| Project/Area Number |
01480524
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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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 Medical College |
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Principal Investigator |
KAGAMIYAMA Hiroyuki Osaka Medical College, Medical School, Professor, 医学部, 教授 (80028555)
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| Project Period (FY) |
1989 – 1991
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| Project Status |
Completed (Fiscal Year 1991)
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| Budget Amount *help |
¥6,500,000 (Direct Cost: ¥6,500,000)
Fiscal Year 1991: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1990: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1989: ¥4,400,000 (Direct Cost: ¥4,400,000)
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| Keywords | Mutagenesis / Transaminase / Aspartate Aminotransferase / Enzyme Mechanism / Pyridoxal Phosphate / Active Site / Protein Engineering / 部位特異性突然変異 / アスパラギン酸アミノ基転移酵素 / ピリドキサル燐酸 / 活性中心 / 酵素の活性中心 / 基質認識 |
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| Research Abstract |
We have examined the functional role of some active site residues of E. coli aspartate aminotransferase by using kinetic analysis on mutant enzymes produced by the site-directed mutagenesis. Aspartate aminotransferase has been the most extensively studied representative of many transaminases ; X-ray analysis has been done. Our findings are as follows :
1. Lys258 is essential for catalysis, acting as a catalytic base to withdraw an a -proton from the amino acid substrate, which is a prerequisite for the catalysis.
2. A negative charge at position 222, and a hydrogen bond between the hydroxyl group of Tyr225 and the unprotonated hydroxyl group of the coenzyme probably help in lowering the electron density of the coenzyme to facilitate the a- proton removal.
3. Arg292 and Arg386 are essential for the recognition of the dicarboxylic substrates.
4. Substitution of Arg292 to uncharged residues greatly enhanced the catalytic efficiency of the transamination of neutral amino acid without. any effect on the binding.
5. The endole ring of Trpl4O not only regulates the rotational movement of the coenzyme ring during the catalysis, but it may be also involved in the binding of the carboxyl side chain of the dicarboxylic acid substrates.
6. The phenol group of Tyr7O is essential for the stabilization of the transition states with all substrates. The presence of the benzen ring at position 70 is necessary to recognize the glutanate-2-oxoglutarate substrate pair.
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