The regulation of oat coleoptile phosphoenolpyruvate carboxylase and malic enzyme activities by H+ and metabolites. Kinetic evidence for and against a metabolic pH-stat

Abstract

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) and malic enzyme (EC 1.1.1.40) activities in soluble protein extracts of Avena coleoptiles were investigated as functions of pH. The presence of malic enzyme activity was confirmed by radiochemical assays which identified the product of the forward reaction and spectrophotometric assays which demonstrated that reduction or oxidation of NADP required all the substrates and cofactors necessary for the forward or reverse reactions. In neither assay could NADP or Mn2+ be replaced by NAD or Mg2+. Activity was independent of pH (6.0 to 7.7) at malate concentrations less than 0.1 mM. At higher concentrations the pH increase raised activity by at least 100%, and substrate inhibition by malate resulted in activity at 0.97 mM malate which was less than that at 0.5 mM malate. The increase in malic enzyme activity with rising pH can be attributed to a decrease in substrate inhibition and a decrease in Km,app (Mn2+). pH increases between 7.0 and 7.6 increased Vmax and phosphoenolpyruvate limited phosphoenolpyruvate carboxylase (PEPC) activity by over 200%, and decreased the Km,app (Mg2+). Inhibition of PEPC and malic enzyme by various carboxylic acids and phosphorylated sugars decreased as the assay pH rose. The results are discussed in light of contrasting suggestions concerning the role of these enzymes in cytosol pH regulation.