Thermodynamic characterization of 5'-AMP binding to bovine liver glycogen phosphorylase alpha

The binding of adenosine 5'-monophosphate to liver glycogen phosphorylase a (EC 2.4.1.1) has been studied by size exclusion high performance liquid chromatography and isothermal titration microcalorimetry at pH 6.9 over a temperature range of 25 to 35 degrees C. The results are compared with those of the binding of the same nucleotide to the muscle isozyme and to liver phosphorylase b. Calorimetric measurements in various buffer systems with different ionization heats suggest that protons are released during the binding of the nucleotide. The dimer of liver glycogen phosphorylase a has been shown to have two equal and independent sites for 5'-AMP, which would correspond to the activator sites identified in the muscle isozyme. The binding constants as well as the changes in Gibbs energy, enthalpy, and entropy per site for 5'-AMP binding were calculated at each temperature. The results show that the major contribution to the negative value of Delta G(0) stems from the value of Delta H in the range of 25 to 35 degrees C. The enthalpy change of binding is strongly temperature-dependent, arising from a large negative Delta C-p of binding equal to -1.45 +/- 0.02 kJ K-1 (mol of 5'-AMP bound)(-1), which suggests significant changes in the polar and apolar surfaces accessible to the solvent.