Re-examination of the roles of PEP and Mg2+ in the reaction catalysed by the phosphorylated and non-phosphorylated forms of phosphoenolpyruvate carboxylase from leaves of Zea mays

Abstract

To study the effects of phosphoenolpyruvate (PEP) and Mg2+ on the activity of the non-phosphorylated and phosphorylated forms of phosphoenolpyruvate carboxylase (PEPC) from Zea mays leaves, steady-state measurements have been carried out with the free forms of PEP (fPEP) and Mg2+ (fMg2+), both in a near-physiological concentration range. At pH 7.3, in the absence of activators, the initial velocity data obtained with both forms of the enzyme are consistent with the exclusive binding of MgPEP to the active site and of fPEP to an activating allosteric site. At pH 8.3, and in the presence of saturating concentrations of glucose 6-phosphate (Glc6P) or Gly, the free species also combined with the active site in the free enzyme, but with dissociation constants at least 35-fold that estimated for MgPEP. The latter dissociation constant was lowered to the same extent by saturating Glc6P and Gly, to approx. one-tenth and one-sixteenth in the non-phosphorylated and phosphorylated enzymes respectively. When Glc6P is present, fPEP binds to the active site in the free enzyme better than fMg2+, whereas the metal ion binds better in the presence of Gly. Saturation of the enzyme with Glc6P abolished the activation by fPEP, consistent with a common binding site, whereas saturation with Gly increased the affinity of the allosteric site for fPEP. Under all the conditions tested, our results suggest that fPEP is not able to combine with the allosteric site in the free enzyme, i.e. it cannot combine until after MgPEP, fPEP or fMg2+ are bound at the active site. The physiological role of Mg2+ in the regulation of the enzyme is only that of a substrate, mainly as part of the MgPEP complex. The kinetic properties of maize leaf PEPC reported here are consistent with the enzyme being well below saturation under the physiological concentrations of fMg2+ and PEP, particularly during the dark period; it is therefore suggested that the basal PEPC activity in vivo is very low, but highly responsive to even small changes in the intracellular concentration of its substrate and effectors.

Molecular Basis of Aluminium Toxicity in Plants: A Review

“…But for longterm efflux of organic acid anions, continuous synthesis of organic acids inside the root cells is required [162] and for organic acid synthesis and metabolism, cytoplasmic Mg 2+ activity plays important role in activation of many enzymes, such as, citrate synthase [163], malate dehydrogenase [164], malate synthase [165], iso-citrate dehydrogenase [166,167], malic enzyme [168], PEPC (phosphor enol pyruvate carboxylase) [169][170][171], and pyruvate kinase [172]. Even alleviation in Al 3+ toxicity was found by addition of miliM concentrations of Mg in the external medium, by enhancingexudation of citrate in soybean [173] and rice bean [174].…”

Light-enhanced dark respiration in leaves, isolated cells and protoplasts of various types of C4 plants

“…Such a result was unexpected, because the capacity for glycine oxidation of bundle sheath mitochondria, where glycine-oxidizing enzymes are located but in amounts much smaller than in leaves of C 3 species (Morgan et al, 1993), is relatively weak as compared to oxidation of, for example, malate (Ohnishi and Kanai, 1983;Petit and Cantrel, 1986). It is known, however, that glycine in mesophyll cells of C 4 plants can stimulate the synthesis of malate by activation of phosphoenolpyruvate carboxylase (PEPC) (Uedan and Sugiyama, 1976;Gillinta and Grover, 1995;Tovar-Méndez et al, 1998), and The data represent the mean values7SE of five to seven independent experiments. Oxygen evolution rate was measured in the presence of bicarbonate at first (control) and then, in separate experiments, after addition of metabolites.…”

Photosynthetic and Other Phosphoenolpyruvate Carboxylase Isoforms in the Single-Cell, Facultative C4System of Hydrilla verticillata

“…The H. verticillata data parallel this, in that the C 4 leaf PEPC was strongly homotropic with PEP acting as a positive modulator. A similar situation exists with maize (Tovar-Mendez et al, 1998). The in vivo role for allosteric regulation of the C 4 photosynthetic isoform is undetermined.…”