Regulation of Phosphorylase b by AMP

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Abstract
The enthalpies of binding of AMP to phosphorylase b have been measured as a function of enzyme concentration in glycylglycine buffer, pH 6.9. The results show how a conformational transition, which takes place in the concentration range of 1.7 to 2.5 mg/ml of phosphorylase b, affects the enthalpies of the two binding sites per monomer for the allosteric activator AMP. The enthalpies of the AMP interaction with its higher and lower affinity binding sites are −220 and −640 kJ (mol monomer)−1 at an enzyme concentration of 1 mg/ml, and −120 and −360 kJ (mol monomer)−1 at 2.7 mg/ml. The conformational transition of phosphorylase b alters the reactivity of the slow —SH groups of the enzyme with 5, 5'-dithiobis(2-nitrobenzoic acid), suggesting that the environments of these groups are affected by the process. On the other hand, kinetic data show that the saturation of both classes of AMP binding sites affects the catalytic behavior and that their specific effects on the catalytic process are altered by the enzymatic transition dependent on the enzyme concentration. Thus, a strong inhibition is associated with the saturation of the weaker affinity AMP binding sites at 3 mg/ml while the saturation of these weaker affinity binding sites at 1 mg/ml produces an important activation of the enzyme.