| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2005: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2004: ¥1,900,000 (Direct Cost: ¥1,900,000)
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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-
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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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