Acetyl-CoA-dependent pyruvate carboxylase from the photosynthetic bacterium Rhodobacter capsulatus: rapid and efficient purification using dye-ligand affinity chromatography.
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.
Modak
H V
HV
Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, UK.
Kelly
D J
DJ
D000105:Acetyl Coenzyme A / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001224:Aspartic Acid / Q000494:pharmacology; D001360:Avidin / Q000494:pharmacology; D002245:Carbon Dioxide / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D004396:Coloring Agents; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D007656:Ketoglutaric Acids / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D009154:Mutation; D011766:Pyruvate Carboxylase / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D016354:Rhodobacter capsulatus / Q000201:enzymology* / Q000235:genetics; D012685:Sepharose / Q000031:analogs & derivatives; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid
D000596:Amino Acids; D004396:Coloring Agents; D004791:Enzyme Inhibitors; D007656:Ketoglutaric Acids; D008024:Ligands; D001360:Avidin; D002245:Carbon Dioxide; D001224:Aspartic Acid; D000244:Adenosine Diphosphate; C047264:sepharose CL 6B; D000105:Acetyl Coenzyme A; D012685:Sepharose; D011766:Pyruvate Carboxylase