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J. Biol. Chem., Vol. 275, Issue 45, 35408-35412, November 10, 2000

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Steady State Kinetic Model for the Binding of Substrates and Allosteric Effectors to Escherichia coli Phosphoribosyl-diphosphate Synthase^*

Martin Willemoës^§, Bjarne Hove-Jensen^¶, and Sine Larsen

From the  Centre for Crystallographic Studies, Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark and the ^¶ Center for Enzyme Research, Institute of Molecular Biology, University of Copenhagen, Sølvgade 83H, DK-1307 Copenhagen, Denmark

A steady state kinetic investigation of the P_i activation of 5-phospho-D-ribosyl -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.

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