Kinetic properties of bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from spinach leaves.
A cDNA encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was isolated from a Spinacia oleracea leaf library and used to express a recombinant enzyme in Escherichia coli and Spodoptera frugiperda cells. The insoluble protein expressed in E. coli was purified and used to raise antibodies. Western blot analysis of a protein extract from spinach leaf showed a single band of 90.8 kDa. Soluble protein was purified to homogeneity from S. frugiperda cells infected with recombinant baculovirus harboring the isolated cDNA. The soluble protein had a molecular mass of 320 kDa, estimated by gel filtration chromatography, and a subunit size of 90.8 kDa. The purified protein had activity of both 6-phosphofructo-2-kinase specific activity 10.4-15.9 nmol min(-1) x mg protein (-1) and fructose-2,6-bisphosphatase (specific activity 1.65-1.75 nmol x mol(-1) mg protein(-1). The 6-phosphofructo-2-kinase activity was activated by inorganic phosphate, and inhibited by 3-carbon phosphorylated metabolites and pyrophosphate. In the presence of phosphate, 3-phosphoglycerate was a mixed inhibitor with respect to both fructose 6-phosphate and ATP. Fructose-2,6-bisphosphatase activity was sensitive to product inhibition; inhibition by inorganic phosphate was uncompetitive, whereas inhibition by fructose 6-phosphate was mixed. These kinetic properties support the view that the level of fructose 2,6-bisphosphate in leaves is determined by the relative concentrations of hexose phosphates, three-carbon phosphate esters and inorganic phosphate in the cytosol through reciprocal modulation of 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities of the bifunctional enzyme.
Markham
Jonathan E
JE
Department of Plant Sciences, University of Oxford, South Parks Road, Oxford, OX1 3RB, UK.
Kruger
Nicholas J
NJ
D000595:Amino Acid Sequence; D018076:DNA, Complementary; D017343:Genes, Plant; D007700:Kinetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D025481:Phosphofructokinase-2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018515:Plant Leaves / Q000201:enzymology*; D012333:RNA, Messenger / Q000302:isolation & purification; D018749:RNA, Plant / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D018724:Spinacia oleracea
D018076:DNA, Complementary; D009097:Multienzyme Complexes; D012333:RNA, Messenger; D018749:RNA, Plant; D011994:Recombinant Proteins; D025481:Phosphofructokinase-2