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 micromol 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.
Smith
C R
CR
Departments of Biochemistry and Biology, Queen's University, Kingston, Ontario, Canada.
Knowles
V L
VL
Plaxton
W C
WC
D000244:Adenosine Diphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D001224:Aspartic Acid / Q000378:metabolism; D001937:Brassica / Q000201:enzymology*; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D003600:Cytosol / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D018698:Glutamic Acid / Q000378:metabolism; D006019:Glycolysis; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology; D008954:Models, Biological; D009584:Nitrogen / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D011770:Pyruvate Kinase / Q000737:chemistry* / Q000302:isolation & purification*; D013696:Temperature
D010940:Plant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D000244:Adenosine Diphosphate; D011770:Pyruvate Kinase; D008274:Magnesium; D009584:Nitrogen; D011188:Potassium