Reaction Mechanism of Aspartate Aminotransferase
| Project/Area Number |
02680138
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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 |
物質生物化学
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| Research Institution | Osaka City University |
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Principal Investigator |
HIROTSU Ken City Univ., Faculty of Science, Assoc. Prof., 理学部, 助教授 (10047269)
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| Co-Investigator(Kenkyū-buntansha) |
KURAMITSU Seiki Osaka Univ., Faculty of Science, Prof., 理学部, 教授 (60153368)
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| Project Period (FY) |
1990 – 1991
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| Project Status |
Completed (Fiscal Year 1991)
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| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1991: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1990: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Keywords | aminotransferase / reaction mechanism / X-ray structure / mutant enzyme / 人工変異体 / アミノ基転位酵素 / 高次構造 |
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| Research Abstract |
E. coli aspartate aminotransferase (AspAT) binds the coenzyme pyridoxal phosphate (PLP), in its active site and catalyzes the reversible reaction of transfer of amino group of aspartate to PLP, giving pyridoxamine phosphate and oxaloacetate. X-ray crystallographic study of AspAT and its mutant enzymes was undertaken and the reaction mechanism of this enzyme was analyzed by using the results of X-ray works and enzyme kinetics. AspAT and its complexes with the substrate analogue were crystallized and diffraction data were collected using the synchrotron radiation. The structures of AspAT and the complex were determined at high resolution. The binding of the substrate to AspAT induces the large conformational change of the overall molecule. The precise analysis of the active site structures shows thathow the substrate specifity and the stereospecifity of the reaction are made possible. The driving force of the large conformational change, on binding the substrate to AspAT, will be the inc
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rease of entropy by the release of many water molecules. The amino acid residues which play an important roles on catalytic reaction in the active site are located around PLP. The activity of AspAT decreases markedly when these important amino acid residues are replaced by other residues. In order to elucidate the roles of these important residues, the X-ray structure determination of the mutant enzymes were undertaken. The overall structure of Y70F is similar to that of the wild type AspAT, and the position of the benzene ring of F70 is the same as that of Y70. The fitting Of C_5 substrate into the active site by the graphic method indicates the possibility of the recognition of C_5 substrate by this benzene ring. In Y70W, the substrate analogue binds the active site but does not make schiff base with PLP. This means the Michaelis complex is trapped. The overall structure of R292V is similar to that of the wild type AspAT. Surprisingly, in spite of the exchange of the charged R292 by the neutral V292, the side chain of V292 is located at the similar position to that of R292. X-ray analysis of Y225F shows that the hydrogen bond between OH group of Y225 and PLP are important to the catalytic reaction. Less
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Report
(3 results)
Research Products
(15 results)
