Acetyl-CoA-dependent pyruvate carboxylase from the photosynthetic bacterium Rhodobacter capsulatus: rapid and efficient purification using dye-ligand affinity chromatography

H. V. Modak, D. J. Kelly
1995 Microbiology  
Pyruvate carboxylase (PC) was purified to homogeneity from an overexpressing strain of the purple photosynthetic bacterium Rhodobacter capsulatus using a rapid dye-ligand affinity chromatography procedure, in which dye-bound enzyme was specifically eluted with a low concentration of acetyl-CoA, an allosteric activator of the enzyme. The enzyme purified by this method was obtained in 75% yield with a specific activity of 40 U (mg protein)-'. In contrast, affinity chromatography on a monomeric
more » ... y on a monomeric avidin column, commonly used in the purification of biotin-containing carboxylases, resulted in a yield of < 40%, with a specific activity of 10 U (mg protein)-'. The enzyme purified by the dye-linked procedure had a subunit molecular mass of 140000 Da and was absolutely dependent on acetyl-CoA for activity. Acetyl-CoA was also effective in protecting the enzyme from thermal denaturation. The enzyme was inhibited by 2-oxoglutarate and, to a lesser extent, L-aspartate, with sigmoidal kinetics with respect to acetyl-CoA concentration. The amino acid composition, pH optimum and kinetic constants for pyruvate, ATP and bicarbonate were determined. An N-terminal sequence of 26 residues was obtained, which was homologous to the N-terminal regions of several eukaryotic PCs, propionyl-CoA carboxylases and acetyl-CoA carboxylase. Abbreviations: HRP, horseradish peroxidase; ICM, intra-cytoplasmic membrane; PC, pyruvate carboxylase. pc from Psetldomonas citronelloh (Suebert 0001-9931 0 1995 SGM
doi:10.1099/13500872-141-10-2619 pmid:7582022 fatcat:m5643ieuqjcnbgcuvrkxd7kuem