Purification and characterization of phosphoenolpyruvate carboxykinase from the parasitic helminth Ascaris suum.
Phosphoenolpyruvate carboxykinase has been purified from homogenates of Ascaris suum muscle strips to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purification is a three-step procedure which yields pure enzyme in milligram quantities with good yield. The subunit molecular weight of the Ascaris enzyme is between 75,000 and 80,000. The native molecular weight is 83,000 as determined by gel filtration. The kinetic constants for substrates of the carboxylation reaction were determined and compared to those measured for the avian liver enzyme. From kinetic studies it appears likely that two separate roles for divalent metal ions exist in the catalytic process. Studies conducted with Mn2+ or with micromolar concentrations of Mn2+, in the presence of millimolar concentrations of Mg2+ suggest that Mn2+ but not Mg2+ binds directly to and activates the enzyme while either Mn2+ or Mg2+ may bind to the nucleotide resulting in the metal-nucleotide complex. The metal-nucleotide is the active form of the substrate for the reaction. In the presence of Mg2+, an increase in the Mn2+ concentration results in a decrease in the Km for P-enolpyruvate suggesting a direct role for Mn2+ stimulation and regulation of activity. The concentrations of Mn2+ and Mg2+ in Ascaris muscle strips were determined by atomic absorption spectroscopy and support the proposed hypothesis of a specific Mn2+ activation of the enzyme. The nucleotides ATP and ITP act as competitive inhibitors against GTP with KI values of 0.50 and 0.75 mM, respectively. ITP is a competitive inhibitor against both IDP and P-enolpyruvate, suggesting overlapping binding sites for the two substrates on the enzyme.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001200:Ascaris / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D007293:Inosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000302:isolation & purification*
D007293:Inosine Triphosphate; D008345:Manganese; D010728:Phosphoenolpyruvate; D000255:Adenosine Triphosphate; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D008274:Magnesium