Fructose 1,6-bisphosphate-activated pyruvate kinase from E. coli: ligand promoted conformational changes.

The ligand-dependent susceptibility to heat inactivation and to tryptic digestion and the intrinsic fluorescence of the fructose 1,6-bisphosphate-activated pyruvate kinase from Escherichia coli were investigated in the absence and in the presence of physiological ligands. With respect to the enzyme alone, binding of the allosteric activator fructose 1,6-bisphosphate makes the protein sensitive to tryptic attack and thermolabile, while binding of phosphoenolpyruvate and Mg2+, but not of either ligand separately, induces in the enzyme a highly thermostable conformation, the attainment of which does not require an ordered binding sequence of the two ligands. The apparent loosening of the enzyme structure induced by fructose bisphosphate suggests that the activation it exerts at low phosphoenolpyruvate concentration might be due to an increased accessibility of substrate to the active site.