| Project/Area Number |
10044095
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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 Field |
Structural biochemistry
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| Research Institution | Tokyo University of Pharmacy & Life Science |
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Principal Investigator |
OSHIMA Tairo School of Life Science, Tokyo University of Pharmacy & Life Science Professor, 生命科学部, 教授 (60167301)
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| Co-Investigator(Kenkyū-buntansha) |
TANAKA Nobuo Tokyo Institute of Technology, Faculty of Bioscience & Biotechnology Professor, 生命理工学部, 教授 (50032024)
YAMAGISHI Akihiko School of Life Science Associate Professor, 生命科学科, 助教授 (50158086)
ZAVODOSZKY P ハンガリー科学アカデミー, 酵素研, 教授
PETSKO Greg ブランダイス大学, 生物化学科, 教授
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1999: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1998: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | isopropylmalate dehydrogenase / reversible denaturation / free energy change / temperature jump / denaturation / Laue method / 高度好熱菌 / 進化分子工学 / 実験室内進化 / 熱安定酵素 |
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| Research Abstract |
The present study has been done using isopropylmalate dehydrogenase as a model enzyme. A variety of mutant enzymes were constructed using the dehydrogenase from an extreme thermophile, Thermus thermophilus, and their structures, stability and flexibility, and catalytic activities were nalyzed. Especially thermodynamics of thermal denaturation reaction of these mutants and the wild type enzyme were investigated in 1999. To find out conditions under which the thermophile enzyme denatures reversibly, has been difficult, but we succeeded to overcome this problem ; we found that the enzyme denatures reversibly in the presence of guanidine chloride and some specific detergents. The enzyme denatures with an intermediate and the intermediate has no catalytic activity. We co-operated with Prof. Tanaka of Tokyo Institute of Technology, and deviced a new method for analyzing 3D structure of the intermediate using Laue method and temperature jump. By using this method, we condluded that the denaturation starts from two loop regions of the enzyme.
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