microbiology, Vol 141, 2619-2628, Copyright © 1995 by Society for General Microbiology

ARTICLES

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

HV Modak and DJ Kelly
Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, UK.

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)-1. In contrast, affinity chromatography 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)-1. The enzyme purified by the dye-linked procedure had a subunit molecular mass of 140,000 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.

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