Effects of ions on ligand binding to pyruvate kinase: mapping the binding site with infrared spectroscopy

The effects of mono- and divalent ions (Li(+), K(+), Na(+), Cs(+), Mg(2+), Ca(2+), Mn(2+), Zn(2+)) on the binding of phosphoenolpyruvate (PEP) to rabbit muscle pyruvate kinase (PK) were studied by attenuated total reflection infrared spectroscopy in combination with a dialysis accessory. The experiments assessed the structural change of the protein as well as the binding mode of PEP. They indicated that a signal at 1638 cm(-1) assigned to a β sheet was perturbed differently with Na(+) as compared to the other monovalent ions. Otherwise, we obtained similar conformational changes in the presence of different monovalent cations, and therefore, it seems unlikely that the ion effects on activity are due to an ion effect on the structure of the PEP:PK complex. With different divalent cations, a particularly large conformational change was observed with Mn(2+) and attributed to a more closed conformation of the complex. The absorption of bound PEP was also detected. The antisymmetric stretching vibration of the carboxylate group of bound PEP indicates a more homogeneous binding mode for Mn(2+) compared to the other divalent ions. The symmetric stretching vibration depends on both monovalent and divalent ions, indicating that the dihedral angle O-C(1)-C(2)-O is affected by the ions in the catalytic site. Little change in the bond strengths of PEP is observed, indicating that the PEP:PK complex does not adopt a reactive conformation.