| Project/Area Number |
03660138
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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 |
製造化学・食品
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
ODA Jun'ichi INST. CHEM. RES., KYOTO UNIV., PROFESSOR, 化学研究所, 教授 (50027041)
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| Co-Investigator(Kenkyū-buntansha) |
TANAKA Takuji INST. CHEM. RES., KYOTO UNIV., INSTRUCTOR, 化学研究所, 助手 (40227145)
KATO Hiroaki INST. CHEM. RES., KYOTO UNIV., INSTRUCTOR, 化学研究所, 助手 (90204487)
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| Project Period (FY) |
1991 – 1992
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| Project Status |
Completed (Fiscal Year 1992)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1992: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1991: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Keywords | GLUTATHIONE SYNTHETASE / PROTEIN ENGINEERING / PEPTIDE SYNTHESIS / SITE-DIRECTED MUTAGENESIS / SUBSTRATE RECOGNITION |
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| Research Abstract |
We have been studying the crystal structure of glutathione synthetase (GSHase) -substrates complex. These studies showed that the binding site of ATP is between two anti-parallel beta-sheets and that some residues interact with the bound ATP. However, the binding site of gamma-Glu- Cys is not clear because gamma-Glu-Cys gave poor electron density. We assumed some residues in the putative binding site of gamma-Glu-Cys and investigated their properties on the catalysis. Some mutant GSHases in which the residues around proposed binding site of gamma-Glu-Cys are replaced by site- directed mutagenesis are prepared and investigated in their properties. The analysis of the mutant GSHases shows that gamma-Glu-Cys was binding between Arg86 and Arg210 and that the thiol group of gamma-Glu-Cys was recognized by Thr288.
The binding site of ATP was confirmed by the technique of affinity labeling. The crystallography of the labeled GSHase showed that the binding site of the modifier (adenosine-5'-tetraphospho-5'-pyridoxal) is the same as that of ATP.
Crystallography of glutathione synthetase showed that a loop from Ile226 to Gly242 is above the binding sites and gave no electron density because of its flexibility. The position of the loop suggested that the loop have some roles on the catalysis. The analysis of the loop indicated that the loop moved over the active site to protect a labile intermediate from hydrolysis, controlled the recognition of glycine, and accelelared the catalysis by aligning the substrates in proper position.
These results suggest that the mutation of residues around the binding sites and of the loop give new enzymes which catalyze synthesis of a novel peptide.
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