| Project/Area Number |
12680643
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Functional biochemistry
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| Research Institution | Kinki University |
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Principal Investigator |
TONOMURA Benichiro Kinki University, School of Biology Oriented Science and Technology, Professor, 生物理工学部, 教授 (20026545)
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| Co-Investigator(Kenkyū-buntansha) |
TAKITA Teisuke Kyoto University Graduate School, Division of Agricultural Science, Assistant Prof., 大学院・農学研究科, 助手 (70263126)
MORIMOTO Koichi Kinki University, School of Biology Oriented Science and Technology, Lecturer, 生物理工学部, 講師 (10319741)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 2001: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2000: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Keywords | aminoacyl-tRNA synthetase / lysyl-tRNA synthetase / protein crystallization / mechanism of protein- substrate recognition / protein fluorescence |
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| Research Abstract |
(1) The base sequence of the cloned gene of the lysyl-tRNA synthetase from a mesothermophile, Bacillus stearothermophilus was determined. The primary structure of the enzyme (B.s.LysRS) was deduced from the base sequence of the gene. Comparative studies were made on the primary structures of the gene and the protein of B.s.Lys RS. and the strategy for thermostability of the enzyme was discussed.
(2) A highly purified preparation of B.s.LysRS was subjected to crystallization for the structural analysis. An orthorhombic crystal suitable for the X-ray analysis was obtained. The X-ray diffraction on this crystal gave 2.63A resolution. The model building of B.s.LysRS molecule is underway with these diffraction data by the molecular replacement method with the structure of E. coli LysRS(U) as reference.
(3) Functional analysis of B.s.LysRS : Among the amino acid residues that constitute the substrate binding site, those which locate in the position being able to interact with the substrate L-lysine were deduced from the analogy to the structure of E.coli LysRS. Those residues were replaced by other amino acid by the method of site-directed mutagenesis. The kinetic parameters of those artificial mutant LysRS in the ATP-PPi exchange reaction and the equilibrium parameters in the binding of L-lysine and the mutant enzyme, as measured with the protein fluorescence change as probe, were determined and compared with those of the wild type enzyme, The functions of the replaced amino acid residues were discussed.
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