Calorimtric study of the interactions between phophorylase b and its nucleotide activators

Interactions of glycogen phosphorylase b with AMP and IMP have been studied by calorimetry at two different temperatures: 18 and 25°. Calorimetric titration of the enzyme with AMP shows a biphasic curve suggesting that the enzyme contains two sets of AMP binding sites with different affinity. The high affinity sites are essentially filled at 0.3 mM AMP whereas significant nucleotide binding at the low affinity sites occurs only when AMP concentration exceeds 1 mM, Since phosphorylase b is maximally activated at 1 mM AMP, the low affinity sites are probably not directly involved in the AMP activation of the enzyme. The heat of interaction between phosphorylase b and 0.5 mM AMP has been measured calorimetrically as a function of the enzyme concentration. This heat per mole of the enzyme increases as the enzyme concentration increases at 18° but not at 25°. Sedimentation velocity experiments have shown that phosphorylase b, in the presence of 0.5 mM AMP, undergoes reversible changes between a dimeric and a tetrameric species. These results suggest that the heat of phosphorylase 6–AMP interaction at 18° consists of the heat of AMP binding and that of the tetramer formation. The enthalpies of the AMP binding and the tetramer formation, as well as the equilibrium constant for the enzyme association calculated from the calorimetric data at 18°, are respectively 2.6 kcal/mol, –85 kcal/mol, and 2.1 × 105 M–1. At 25°, the enthalpy of AMP binding to phosphorylase b is found to be –13.2 kcal/mol. In contrast to AMP, IMP does not induce phosphorylase b association either at 25 or at 18°. The enthalpy of IMP binding to phosphorylase b is about -11 kcal/mol, independent of the temperature. Changes in free energy, enthalpy, and entropy accompanying the AMP induced phosphorylase b association at 18° have been calculated from the calorimetric data to be respectively –7.1 kcal/mol, –85 kcal/mol, and 268 eu. These thermodynamic properties indicate that the driving force for the AMP induced enzyme association is enthalpic. Thermodynamic parameters for bindings of AMP and IMP to phosphorylase b, both at 18 and 25° have also been calculated. The results indicate that conformational changes in phosphorylase b induced by AMP at 18° are different from those induced by IMP and those induced by AMP at 25°.