Thermodynamics of the binding of AMP to glycogen phosphorylase a

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.