1988 Fiscal Year Final Research Report Summary
Dynamic Analysis of Enzyme Functions
| Project/Area Number |
60430027
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| Research Category |
Grant-in-Aid for General Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Research Field |
応用生物化学・栄養化学
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| Research Institution | Kyoto University |
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Principal Investigator |
HIROMI Keitaro Kyoto University, Faculty of Agriculture, 農学部, 教授 (50025425)
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| Co-Investigator(Kenkyū-buntansha) |
AKASAKA Kazuyuki Kyoto University, Faculty of Science, 理学部, 助教授 (50025368)
NAKATANI Hiroshi Kyoto University, Faculty of Agriculture, 農学部, 助手 (00026577)
OHNISHI Masatake Kyoto University, Faculty of Agriculture, 農学部, 助手 (90026576)
TONOMURA Ben'ichiro Kyoto University, Faculty of Agriculture, 農学部, 助教授 (20026545)
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| Project Period (FY) |
1985 – 1988
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| Keywords | Proteases / Aminoacyl-tRNA synthetases / Amylases / Protease inhibitors / Enzyme-substrate interactions / Fluorescence titration / Stopped-flow method / 核磁気共鳴 |
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| Research Abstract |
This research project has been focused particularly on the study of molecular recognition of substrate by enzymes among many other aspects. The study was conducted by means of absorption spectroscopy, fluorescence spectroscopy, and nuclear magnetic resonance (NMR) through equilibrium and kinetic analyses with about 10 kinds of enzymes. Satisfactory results were obtained. A micro-stopped flow apparatus which, as compared with the conventional apparatus, exhibits the equal degree of performance with only one tenth volume of the sample, and a temperaturejump apparatus which enables one to change the temperature either jumping-up or-down over a relatively wide defference by using a rapid-temperature exchange device, were constructed. Kinetic analysis of rapid chemical modification by Nbromosuccinimide was proved to be useful for investigating the state of tryptophan residues at the reactive site of enzyme. Flucuation of a protein molecule was discussed according to the results of NMR study on hydrogen-deuterium exchange. Rapid-kinetic analyses of all those enzyme systems suggested it universal that binding of an enzyme and the substrate proceeds with at least two steps. In this two step mechanism a bi-molecular process to form a loosely bound intermediate is followed by a uni-molecular isomerization process which results in the formation of a specific, strongly-bound complex. Internal fluctuation of the enzyme molecule is believed to play an important role in the second process.
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