Marked modulation by phosphate of phosphoenolpyruvate carboxylase in leaves of Amaranthus hypochondriacus, a NAD-ME type C₄ plant: decrease in malate sensitivity but no change in the phosphorylation status

Abstract

The effect of Pi on the properties of phosphoenolpyruvate carboxylase (PEPC) from Amaranthus hypochondriacus, a NAD-ME type C₄ plant, was studied in leaf extracts as well as with purified protein. Efforts were also made to modulate the Pi status of the leaf by feeding leaves with either Pi or mannose. Inclusion of 30 mM Pi during the assay enhanced the enzyme activity in leaf extracts or of purified protein by >2-fold. The effect of Pi on the enzyme purified from dark-adapted leaves was more pronounced than that from light-adapted ones. The K_i for malate increased >2.3-fold and >1.9-fold by Pi in the enzyme purified from dark-adapted leaves and light-adapted leaves, respectively. Pi also induced an almost 50-60% increase in K_m for PEP or K_a for glucose-6-phosphate. Feeding the leaves with Pi also increased the activity of PEPC in leaf extracts, while decreasing the malate sensitivity of the enzyme. On the other hand, Pi sequestering by mannose marginally decreased the activity, while markedly suppressing the light activation, of PEPC. There was no change in phosphorylation of PEPC in leaves of A. hypochondriacus due to the feeding of 30 mM Pi. However, feeding with mannose decreased the light-enhanced phosphorylation of PEPC. The marked decrease in malate sensitivity of PEPC with no change in phosphorylation state indicates that the changes induced by Pi are independent of the phosphorylation of PEPC. It is suggested here that Pi is an important factor in regulating PEPC in vivo and could also be used as a tool to analyse the properties of PEPC.