Binding of divalent magnesium by Escherichia coli phosphoribosyl diphosphate synthetase.
The mechanism of binding of the substrates Mg x ATP and ribose 5-phosphate as well as Mg2+ to the enzyme 5-phospho-D-ribosyl (alpha-1-diphosphate synthetase from Escherichia coli has been analyzed. By use of the competive inhibitors of ATP and ribose 5-phosphate binding, alpha,beta-methylene ATP and (+)-1-alpha,2-alpha,3-alpha-trihydroxy-4-beta-cyclopentanemethanol 5-phosphate, respectively, the binding of Mg2+ and the substrates were determined to occur via a steady state ordered mechanism in which Mg2+ binds to the enzyme first and ribose 5-phosphate binds last. Mg2+ binding to the enzyme prior to the binding of substrates and products indicated a role of Mg2+ in preparing the active site of phosphoribosyl diphosphate synthetase for binding of the highly phosphorylated ligands Mg x ATP and phosphoribosyl diphosphate, as evaluated by analysis of the effects of the inhibitors adenosine and ribose 1,5-bisphosphate. Calcium ions, which inhibit the enzyme even in the presence of high concentrations of Mg2+, appeared to compete with free Mg2+ for binding to its activator site on the enzyme. Analysis of the inhibition of Mg2+ binding by Mg x ADP indicated that Mg x ADP binding to the allosteric site may occur in competition with enzyme bound Mg2+. Ligand binding studies showed that 1 mol of Mg x ATP was bound per mol of phosphoribosyl diphosphate synthetase subunit, which indicated that the allosteric sites of the multimeric enzyme were not made up by inactive catalytic sites.
Willemoës
M
M
Center for Enzyme Research, Institute of Molecular Biology, University of Copenhagen, Denmark.
Hove-Jensen
B
B
D000241:Adenosine / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D010428:Pentosephosphates / Q000378:metabolism; D011485:Protein Binding; D012268:Ribose-Phosphate Pyrophosphokinase / Q000378:metabolism*; D012267:Ribosemonophosphates / Q000378:metabolism
D010428:Pentosephosphates; D012267:Ribosemonophosphates; C031154:ribose 1-phosphate; C050562:ribose-1,5-bisphosphate; D000249:Adenosine Monophosphate; C031626:ribose-5-phosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D012268:Ribose-Phosphate Pyrophosphokinase; D008274:Magnesium; D000241:Adenosine; D002118:Calcium