| Project/Area Number |
13125209
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| Research Category |
Grant-in-Aid for Scientific Research on Priority Areas
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| Allocation Type | Single-year Grants |
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| Review Section |
Science and Engineering
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| Research Institution | Osaka Medical College |
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Principal Investigator |
HAYASHI Hideyuki Osaka Med. Col., Dept. Biochemistry, Associate Professor, 医学部, 助教授 (00183913)
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| Co-Investigator(Kenkyū-buntansha) |
YANO Takato Osaka Med. Col., Dept. Biochemistry, Assistant Professr, 医学部, 講師 (40239827)
KURAMITSU Seiki Osaka Univ., Graduate School of Science, Professor, 大学院・理学研究科, 教授 (60153368)
KAGAMIYAMA Hiroynki Osaka Med. Col., Dept. Biochemistry, Professor, 医学部, 教授 (80028555)
MIZUGUCHI Hiroyuki Osaka Med. Col., Dept. Biochemistry, Research Associate, 医学部, 助手 (40247838)
石井 誠志 大阪医科大学, 医学部, 助手 (10247851)
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| Project Period (FY) |
2001 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥23,600,000 (Direct Cost: ¥23,600,000)
Fiscal Year 2003: ¥8,400,000 (Direct Cost: ¥8,400,000)
Fiscal Year 2002: ¥8,600,000 (Direct Cost: ¥8,600,000)
Fiscal Year 2001: ¥6,600,000 (Direct Cost: ¥6,600,000)
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| Keywords | proton-transfer / pKa / catalytic-mechanism / free-energy / induced-fit / stereochemistry / transferase / composite-biocatalyst / アミノ基転移酵素 / コンフォメーション / スフィンゴ脂質 / ピリドキサールリン酸 / 歪み |
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| Research Abstract |
By analyzing the proton-transfer process of pyridoxal enzymes, we elucidated the refined catalytic reaction mechanisms of them, thereby presenting the molecular understanding of the "induced fit" and "multisubstrate recognition". In aspartate aminotransferase (AAT), the conformational hange to the closed form induces an electrostatic repulsion between the main chain carbonyl group of Gly38 and the Schiff base, thereby raising the ree energy level of the Michaelis complex and increasing the k_<cat> value. Kinetic and structural analysis on the reaction of AAT with C5 substrates in comparison with C4 substrates revealed that in the case of AAT-C5 the Michaelis complex takes the open conformation and the external aldimine complex takes the closed conformation, while in AAT-C4, both complexes take the closed conformation. In the closed conformation, There is a hydrophobic interaction between the residues that is located at the active site entrance and covers the active site after the substrate binding. In the Michaelis complex with C5, the loss of this hydrophobic interaction increases the free energy level of the Michaelis complex, again contributing to the increase in the k_cat Value for C5 substrates. In this way, we revealed the systematic interactions between the parts--catalytic part, recognition part, regulation part, and so on--of composite biocatalysts.
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