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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 DeltaG0 stems from the value of DeltaH in the range of 25 to 35 degrees C. The enthalpy change of binding is strongly temperature-dependent, arising from a large negative DeltaCp 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.

The binding of AMP to rabbit muscle glycogen phosphorylase a (EC 2.4.1.1.) has been studied by equilibrium dialysis and isothermal microcalorimetry at pH 6.9 over a temperature range of 25 degrees C to 35 degrees C. Thermal titration experiments were carried out in various buffer systems. We have found by these methods that a certain number of protons are released when the protein binds to the ligand and are taken up by the buffer. The tetramer of phosphorylase a has been shown to have four equal and independent, non-cooperative binding sites for AMP at 25 degrees C, 30 degrees C, and 35 degrees C; these sites can be assigned to the so-called nucleotide or, activator, sites in the protein. The binding constants together with the changes in Gibbs energy, enthalpy, and entropy per site for the AMP binding were calculated at each temperature. A negative delta Cp value of -2.3 +/- 0.2 J K-1 (AMP bound)-1 was obtained for this binding process. The hydrophobic and vibrational contributions of the heat capacity and entropy changes have been resolved by the method described by Sturtevant (Sturtevant, J. M. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 2236-2240). From this analysis, it appears that the binding is, in all cases, enthalpy-driven, the two entropic contributions, hydrophobic and vibrational, having opposing effects.