Purification and characterization of cytosolic pyruvate kinase from Brassica napus (rapeseed) suspension cell cultures - Implications for the integration of glycolysis with nitrogen assimilation
Cr. Smith et al., 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 BIOCH, 267(14), 2000, pp. 4477-4485
Cytosolic pyruvate kinase (PKc) from Brassica napus suspension cells was pu
rified 201-fold to electrophoretic homogeneity and a final specific activit
y of 51 mu mol phosphoenolpyruvate utilized per min per mg protein. SDS/PAG
E 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 r
elatively heat-stable and displayed a broad pH optimum of pH 6.8. PKc activ
ity was absolutely dependent upon the simultaneous presence of a bivalent a
nd univalent cation, with Mg2+ and K+ fulfilling this requirement. Hyperbol
ic 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). Al
though the enzyme utilized UDP, CDP and IDP as alternative nucleotides, ADP
was the preferred substrate. L-Glutamate, oxalate, and the flavonoids ruti
n 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 reversin
g the inhibition of PKc by L-glutamate. Reciprocal control by l-aspartate a
nd L-glutamate is specific for these amino acids and provides a rationale f
or 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 hig
hlights the pivotal role of L-aspartate and L-glutamate in the coordinate r
egulation of these key phosphoenolpyruvate utilizing cytosolic enzymes.