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

Eur J Biochem. 2000 Jul;267(14):4477-85. doi: 10.1046/j.1432-1327.2000.01494.x.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Diphosphate / metabolism
  • Allosteric Regulation
  • Aspartic Acid / metabolism
  • Brassica / enzymology*
  • Cells, Cultured
  • Chromatography, Affinity
  • Chromatography, Liquid
  • Cytosol / enzymology*
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Activation
  • Glutamic Acid / metabolism
  • Glycolysis
  • Hydrogen-Ion Concentration
  • Immunoblotting
  • Kinetics
  • Magnesium / metabolism
  • Mitochondria / enzymology
  • Models, Biological
  • Nitrogen / metabolism*
  • Plant Proteins / metabolism
  • Potassium / metabolism
  • Pyruvate Kinase / chemistry*
  • Pyruvate Kinase / isolation & purification*
  • Temperature

Substances

  • Plant Proteins
  • Aspartic Acid
  • Glutamic Acid
  • Adenosine Diphosphate
  • Pyruvate Kinase
  • Magnesium
  • Nitrogen
  • Potassium