Purification and characterization of cytosolic pyruvate kinase from Brassica napus (rapeseed) suspension cell cultures

Implications for the integration of glycolysis with nitrogen assimilation

Authors


W. C. Plaxton, Department of Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6. Fax: + 1613 533 6617, Tel.: + 1613 533 6150, E-mail: plaxton@biology.queensu.ca

Abstract

Cytosolic pyruvate kinase (PKc) from Brassica napus suspension cells was purified 201-fold to electrophoretic homogeneity and a final specific activity of 51 µmol 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 Km 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 (I50 values = 4, 0.3, 0.07, and 0.10 mm, respectively).l-Aspartate functioned as an activator (Ka = 0.31 mm) by causing a 40% increase in Vmax 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 NH +4-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.

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