Binding of divalent magnesium by Escherichia coli phosphoribosyl diphosphate synthetase

The mechanism of binding of the substrates Mg·ATP and ribose 5- phosphate as well as Mg ²⁺ to the enzyme 5-phospho-D-ribosyl α-1- diphosphate synthetase from Escherichia coli has been analyzed. By use of the competive inhibitors of ATP and ribose 5-phosphate binding, α,β-methylene ATP and (+)-1-α,2-α,3-α-trihydroxy-4-β-cyclopentanemethanol 5-phosphate, respectively, the binding of Mg ²⁺ and the substrates were determined to occur via a steady state ordered mechanism in which Mg ²⁺ binds to the enzyme first and ribose 5-phosphate binds last. Mg ²⁺ binding to the enzyme prior to the binding of substrates and products indicated a role of Mg ²⁺ in preparing the active site of phosphoribosyl diphosphate synthetase for binding of the highly phosphorylated ligands Mg·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 Mg ²⁺, appeared to compete with free Mg ²⁺ for binding to its activator site on the enzyme. Analysis of the inhibition of Mg ²⁺ binding by Mg·ADP indicated that Mg·ADP binding to the allosteric site may occur in competition with enzyme bound Mg ²⁺. Ligand binding studies showed that 1 mol of Mg·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.