy, Professor, 薬学部, 教授 (00012675)
IWAMA Masanori  Hoshi College of Pharmacy, Lecturer, 薬学部, 講師 (50130753)
OHGI Kazuko  Hoshi College of Pharmacy, Associate professor, 薬学部, 助教授 (90061291)
Project Period (FY) 1993 – 1995
Keywordsribonuclease / RNase / gene engeneering / X-ray crystallography / site directed mutagenesis / active site
Research Abstract

1. The primary structure of base non-specific RNases from various origins were elucidated. They were RNases from Irpex lacteus, oyster, squid liver, bovine spleen and bullfrog liver enzymes. The primary structures of the former four enzymes were elucidated completely, and those of the rest of RNases were mostly elucidated. The amino acid residues which are important for catalysis are concentrated in two segments like cassettes. The N-terminal structure of animal RNases and the location of cysteine residues are closely related to plant RNases rather than fungal RNases. 2. X-ray crystallography of RNase Rh-2'-AMP and-d (ApC) revealed that B1 site was composed of Tyr57, Trp49 and Asp51. The base moiety of substrate stacked with two aromatic amino acid side chaines, Try57 and Trp49, and Asp51 hydrogen bonded directly with base. Lys108 was located very closed to P^<, > atom. B2 base stacked with Phe101 and Gln95 seems to function as lid of base recognition site. 3. The amino acid residues o … More f Lys108 and base recognition site of RNase Rh from Rhizopus niveus was engineered by site directed mutagenesis, and the enzymatic properties of mutant enzymes were studied. The results indicated that replacement of Lys108 by Leu decreased the enzymatic activity considerably, and the replacement by Arg, Thr, and Ser are partially effective. Replacement of Asp51 by Glu, Gln and Asn changed the base preference of RNase Rh from adenine to guanine. Thus it was concluded that Asp51 is very crucial for the adenine preference of fungal RNases. Trp49 was very important to keep the active conformation of RNase Rh and another amino acid residues responsible for base recognition, Tyr57 could be replaced by aromatic amino acids showing relatively high activity. However, the enzymes in which Tyr57 was replaced by aliphatic amino acids are also fairly active, but the enzymatic activities of the mutants showed lower activity as compared to that with aromatic amino acid mutants. 4. We could expressed tomato RNase LE from yeast cells in a good yield. We also crystallized it and X-raycrystallographic analysis of the crystal is now in progress. Less

  • Research Products

    (14 results)

All Other

All Publications (14 results)

  • [Publications] Watanabe,H.,Narumi,H.,Inaba,T.,Ohgi,K. &Irie,M.: "Purification,some properties and primary structure of a base non-specific ribonuclease from oyster(Crussdstrea grigus)." J. Biochem.114. 800-807 (1993)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Irie,M.,Ohgi,K. Watanabe,H.,Iwama,M.,Nakamura,T. K.,et. al.: "pH-profile of kinetic constants of RNase Rh from Rhizopus niveus and its mutant enzymes towards UpU and possible mechanisms of RNase Rh." J. Biochem.115. 1083-1087 (1994)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Ohgi,K.,Iwama,M. Tada,K.,Takizawa,R. & Irie,M.: "Role of Lys108 in the enzymatic activity of RNase Rh from Rhizopus niveus." J. Biochem.117. 27-33 (1995)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Yagi,H.,Kobayashi,H.,Inokuchi,N.,Koyama,T. & Irie,M.: "Purification and properties of a base non-specific acid ribonuclease from bullfrog(Rana catesbeiana)." Biol. Pharm. Bull.18. 219-222 (1995)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Watanabe,H.,Hamid Fauzi,Iwama,M.,Onda,T.,Ohgi,K. & Irie,M.: "Base non-specific acid ribonuclease from Irpex lacteus. Primary structure,phylogenetic relationship in RNase T2 family enzyme." Biosci. Biotech. Biochem.59. 2097-2103 (1995)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Ohgi,K.,Takeuchi,M.,Iwama,M. & Irie,m.: "Enzymatic properties of mutant enzymes at Trp49 and Tyr57 of RNase Rh from Rhizopus niveus." J. Biochem.119. 9-15 (1996)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Ohgi,K.,Takeuchi,M.,Iwama,M. & Irie,M.: "Enzymatic properties of mutant forms of RNase Rh from Rhizopus niveus at to Asp51." J. Biochem.119(in press). (1996)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Watanabe, H.: "Purification, some properties and primary structure of a base non-specific ribonuclease from oyster (Crussdstrea grigus)." J.Biochem.114. 800-807 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Irie, M.: "pH-profile of kinetic constants of RNase Rh from Rhizopus niveus and its mutant enzymes towerds UpU and possible mechanisms of RNase Rh." J.Biochem.115. 1083-1087 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Ohgi, K.: "Role of Lys108 in the enzymatic activity of RNase Rh from Rhizopus niveus." J.Biochem.117. 27-33 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Yagi, H.: "Purification and properties of a base nonspecific acid ribonuclease from bullfrog (Rana catesbeiana)" Biol. Pharm. Bull.18. 219-222 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Watanabe, H.: "Base non-specific acid ribonuclease from Irpex lacteus. Primary structure, phylogenetic relationship in RNase T2 family enzyme." Biosci. Biotech. Biochem.59. 2097-2103 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Ohgi, K.: "Enzymatic properties of mutant enzymes at Trp49 and Tyr57 of RNase Rh from Rhizopus niveus." J.Biochem.119. 9-15 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Ohgi, K.: "Enzymatic properties of mutant forms of RNase Rh from Rhizopus niveus as to Asp51." J.Biochem.119 (in press). (1996)

    • 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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