Purification and characterization of cytosolic pyruvate kinase from Brassica napus (rapeseed) suspension cell cultures -: Implications for the integration of glycolysis with nitrogen assimilation

Cytosolic pyruvate kinase (PKc) from Brassica napus suspension cells was purified 201-fold to electrophoretic homogeneity and a final specific activity of 51 mu mol phosphoenolpyruvate utilized per min per mg protein. SDS/PAGE and gel filtration analyses of the final preparation indicated that this PKc is a 220-kDa homotetramer composed of 56-kDa subunits. The enzyme was relatively heat-stable and displayed a broad pH optimum of pH 6.8. PKc activity was absolutely dependent upon the simultaneous presence of a bivalent and univalent cation, with Mg2+ and K+ fulfilling this requirement. Hyperbolic saturation kinetics were observed for phosphoenolpyruvate, ADP, Mg2+ and K+ (apparent K-m values = 0.12, 0.075, 0.21 and 0.48 mm, respectively). Although the enzyme utilized UDP, CDP and IDP as alternative nucleotides, ADP was the preferred substrate. L-Glutamate, oxalate, and the flavonoids rutin and quercetin were the most effective inhibitors (I-50 values = 4, 0.3, 0.07, and 0.10 mm, respectively)(.) L-Aspartate functioned as an activator (K-a = 0.31 mm) by causing a 40% increase in V-max while completely reversing the inhibition of PKc by L-glutamate. Reciprocal control by l-aspartate and L-glutamate is specific for these amino acids and provides a rationale for the in vivo activation of PKc that occurs during periods of enhanced NH4+-assimilation. Allosteric features of B. napus PKc are compared with those of B. napus phosphoenolpyruvate carboxylase. A model is presented that highlights the pivotal role of L-aspartate and L-glutamate in the coordinate regulation of these key phosphoenolpyruvate utilizing cytosolic enzymes.