γ-Aminobutyrate:α-Ketoglutarate aminotransferase from Pseudomonas sp. F-126: Purification, crystallization, and enzymologic properties

Abstract

γ-Aminobutyrate:α-ketoglutarate aminotransferase was purified from Pseudomonas sp. F-126 and crystallized. The crystalline enzyme is homogenous by the criteria of disc electrophoresis and ultracentrifugation ( s 0 20,w = 8.8 S). Molecular weights of 176,000 and 178,000 were obtained by gel filtration and ultracentrifugal sedimentation equilibrium methods, respectively. The enzyme exhibits absorption maxima at 415, 278, and 258 nm with shoulders at 284, 270, and 265 nm. The enzyme catalyzes the transamination of various ω-amino acid with α-ketoglutarate; γ-aminobutyrate is the best amino donor. The Michaelis constants are 2.8 m m for α-ketoglutarate and 4.1 m m for γ-aminobutyrate. In addition to ω-amino acids both isomers of ornithine and lysine and their decarboxylated product; i.e., putrescine and cadaverine, can serve as amino donors. No activity was observed with β-alanine. The enzyme has a maximum activity in the pH range of 8.5–9.0 and at 60 °C. The enzyme is stable at pH 6.0–10.0 at temperatures up to 65 °C. Pyridoxal 5′-phosphate protects the enzyme from heat inactivation. Carbonyl reagents and sulfhydryl reagents inhibit the enzyme activity but the enzyme inactivated by these reagents was reactivated by incubation with pyridoxal 5′-phosphate and thiol compounds, respectively. Chelating agents, nonsubstrate l- and d-α-amino acids, and metal ions except cupric ion showed no effect on the enzyme activity.