2005 Fiscal Year Final Research Report Summary
Studies on the proton transfer processes in pyridoxal enzymes
| Project/Area Number |
16570125
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Functional biochemistry
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| Research Institution | Osaka Medical College |
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Principal Investigator |
HAYASHI Hideyuki Osaka Medical College, Faculty of Medicine, Professor, 医学部, 教授 (00183913)
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| Co-Investigator(Kenkyū-buntansha) |
YAMAMOTO Daisuke Osaka Medical College, Faculty of Medicine, Assistant Professor, 医学部, 講師 (50240106)
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| Project Period (FY) |
2004 – 2005
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| Keywords | pyridoxal 5'-phosphate / proton transfer / driving force / tunneling / aminotransferase / amine oxidase / energy barrier / catalytic mechanism |
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| Research Abstract |
The reaction of aspartate aminotransferase (AAT) with C5 substrate was studied kinetically and structurally. The Michaelis complex between AAT and C5 substrate takes the open conformation, and a transition to the closed conformation occurs when the external aldimine complex is formed from the Michaelis complex by transaldimination. The Michaelis complex can take 4 types of protonation forms, while the external aldimine has only 2 types of protonation forms. The structural basis for controlling the protonation was solved ; the open conformation of the Michaelis complex allows the binding of glutamate in such a way that the α-amino group is oriented to the opposite direction to the PLP (pyridoxal 5'-phosphate)-Lys258 Schiff base, enabling the multiple protonation patterns of the α-amino group and the Schiff base. It was also found that during the conformational transition to the closed form the substrate glutamate is forced to take an unfavorable conformation (cis-conformation of the C1-
… More
Cα-Cβ-Cγ). That is, the "induced-fit" occurs not only to the enzyme but also to the substrate. This is a new notion of the substrate recognition mechanism. Through this study, the entire reaction mechanism of AAT has been established.
In order to know the quantum-chemical basis for the proton transfer, a quantum-chemical calculation was performed on the PLP-Lys258 Schiff base of AAT. The active site was excised from the X-ray structure, and the main chain atoms were fixed while other side chain atoms were allowed to move. The calculation at the level of B3LYP 6-31G(d,p) resulted in the "strain" energy 15 kJ/mol of the protonated form of the PLP-Lys258 Schiff base. This was very closed to the experimental value of 16 kJ/mol. In this way, the method for calculating the proton-transfer energy of the essential catalytic group of the active site has been successfully established.
In addition to the reaction mechanism of PLP enzymes, the proton transfer process in the Cu^<2+>-containing amino oxidase, which is mechanistically closely related to PLP-dependent aminotransferase, has been studied. Using temperature-dependent kinetic analysis on the prototropic shift process and deuterated substrates, the proton transfer of tyramine proceeds through a proton-tunneling mechanism.
In summary, the studies shown above collectively shows the way in which the various research methods, spectroscopy, crystallography, reaction kinetics, theoretical calculation, and others are organized to clarify the proton transfer process in enzymes. Less
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[Journal Article] Kinetic and Structural Studies on the Catalytic Role of the Aspartic Acid Residue Conserved in Copper Amine Oxidase.2006
Author(s)
Chiu, Y.C., Okajima, T., Murakawa, T., Uchida, M., Taki, M., Hirota, S., Kim, M., Yamaguchi, H., Kawano, Y., Kamiya, N., Kuroda, S., Hayashi, H., Yamamoto, Y., Tanizawa, K.
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Journal Title
Biochemistry 45
Pages: 41055-4120
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Quantum mechanical hydrogen tunneling in bacterial copper amine oxidase reaction.2006
Author(s)
Murakawa, T., Okajima, T., Kuroda, S., Nakamoto, T., Taki, M., Yamamoto, Y., Hayashi, H., Tanizawa, K.
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Journal Title
Biochem Biophys Res Commun 342
Pages: 414-423
Description
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[Journal Article] Molecular cloning, expression, and characterization of pyridoxamine-pyruvate aminotransferase.2006
Author(s)
Yoshikane, Y., Yokochi, N., Ohnishi, K., Hayashi, H., Yagi, T.
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Journal Title
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Binding of C5-dicarboxylic substrate to aspartate aminotransferase : implications for the conformational change at the transaldimination step.2005
Author(s)
Islam, M.M., Goto, M., Miyahara, I., Ikushiro, H., Hirotsu, K., Hayashi, H.
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Journal Title
Biochemistry 44
Pages: 8218-8229
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Crystal structure of a putative aspartate aminotransferase belonging to subgroup IV.2004
Author(s)
Katsura, Y, Shirouzu, M., Yamaguchi, H., Ishitani, R., Nureki, O., Kuramitsu, S., Hayashi, H., Yokoyama, S.
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Journal Title
Proteins 55
Pages: 487-492
Description
「研究成果報告書概要(欧文)」より
