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 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.
Willemoës
M
M
Centre for Crystallographic Studies, Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark. martin@xray.ki.ku.dk
Hove-Jensen
B
B
Larsen
S
S
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000495:Allosteric Site; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008024:Ligands; D008274:Magnesium / Q000494:pharmacology; D008954:Models, Biological; D011485:Protein Binding; D012268:Ribose-Phosphate Pyrophosphokinase / Q000737:chemistry / Q000378:metabolism*; D012267:Ribosemonophosphates / Q000494:pharmacology
D008024:Ligands; D012267:Ribosemonophosphates; C031626:ribose-5-phosphate; D000244:Adenosine Diphosphate; D012268:Ribose-Phosphate Pyrophosphokinase; D008274:Magnesium