Regulation of Phosphoenolpyruvate Carboxylase in Zea mays by Protein Phosphorylation and Metabolites and Their Roles in Photosynthesis

Abstract

The effects of metabolites, protein phosphorylation and malate inhibition on phosphoenolpyruvate carboxylase (PEPC) activity were investigated at pH 7.0 in partially purified enzyme from maize leaves. Glycine, glucose 6-phosphate or alanine stimulated the activity two- to three-fold. Glycine and glucose 6-phosphate increased the affinity for PEP by factors of eight and four respectively. These metabolites changed the response of the enzyme activity to pH. Activity increased between pH 6.8 and 8.0 by 10-fold in the absence and 26% in the presence of these metabolites. In vitro phosphorylation of PEPC increased the activity two-fold in the absence but not in the presence of these metabolites. Malate was a strong inhibitor of PEPC, the KI value being 0.25-0.5 mM. Protein phosphorylation and the above metabolites increased the Ki value by factors of three and 12 respectively, but they synergistically increased the Ki 50-fold, thus providing maximal protection against malate inhibition. In the crude extracts from light- and dark-adapted leaves in the presence of a physiological concentration of malate (20 mM), PEPC activity comparable to the photosynthetic rate was obtained only from the light-adapted leaves in the presence of metabolites indicating that both light-induced protein phosphorylation and metabolic activators were essential for PEPC activation during photosynthesis. We propose that both these factors act synergistically to modulate PEPC during photosynthesis in maize.

Carbon fixation in Pinus halepensis submitted to ozone. Opposite response of ribulose‐1,5‐bisphosphate carboxylase/oxygenase and phosphoenolpyruvate carboxylase

“…This phosphorylation motif has previously been found in spruce by Relle and Wild (1996). Moreover, this phosphorylation state was reported to account for the modulation of some properties of the enzyme ( Duff et al 1995; Gao and Woo 1996). Particularly, phosphorylated PEPC was shown to be both more active and less sensitive to feedback inhibition by malate ( Nimmo et al 1987).…”

The regulatory role of residues 226–232 in phosphoenolpyruvate carboxylase from maize

“…Due to the reported regulatory interactions between malate and glycine it is noteworthy that R226Q and E229A are also desensitized to the activator glycine. Kinetic studies have shown that glycine can effectively overcome malate inhibition, raising the I 0.5 value for malate significantly (Gillinta and Grover 1995;Gao and Woo 1996;Tovar-Mendez et al 2000). Indeed, kinetic data suggest that glycine and malate bind in a mutually exclusive manner (Tovar-Mendez et al 2000).…”

Site‐directed mutagenesis of Lys600 in phosphoenolpyruvate carboxylase of Flaveria trinervia: its roles in catalytic and regulatory functions

“…The lysate was fractionated with 5-12% PEG-8000. The pellet was resuspended in buffer A for fast protein liquid chromatography (20 mM Tris, pH 8.0, 10% glycerol, 1 mM DTT, 0.1 mM EDTA) and purified on a Mono-Q column equilibrated with the buffer A [19,20]. The fractions (0.5 ml) containing high PEPC activity were pooled, desalted on a Sephadex-G-25 column and stored in 50% glycerol at -20°C.…”