Biochemistry, Department of


Date of this Version



Published in European Journal of Biochemistry 269 (2002), pp. 1267–1277. Copyright © 2002 FEBS; published by Wiley-Blackwell. Used by permission.


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.