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 bad 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.