|Budget Amount *help
¥2,600,000 (Direct Cost : ¥2,600,000)
Fiscal Year 1997 : ¥1,200,000 (Direct Cost : ¥1,200,000)
Fiscal Year 1996 : ¥1,400,000 (Direct Cost : ¥1,400,000)
Aspartate aminotransferase catalyzes the reversible interconversion of aspartate and 2-oxoglutarate to oxalacetate and glutamate.X-ray crystallographic data showed that each subunit of the dimeric enzymes consist of three parts ; a floppy amino-terminal region (residues 1-14) , the small domain that comprises the amino terminal residues15-48) and the carboxyl terminal residues 326-410, and the large domain (residues 49-325) that provides the binding site fpr the conezyme. Peptide composed of Ala227-Phe228-Gly229 are in the domain-domain interface and has two conformations that depend on the "induced fit" movement.
Phe228 was replaced by a less bulky residue, Ala, and by a bulky one, Tyr.Phe228Ala and Phe228Tyr mutant enzyme showed a decrease in the affinity for the substrate. Phe228Leu mutant enzyme showed a slight decrease in both the catalytic rate and affinity for the substrate to compare with the wild type enzyme. Gly227Ala and Phe228Tyr mutant enzyme revealed increase of k_<cat>/K_
m, values for the substrate due to the increase of affinity. The catalytic competence, k_<cat>/K_m, of Ala229Gly mutant enzyme retained almost 50% reactivity for that of wild type enzyme.
pH titration of wild-type and mutant enzymes showed different pKa values of the internal aldimine individually. The pKa valurs for Phe228Leu, Ala229Gly and Ala229Val were higher by 0.6,0.3 and 0.35 Ph unit, respectively, than that for the wild-type enzyme. The pKa values for Phe228Tyr were lower by 0.5 pH unit.These results indicate that the single mutation of inter-domain residue may affect the state of bound pyridoxal 5'-phosphate.
To analyze the state of internal aldimine bond, we synthesized substrate analog, [^<15>N] 2-methylaspartate. Nitrogen signal was assigned on the ^<15>N NMR and the pKa value of amino group of substrate analog was determined as 10.6. The ^1H and ^<15>N chemical shifts of the aldimine bond NH group in Schiff's bases of pyridoxal 5'-phosphate and L-aspartate, 2-methyl-DL-aspartate and L-alanine were analyzed using one-and two-dimensional NMR techniques.