Steady state kinetic model for the binding of substrates and allosteric effectors to Escherichia coli phosphoribosyl-diphosphate synthase

A steady state kinetic investigation of the P_i activation of 5-phospho-D-ribosyl α-1-diphosphate synthase from Escherichia coli suggests that P_i can bind randomly to the enzyme either before or after an ordered addition of free Mg²⁺ and substrates. Unsaturation with ribose 5-phosphate increased the apparent cooperativity of P_i activation. At unsaturating P_i concentrations partial substrate inhibition by ribose 5-phosphate was observed. Together these results suggest that saturation of the enzyme with P_i directs the subsequent ordered binding of Mg²⁺ and substrates via a fast pathway, whereas saturation with ribose 5-phosphate leads to the binding of Mg²⁺ and substrates via a slow pathway where P_i binds to the enzyme last. The random mechanism for P_i binding was further supported by studies with competitive inhibitors of Mg²⁺, MgATP, and ribose 5-phosphate that all appeared noncompetitive when varying P_i at either saturating or unsaturating ribose 5-phosphate concentrations. Furthermore, none of the inhibitors induced inhibition at increasing P_i concentrations. Results from ADP inhibition of P_i activation suggest that these effectors compete for binding to a common regulatory site.