| Project/Area Number |
06660116
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | Saga University |
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Principal Investigator |
WATANABE Keiichi Saga University, Department of Applied Biological Sciences, Associate Professor, 農学部, 助教授 (40191754)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1995: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1994: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Keywords | RNA N-glycosidase, / Ribosome-inactivating protein, / X-ray crystallography, / Ricin A-chain, / Pokeweed antiviral protein, / Protein engineering. / RNAN-グリコシダーゼ / リボソーム不活性化タンパク質 |
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| Research Abstract |
Ricin A-chain is an RNA N-glycosidase that inactivates ribosomes by depurination of a highly conserved adenosine in 28S rRNA.Chemical modification studies suggested that certain arginine residues, located outside the active site cleft, are also involved in the enzymatic activity. To confirm this suggestion, site-directed mutagenesis was performed. Both the wild-type and mutant proteins were expressed in Escherichia coli, purified and tested for enzymatic activity. Conversion of Arg213 to Ser reduced kcat 15-fold without effect on Km, suggesting that Arg213 plays a role in catalysis. Conversion of Arg196 to Gln reduced the activity 330-fold, and the individual conversion of Arg235 to Ser caused a more drastic loss (17000-fold) in activity. Circular dichroism and fluorescence analyzes of the mutant proteins suggested that neither the secondary structure nor the environment of Trp211 at the bottom of the cleft was affected upon each mutation. The side chain of Arg213 is adjacent to the edge of the cleft. Arg196 and Arg235 are located on the other side of the cleft. These results support the idea that the Arg residues outside the active site cleft are also involved in the enzymatic action, providing valuable information to understand the mechanism of enzymatic action of ricin A-chain.
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