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 ·mg protein −1 ) and fructose‐2,6‐bisphosphatase (specific activity 1.65–1.75 nmol·min −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.