| Project/Area Number |
04044103
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| Research Category |
Grant-in-Aid for international Scientific Research
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| Allocation Type | Single-year Grants |
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| Section | Joint Research |
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| Research Institution | Osaka University |
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Principal Investigator |
KATUBE Yukiteru Institute for Protein Research, Osaka University, 蛋白質研究所, 教授 (20032013)
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| Co-Investigator(Kenkyū-buntansha) |
HAJDU J. オックスフォード大学, 分子生物学科, 講師
PETSKO G. ブランダイス大学, 基礎医学研究センター, 教授
KATO Hiroaki Institute for Chemical Research, Kyoto University, 化学研究所, 助手 (90204487)
HATA Yasuo Institute for Chemical Research, Kyoto University, 化学研究所, 助教授 (10127277)
NISHIOKA Takaaki Institute for Chemical Research, Kyoto University, 化学研究所, 助教授 (80026559)
ODA Jun'ichi Institute for Chemical Research, Kyoto University, 化学研究所, 教授 (50027041)
勝部 幸輝 大阪大学, 蛋白質研究所, 教授 (20032013)
HAJDU Janos Laboratory of Molecular Biophysics, Oxford University
PETSKO Gregory A. Basic Medical Sciences Research Center, Brandeis University
KATUBE Yukiteru Institute for Protein Research, Osaka University
J Hajdu 英国オックスフォード大学, 分子生物学科, 講師
G Petsko 米国ブランダイス大学, 基礎医科学研究センター, 教授
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| Project Period (FY) |
1992 – 1993
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| Project Status |
Completed (Fiscal Year 1993)
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| Budget Amount *help |
¥8,100,000 (Direct Cost: ¥8,100,000)
Fiscal Year 1993: ¥4,100,000 (Direct Cost: ¥4,100,000)
Fiscal Year 1992: ¥4,000,000 (Direct Cost: ¥4,000,000)
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| Keywords | Time Resolve X-ray Structure Analysis / Glutatuione Synthetase / Laue Method in Time Resolution / Kinetic Protein-Crystallography / 時間分割結晶構造解析 / ラウエ法 / 放射光 / フローセル / フラッシュ光反応 / 変異体酵素 / ATP |
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| Research Abstract |
One of the major goals of structural biology should perhaps be to achieve 4-dimensional structure determination (with time being the 4th dimension).
In order to apply time resolved crystallography to glutathione synthetase from E.coli, we determined a crystal structure of the enzyme by X-ray diffraction method. The enzyme is a tetramer with four identical Subunits of 316 amino acid residues. The loop from Ile-226 to Arg-241 in the enzyme is rich in glycine and alanine and too flexible to take a fixed conformation. The apparent function of the loop is to serve as a lid that shields the reaction intermediate, gamma-glutamylcysteinylphosphate, from the solvent water.
Kinetic diffraction studies require the fast measurement of a sequence of 3-dimensional sets of structure factors with an accuracy and completeness that is sufficient to describe the structures during the reaction in the crystal. This is not easy requirement because of fast catalytic reaction-rate of the enzyme, msec-mu sec. To restrict the catalytic reaction-rate, we prepared some mutants by site-directed mutagenesis. The Km value of R210K mutant was similar to that of the wild-type, but its kcat value drastically decreased. Laue data of R210K by synchrotron radiation were processed using the program LAUEMAD. The power and limitations of time resolved X-method by using Laue diffraction were investigated. In particular, the effects of crystalline disorder, imperfect wavelength scaling, and a systematic lack of completeness in Laue structure factor sets at low and medium resolution were analyzed.
As results of the above investigation, we learned that R210K would be a good target of time resolved X-ray structure study combined Laue and monochromatic diffraction techniques.
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