Roles of the amino acid residues at β-strand Ⅲ of D-amino acid aminotransferase in the catalytic property of the enzyme = D-amino acid aminotransferase 의 β-strand Ⅲ 부위의 아미노산들의 효소촉매작용에 대한 역할

Abstract

A PCR-based random mutagenesis was performed to study the active site of the D-amino acid aminotransferase of Bacillus sp. YM-1. A mutant enzyme showing higher affinity to D-valine was selected and the DNA sequence was determined. It was found that the mutation of the $Val^{33}$ residue to alanine was responsible for the enhanced affinity to the branched substrate. This residue was observed in the b-strand III of D-AAT which formed by the amino acid residues from the $Val^{30}$ to the $Val^{36}$ residues and was located across the active site of D-AAT. The odd-numbered amino acids ($Tyr^{31}$, $Val^{33}$, and $Lys^{35}$) in the strand were revealed to be directed toward the active site. Interestingly, the $Glu$^{32}$ was also directed the active site. Studies were focused on the involvement of these amino acid residues in catalysis by alanine scanning mutagenesis. The Y31A and E32A mutant enzymes showed a remarkable decrease in the $k_{cat}$ value, retaining less than 1% of the wild-type enzyme activity. The $k_{cat}$ values of V33A and K35A changed only slightly, but the $K_m$ of K35A for α-ketoglutarate increased to 35.6mM, compared to the $K_m$ value of 2.5mM of the wild-type enzyme. These results suggested that the positive charge at $Lys^{35}$ interacted electrostatically with the negative charge at the side chain of α-ketoglutarate. Site-directed mutagenesis of the $Glu^{32}$ residue was conducted to demonstrate the role of this residue in detail. From the kinetic and spectral characteristics of the $Glu^{32}$-substituted enzymes, the $Glu^{32}$ residue seemed to interact with the positive charge at the Schiff base formed between the aldehyde group of pyridoxal 5``-phosphate (PLP) and the ε-amino group of $Lys^{145}$ residue. Because the charge interaction of the $Glu^{32}$ residue with the $Lys^{145}$ residue was supposed to affect the formation and the stabilization of the enzyme-substrate complexes, studies were proceeded to investigate the changes ...