Binding of ATP to the Fructose-2,6-bisphosphatase Domain of Chicken Liver 6-Phosphofructo-2-kinase/Fructose-2,6-

To understand the mechanism by which the activity of the 6-phosphofructo-2-kinase (6PF-2K) of chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is stimulated by its substrate ATP, we studied two mutants of the enzyme. Mutation of either Arg-279, the penulti-mate basic residue within the Walker A nucleotide-bind-ing fold in the bisphosphatase domain, or Arg-359 to Ala eliminated the activation of the chicken 6PF-2K by ATP. Binding analysis by fluorescence spectroscopy using 2*(3*)-O-(N-methylanthraniloyl)-ATP revealed that the kinase domains of these two mutants, unlike that of the wild type enzyme, showed no cooperativity in ATP bind-ing and that the mutant enzymes possess only the high affinity ATP binding site, suggesting that the ATP bind-ing site on the bisphosphatase domain represents th