A CALORIMETRIC STUDY OF THE BINDING OF AMP TO LIVER-GLYCOGEN PHOSPHORYLASE-B

The energetics of the interaction between liver glycogen phosphorylase b and the adenosine 5'-monophosphate (AMP) have been studied by equil ibrium dialysis and isothermal titration calorimetry (ITC) at 25 degre es C. A concomitant net release of protons with AMP to phosphorylase b inding was detected carrying out calorimetric experiments in three buf fers having different heats of ionization at 25 degrees C. Four bindin g sites were found for AMP in the dimeric enzyme, which would correspo nd to the activator and the inhibitor sites identified in the muscle i sozyme. The affinity of AMP for these four sites is similar. Thus, the binding of AMP to the activator sites seems to be non-cooperative and it does not perform the conformational change necessary to activate t he enzyme. Moreover, the inhibitor sites are occupied almost in the sa me extension that the activator sites, which would impair any activati on of the enzyme.