Purification and kinetics of mouse liver fructose 6-phosphate, 2-kinase

The mouse liver fructose 6-phosphate, 2-kinase was purified by ultracentrifugation, polyethylene glycol precipitation, and subsequently by chromatography on DEAE-Sephadex, Blue-Sepharose and phasphocellulose columns. Gel filtration and SDS polyacrylamide electrophoresis showed that the enzyme has a molecular weight of 110,000 with two identical subunits. Mg2+ is essential for its activity. The activation of the enzyme by Mg2+ showed a positive cooperativity. The substrate saturation curve for fructose 6-phosphate was sigmoidal and for ATP was hyperbolic. The Km's for ATP increased with decrease in concentrations of fructose 6-phosphate indicating that the sequence for the substrates binding was in an ordered mechanism with respect to fructose 6-phosphate prior to ATP. An ionizable residue at the active site with pKa 9.5 was essential for the ATP binding and the pKa shifted to 9.8 after the binding of ATP.