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Phosphoenolpyruvate carboxylase (PEPC) specific activity increased by 250% following 8 to 10 days of Pi starvation of Brassica napus suspension cells. Densitometric scanning of PEPC immunoblots revealed a close correlation between PEPC activity and the amount of the antigenic 104-kDa PEPC subunit. To further assess the influence of Pi deprivation on PEPC, the enzyme was purified from Pi-sufficient (+Pi) and Pi-starved (–Pi) cells to electrophoretic homogeneity and final specific activities of 37–40 µmol phosphoenolpyruvate utilized per min per mg protein. Gel filtration, SDS/PAGE, and CNBr peptide mapping indicated that the +Pi and –Pi PEPCs are both homotetramers composed of an identical 104-kDa subunit. Respective pH–activity profiles, phosphoenolpyruvate saturation kinetics, and sensitivity to l-malate inhibition were also indistinguishable. Kinetic studies and phosphatase treatments revealed that PEPC of the +Pi and –Pi cells exists mainly in its dephosphorylated (l-malate sensitive) form. Thus, up-regulation of PEPC activity in –Pi cells appears to be solely due to the accumulation of the same PEPC isoform being expressed in +Pi cells. PEPC activity was modulated by several metabolites involved in carbon and nitrogen metabolism. At pH 7.3, marked activation by glucose 6-phosphate and inhibition by l-malate, l-aspartate, l-glutamate, dl-isocitrate, rutin and quercetin was observed. The following paper provides a model for the coordinate regulation of B. napus PEPC and cytosolic pyruvate kinase by allosteric effectors. l-Aspartate and l-glutamate appear to play a crucial role in the control of the phosphoenolpyruvate branchpoint in B. napus, particularly with respect to the integration of carbohydrate partitioning with the generation of carbon skeletons required during nitrogen assimilation.
Phosphoenolpyruvate carboxylase (PEPC) is a ubiquitous cytosolic enzyme in vascular plants that is also widely distributed in green algae, cyanobacteria, and bacteria . It catalyzes the irreversible β-carboxylation of phosphoenolpyruvate (PEP) in the presence of and Mg2+to yield oxaloacetate and Pi. The enzyme is particularly abundant in the mesophyll cells of C4 and crassulacean acid metabolism (CAM) leaves where it participates in photosynthesis by catalyzing the initial fixation of atmospheric CO2. Both allosteric mechanisms and covalent modification are involved in the regulation of PEPC activity during C4 and CAM photosynthesis [1–3]. Early work determined that C4 and CAM photosynthetic PEPCs were regulated via a diurnal cycle that served to modulate their sensitivity to l-malate inhibition, without affecting Vmax or the amount of PEPC protein. This has been shown to be due to reversible phosphorylation by an endogenous Ca2+-independent PEPC protein kinase and dephosphorylation by a protein phosphatase type 2 A (PP2A) at a key serine residue located near the N-terminus of the 100- to 110-kDa PEPC subunit [1–3].
In contrast to C4 and CAM leaf PEPCs, the properties, regulation and functions for the enzyme from C3 plants and nonphotosynthetic tissues of C4 and CAM plants are less well understood. Proposed roles for the C3 enzyme are diverse and include: (a) regulation of cellular pH and cation balance, (b) production of dicarboxylic acids used as respiratory substrates by nitrogen-fixing bacteroids of legume root nodules, and/or (c) the anaplerotic replenishment of citric acid cycle intermediates consumed in biosynthesis . The enzyme has been partially or fully purified from several nonphotosynthetic plant tissues including germinating castor seeds , soybean root nodules , and ripening banana fruit . As with the C4 and CAM leaf enzyme, reversible phosphorylation and allosteric effectors have been suggested to be important in the regulation of PEPC from C3 plants [1,4–10]. Radiolabeling studies with 32Pi have demonstrated the in vivo phosphorylation of PEPC in soybean root nodules , banana fruit , wheat leaves , Vicia faba guard cells , and germinating wheat seeds . In banana fruit, an endogenous PEPC kinase forms a tight complex with its target enzyme . However, the regulatory properties of plant PEPC that mediate glycolytic flux and PEP partitioning during nutritional Pi deprivation have not been elucidated.
Phosphorous is an essential element for normal growth and metabolism, as it plays a central role in virtually all metabolic processes. Plants preferentially absorb phosphorous from the soil in its fully oxidized anionic form, Pi (H2PO4−, orthophosphate). Although widely distributed in the earth's crust, most Pi exists in insoluble mineral forms and, as such, is unavailable to plants . Thus, in many natural environments Pi deficiency is the rule rather than the exception. Plants have evolved to acclimatize to Pi stress to varying degrees through a number of mechanisms, including acid phosphatase (APase) induction . APases can function as intracellular or extracellular Pi salvaging systems that can scavenge Pi from phosphate esters. The plant ‘Pi starvation response’ also involves the induction of PEPC activity, which has been reported for several C3 plants including: Brassica nigra (black mustard) and Catharanthus roseus (periwinkle) suspension cells [12,13], Lupinus albus (lupin), Lycopersicon esculentum (tomato) and Cicer arietinum (chickpea) roots [14,15], and B. napus shoots . PEPC induction has been correlated with increases in in vivo dark 14CO2 fixation and/or levels of PEPC-derived organic acids [13–19]. During Pi deprivation, PEPC (with malate dehydrogenase and NAD-malic enzyme) may provide a metabolic ‘bypass’ to the ADP-limited cytosolic pyruvate kinase (PKc) to facilitate continued pyruvate supply to the citric acid cycle, while concurrently recycling the PEPC by-product Pi for its assimilation into the metabolism of the Pi-starved cells [11,13]. In addition, PEPC induction can promote the synthesis and consequent excretion of large amounts of malic and citric acids from roots during Pi stress. This acidifies the rhizosphere, which therefore increases Pi availability to the plant by solubilizing otherwise inaccessible sources of mineral Pi[11,14,16,19]. Increased levels of PEPC protein and mRNA were correlated with enhanced PEPC activity of proteoid roots of Pi-deficient lupin plants . The possible expression of a separate ‘–Pi inducible’ PEPC isozyme was indicated by the isolation of a PEPC cDNA from the Pi-stressed proteoid lupin roots . In addition, kinetic studies of partially purified PEPC from proteoid lupin roots suggested that during Pi stress the enzyme may be phosphorylated by an endogenous PEPC kinase, resulting in reduced sensitivity of PEPC to inhibition by l-malate .
The initial goal of the current study was to investigate the impact of Pi starvation on the PEPC of B. napus suspension cell cultures. Such cultures represent an ideal model system for examining the influence of Pi nutrition on plant metabolism, because they contain a homogeneous population of cells, with each cell in direct contact with the culture medium. Moreover, relatively large quantities of cells at a precise nutritional and developmental state can be amassed for use in enzyme purification. Our second goal was to compare the kinetic and regulatory features of the purified PEPC from Pi-sufficient B. napus with those being concurrently characterized for the homogeneous PKc from the same cells . Coordinate regulation of these two PEP utilizing enzymes plays a critical role in the regulation of plant cytosolic glycolytic flux, particularly with respect to the integration of carbon partitioning with the generation of 2-oxoglutarate needed for nitrogen assimilation by Gln synthetase/Gln 2-oxoglutarate aminotransferase (GS/GOGAT) [21,22]. PEPC has an additional metabolic function during nitrogen assimilation to produce oxaloacetate for l-Asp synthesis by Asp aminotransferase (AAT). PKc and PEPC appear to be activated (or deinhibited) in vivo following nitrogen resupply to nitrogen limited plant tissues and green algae . This not only generates necessary carbon skeletons for AAT and/or GS/GOGAT, but also serves to reduce PEP levels, thereby relieving PEP inhibition of the ATP-dependent phosphofructokinase , thus stimulating overall glycolytic flux. Despite the tremendous importance of PEPC and PKc in controlling interactions between plant carbon and nitrogen metabolism, few workers have attempted to characterize both enzymes from the same plant tissue or cell type . Indeed, the simultaneous complete purification and thorough comparative analysis of PEPC and PKc from the same vascular plant source has not been described. This is important because vascular plants express tissue- and developmental-specific isozymes of PKc and PEPC that may display very different physical, kinetic, and regulatory properties [1,21–23]. In particular, the combined results of the present and following  papers suggest a pivotal role for l-Asp and l-Glu in the coordinate regulation of B. napus PEPC and PKc.