PMID:10954724
| Citation |
Willemoës, M, Hove-Jensen, B and Larsen, S (2000) Steady state kinetic model for the binding of substrates and allosteric effectors to Escherichia coli phosphoribosyl-diphosphate synthase. J. Biol. Chem. 275:35408-12 |
|---|---|
| Abstract |
A steady state kinetic investigation of the P(i) activation of 5-phospho-d-ribosyl alpha-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 Mg(2+) 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 Mg(2+) and substrates via a fast pathway, whereas saturation with ribose 5-phosphate leads to the binding of Mg(2+) 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 Mg(2+), 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. |
| Links |
PubMed Online version:10.1074/jbc.M006346200 |
| Keywords |
Adenosine Diphosphate/pharmacology; Allosteric Site; Dose-Response Relationship, Drug; Escherichia coli/enzymology; Kinetics; Ligands; Magnesium/pharmacology; Models, Biological; Protein Binding; Ribose-Phosphate Pyrophosphokinase/chemistry; Ribose-Phosphate Pyrophosphokinase/metabolism; Ribosemonophosphates/pharmacology |
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