THERMODYNAMIC CHARACTERIZATION OF 5'-AMP BINDING TO BOVINE LIVER-GLYCOGEN PHOSPHORYLASE-ALPHA

The binding of adenosine 5'-monophosphate to liver glycogen phosphoryl ase 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 c ompared with those of the binding of the same nucleotide to the muscle isozyme and to liver phosphorylase b. Calorimetric measurements in va rious buffer systems with different ionization heats suggest that prot ons are released during the binding of the nucleotide. The dimer of li ver glycogen phosphorylase a has been shown to have two equal and inde pendent sites for 5'-AMP, which would correspond to the activator site s identified in the muscle isozyme. The binding constants as well as t he 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 cha nge 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.