Influence of phosphorylation on the interaction of effectors with rat liver pyruvate kinase.

The binding of the allosteric effector fructose 1,6bisphosphate to rat liver pyruvate kinase type L and the influence of phosphorylation on that binding has been investigated. Pyruvate kinase contains four binding sites for fructose 1,6-bisphosphate and the binding exhibits an overall positive cooperative behavior (nH = 2). Determination of the individual intrinsic binding constants showed the relationship K'l < K'2 < K', > K'4 suggesting that there is a mixture of positive and negative cooperative interactions between the subunits of this tetrameric enzyme. Phosphorylation of pyruvate kinase increases the S0.s for fructose 1,6-bisphosphate binding by 2-fold and decreased K', and K'2 by 50%. Phosphoenolpyruvate increased the affinity and cooperativity of fructose 1,6-bisphosphate binding while the allosteric inhibitor alanine decreased the affinity of binding. The other substrate of pyruvate kinase, ADP, or the allosteric inhibitor ATP, had no effect on the binding of fructose 1,6-bisphosphate. W absorption difference measurements show that phosphoenolpyruvate and fructose 1,6-bisphosphate induced identical spectral changes that resulted from perturbations in what are probably tryptophanyl residues of the enzyme. These ligand-induced spectral differences were quantitatively less for the phosphorylated form of pyruvate kinase than for the dephosphorylated form. These data indicate that phosphorylation results in a decrease in the ability of the enzyme to change its conformation in response to substrate and fructose 1,6-bisphosphate, and together with the fructose 1,6-bisphosphate-binding studies suggest that phosphorylation shifts the equilibrium between the active and inactive forms of the enzyme to favor the inactive form.