Purification and characterization of homo- and hetero-dimeric acetate kinases from the sulfate-reducing bacterium Desulfovibrio vulgaris

J Biochem. 2001 Mar;129(3):411-21. doi: 10.1093/oxfordjournals.jbchem.a002872.

Abstract

Two distinct forms of acetate kinase were purified to homogeneity from a sulfate-reducing bacterium Desulfovibrio vulgaris Miyazaki F. The enzymes were separated from the soluble fraction of the cells on anion exchange columns. One acetate kinase (AK-I) was a homodimer (alpha(S)(2)) and the other (AK-II) was a heterodimer (alpha(S)alpha(L)). On SDS-PAGE, alpha(L) and alpha(S) subunits migrated as bands of 49.3 and 47.8 kDa, respectively, but they had an identical N-terminal amino acid sequence. A rapid HPLC method was developed to directly measure ADP and ATP in assay mixtures. Initial velocity data for AK-I and AK-II were collected by this method and analyzed based on a random sequential mechanism, assuming rapid equilibrium for the substrate binding steps. All kinetic parameters for both the forward acetyl phosphate formation and the reverse ATP formation catalyzed by AK-I and AK-II were successfully determined. The two enzymes showed similar kinetic properties in Mg(2+) requirement, pH-dependence and magnitude of kinetic parameters. These results suggest that two forms of acetate kinase are produced to finely regulate the enzyme function by post-translational modifications of a primary gene product in Desulfovibrio vulgaris.

MeSH terms

  • Acetate Kinase / chemistry
  • Acetate Kinase / isolation & purification*
  • Acetate Kinase / metabolism*
  • Adenosine Diphosphate / metabolism
  • Adenosine Triphosphate / metabolism
  • Amino Acid Sequence
  • Chromatography, High Pressure Liquid
  • Desulfovibrio vulgaris / enzymology*
  • Desulfovibrio vulgaris / metabolism
  • Dimerization
  • Enzyme Stability
  • Hydrogen-Ion Concentration
  • Kinetics
  • Lactic Acid / metabolism
  • Magnesium / pharmacology
  • Molecular Sequence Data
  • Molecular Weight
  • Protein Structure, Quaternary
  • Protein Subunits
  • Sulfates / metabolism

Substances

  • Protein Subunits
  • Sulfates
  • Lactic Acid
  • Adenosine Diphosphate
  • Adenosine Triphosphate
  • Acetate Kinase
  • Magnesium