Illumination increases the affinity of phosphoenolpyruvate carboxylase to bicarbonate in leaves of a C₄ plant, Amaranthus hypochondriacus

Abstract

Illumination increased markedly the affinity to bicarbonate of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) in leaves of Amaranthus hypochondriacus L., a C₄ plant. When leaves were illuminated, the apparent K_m for (HCO₃^-) of PEPC decreased by about 50% concurrent with a 2- to 5-fold increase in V_max and 3- to 4-fold increase in K_i for malate. The inclusion of ethoxyzolamide, an inhibitor of carbonic anhydrase, during the assay had no effect on kinetic and regulatory properties of PEPC indicating that carbonic anhydrase was not involved during light-induced sensitization of PEPC to HCO₃^-. Pretreatment of leaf discs with cycloheximide (CHX), a cytosolic protein synthesis inhibitor, suppressed significantly the light-enhanced decrease in apparent K_m (HCO₃^-). Further, in vitro phosphorylation of purified dark-form PEPC by protein kinase A (PKA) decreased the apparent K_m (HCO₃^-) of the enzyme, in addition increasing K_i (malate) as expected. Such changes, due to in vitro phosphorylation of purified PEPC by PKA, occurred only with wild-type PEPC, but not in the mutant form of maize (S15D) which is already a mimic of the phosphorylated enzyme. These results suggest that phosphorylation of the enzyme is important during the sensitization of PEPC to HCO₃^- by illumination in C₄ leaves. Since illumination is expected to increase the cytosolic pH and the availability of dissolved HCO₃^- in mesophyll cells, the sensitization by light of PEPC to HCO₃^- could be physiologically quite significant.