Studies of Parameters Affecting the Allosteric Nature of Phosphoenolpyruvate Carboxylase of Escherichia coli

Abstract

Abstract Phosphoenolpyruvate carboxylase is an allosteric enzyme known to be activated by acetyl coenzyme A or dioxane and inhibited by aspartate. A study of the experimental parameters affecting the allosteric nature of the enzyme has been made to establish optimal conditions for the enzyme and to understand certain abnormalities in its saturation kinetics. Although the requirement for a metal can be satisfied by either Mg2+ or Mn2+, the latter is more stimulatory except at pH 8.5 or higher. That Mn2+ and acetyl-CoA may act at the same site is indicated by the reduction in the activity of the acetyl-CoA-activated phosphoenolpyruvate carboxylase with the substitution of Mn2+ for Mg2+. Malate has been shown to be as powerful an inhibitor as aspartate. The effect of dioxane is strongly dependent on substrate as well as inhibitor concentrations. In the presence of acetyl-CoA or Mn2+, the saturation curve for phosphoenolpyruvate exhibits a plateau at half-maximal velocity. This phenomenon has been predicted by Koshland's model as being characteristic of a stabilized intermediate conformation of allosteric enzymes in which only half of the available sites are saturated. Thus our evidence points to a sequential alteration of the subunits of phosphoenolpyruvate carboxylase by the substrate, rather than an effect in shifting the equilibrium between two symmetrical conformations of the whole enzyme.