Kinetic Mechanism of Rabbit Muscle Glycogen Phosphorylase a

Isotope-exchange rates at chemical equilibrium were determined for the glycogen-α-d-glucopyranose 1-phosphate-Pi system in the presence of phosphorylase a. Exchange of 32P from α-d-glycopyranose 1-phosphate (glucose-1-P) into Pi and exchange of 14C from glucose-1-P into glycogen were followed simultaneously by the use of glucose-1-P containing both isotopes. Concentrations of glucose-1-P and Pi were varied together in their equilibrium ratio at constant glycogen concentration, and the concentration of glycogen was varied at fixed concentrations of phosphates. Exchange rates for the two isotopes were equal under all conditions (with the possible exception of measurements at the highest concentrations of the phosphates) and gave linear reciprocal plots. One exception was noted in which the reciprocal plot was concave upward at low concentrations of substrate, probably because of allosteric effects. The results support a rapid equilibrium mechanism. Initial velocity of the reaction in the absence of product was determined by the use of an isotopic assay. The results were characteristic of a sequential mechanism. Isotope-exchange rates were also determined under nonequilibrium conditions. Exchange of 32P from glucose-1-P into Pi was followed as a function of glycogen concentration at a fixed concentration of glucose-1-P and several fixed concentrations of Pi. The exchange was also followed as a function of glucose-1-P concentration at fixed glycogen concentration and several fixed Pi concentrations. Similar experiments were done while following the exchange from Pi into glucose-1-P. These experiments are equivalent to conventional product inhibition experiments. The results indicate that the two phosphates are noncompetitive inhibitors with respect to glycogen, but are competitive with respect to one another. Our conclusion is that phosphorylase a has a rapid equilibrium Random Bi-Bi mechanism involving binary complexes of enzyme with glycogen, glucose-1-P, and Pi, and ternary complexes of enzyme with glycogen and glucose-1-P and with glycogen and Pi.