| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2000: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1999: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Research Abstract |
The proton-transfer processes have been studied in detail for three kinds of pyridoxal 5'-phosphate (PLP)-dependent enzymes, aromatic L-amine acid decarboxylase (AADC), aspartate aminotransferase (AAT), and aromatic amino acid aminotransferase (ART). The substrate amino acid with deprotonated a-amine group is preferentially bound to AADC, but the substrate with protonated a-amino group can also be bound to a lesser extent. In the Michaelis complex, the proton on the substrate a-amine group is dissociated because of the electrostatic repulsion with the protonated PLP Schiff base of AADC.In AAT and ART, it was found that *re is a conformational strain in the protonated PLP Schiff base, which is released successively during the course of catalysis. This mechanism is important for regulating the proton-transfer events in the catalytic process. In both AAT and ART electrostatic interactions play minor roles than the Schiff base strain in regulating the pK_a of the active site catalytic group. Altogether, in AADC, the strain between the substrate and the enzyme is considered to increase the k_<cat> value, whereas in AAT and ART, the strain inherent to the enzyme protein but is released in the intermediate including the transition state, increases the k_<cat>/K_m value. This mechanism is most clearly understood in the 3-dimensional energy profiles, which include the "proton number" axis as one of the coordinates. These profiles clearly explain the driving force of proton transfer, showing that the notion of pK_a has a secondary significance in the energetics of catalysis.
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