Calorimetric titration of phosphorylase b with AMP. Anomalous thermal ligand-binding profiles induced by an enzymic impurity.

Calorimetric titrations of glycogen phosphorylase b (EC 2.4.1.1) from rabbit muscle with its allosteric activator, AMP, have been carried out at 25 “C and pH 6.9 in three different buffer systems (glycylglycine, glycerophosphate, and tris(hydroxymethy1)aminomethane). Calorimetric curves of biphasic nature were originally obtained. This biphasic behavior was always concomitant with: (a) a dependency of the saturation heat on the flow rate of the solutions entering the calorimetric unit; (b) an alteration in the spectrophotometric properties of AMP after being mixed with phosphorylase b. After some time the nucleotide displayed spectral properties resembling those of IMP; (c) an anomalous increase in the pH of the AMP-phosphorylase complex in the outflow from the calorimeter. These observations led to the conclusion that glycogen phosphorylase b seems to be contaminated by traces of AMP amino hydrolase (EC 3.5.4.6) which catalyzes the conversion of AMP into IMP with the liberation of ammonia. Calorimetric titrations of phosphorylase b freed from this impurity yield monophasic titration curves, which are practically identical for the three buffer solutions used, thus indicating that no proton uptake or release seems to take place on the AMP binding to phosphorylase b. The heat of the reaction at saturating concentration of AMP for these monophasic curves was totally independent of the calorimetric flow rates. Likewise, the phosphorylase preparations displaying monophasic titration curves did not show any change in pH or W spectrum of AMP on adding this nucleotide. Hence, we concluded that the monophasic curve is the one precisely corresponding to the calorimetric titration of phosphorylase b with AMP. These results cast doubt on previous experimental evidence for a second AMP site per monomer of phosphorylase b at 25 “C.