Kinetic mechanism of phosphorylase b. Rates of initial velocities and of isotope exchange at equilibrium.

In order to assign a kinetic mechanism to phosphorylase b, initial rate studies were conducted in conjunction with isotope exchange studies at equilibrium. Initial velocity rates were measured with varied concentrations of both substrates in each direction, in the presence of saturating levels of AMP. Data were analyzed with double reciprocal plots and secondary replots of intercepts and slopes. The resulting kinetically derived dissociation constants agreed reasonably well with those determined by independent means. These results indicate that the kinetic mechanism of phosphorylase b is rapid equilibrium random bi bi in nature. The rate equation for this mechanism has been modified to take into account the fact that one of the substrates, glycogen, gives rise to a product which is chemically and kinetically indistinguishable under the conditions used to determine initial rates. This phenomenon, equivalent to having one of the two products present at all times, results in the number two being introduced into the rate equation, so the kinetically derived dissociation constant for glycogen is half the true value while the observed K, values for phosphate and glucose l-phosphate are twice the theoretical values. This rate equation should apply wherever an enzyme synthesizes or degrades a homopolymer by 1 unit at a time. To confirm the mechanism suggested by initial velocity experiments, isotope exchange studies at equilibrium were performed for the phosphorylase b system, again in the presence of saturating levels of AMP. The 14C-glucose-l-P s glycogen equilibrium reaction rate increased as the concentrations of either glucose-l-P and orthophosphate or glycogen were increased, and reached a plateau as the concentration of varied substrates became saturating. The same results were obtained for 32Pi = glucose-l-P exchange. Thus, there was no evidence of an inhibition of the exchange of one pair of substrates when the concentration of the other substrate pair was raised. Similar exchange rates were observed in either direction, indicating that rapid equilibrium conditions apply. A reasonable agreement existed between the maximal velocities calculated from the initial rate data