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

Microbiology. 1995 Oct;141 ( Pt 10):2619-28. doi: 10.1099/13500872-141-10-2619.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetyl Coenzyme A / metabolism*
  • Adenosine Diphosphate / pharmacology
  • Allosteric Regulation
  • Amino Acid Sequence
  • Amino Acids / analysis
  • Aspartic Acid / pharmacology
  • Avidin / pharmacology
  • Carbon Dioxide / metabolism
  • Chromatography, Affinity / methods*
  • Coloring Agents
  • Enzyme Activation
  • Enzyme Inhibitors / pharmacology
  • Enzyme Stability
  • Ketoglutaric Acids / pharmacology
  • Kinetics
  • Ligands
  • Molecular Sequence Data
  • Mutation
  • Pyruvate Carboxylase / drug effects
  • Pyruvate Carboxylase / genetics
  • Pyruvate Carboxylase / isolation & purification*
  • Pyruvate Carboxylase / metabolism
  • Rhodobacter capsulatus / enzymology*
  • Rhodobacter capsulatus / genetics
  • Sepharose / analogs & derivatives
  • Sequence Analysis
  • Sequence Homology, Amino Acid

Substances

  • Amino Acids
  • Coloring Agents
  • Enzyme Inhibitors
  • Ketoglutaric Acids
  • Ligands
  • Avidin
  • Carbon Dioxide
  • Aspartic Acid
  • Adenosine Diphosphate
  • sepharose CL 6B
  • Acetyl Coenzyme A
  • Sepharose
  • Pyruvate Carboxylase

Associated data

  • SWISSPROT/P05165
  • SWISSPROT/P11154
  • SWISSPROT/P14882
  • SWISSPROT/P24182
  • SWISSPROT/P32327
  • SWISSPROT/Q05920