Binding of Divalent Magnesium by Escherichia coli Phosphoribosyl Diphosphate Synthetase

Abstract

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.

Class II Recombinant Phosphoribosyl Diphosphate Synthase from Spinach

“…The kinetic mechanism of spinach PRPP synthase isozyme 3 was steady-state ordered Bi Bi with MgATP binding first and PRPP leaving last similar to that shown previously for S. typhimurium and E. coli PRPP synthases (21,28). In addition, spinach PRPP synthase isozyme 3 required free Mg 2ϩ as an activator, similar to what has been shown for the bacterial and human PRPP synthases (8,21,26,29).…”

Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance

“…Steady-state kinetic analysis of the inhibition of S. enterica or E. coli PRPP synthase by substrate analogs revealed an ordered Bi-Bi mechanism with binding of Mg 2ϩ first, followed by MgATP and then ribose 5-phosphate (41,56,57). The ordered kinetic mechanism was further confirmed by equilibrium dialysis.…”

“…The active site must accommodate the substrates ribose 5-phosphate and MgATP. In addition, an overwhelming volume of research data has shown that an additional so-called free Mg 2ϩ is required for activity, because maximal activity is only obtained when Mg 2ϩ is added to the reaction mixture in excess of the MgATP concentration for both bacterial (35,45,(55)(56)(57)(58) and mammalian (59,60) PRPP synthases. Furthermore, most PRPP synthases may accept other divalent metal ions in place of Mg 2ϩ , and thus Mg 2ϩ may be regarded as a pseudosubstrate (45,56,58,61).…”

“…In particular, the latter coordination of Ca 2ϩ causes perturbation of the inline arrangement of the C-1 oxygen of ribose 5-phosphate and the ␤-and ␣-phosphates of ATP described above (54). Previous kinetic analysis revealed that Ca 2ϩ , which does not support PRPP synthase activity alone, is an inhibitor of PRPP synthase activity even in the presence of a very large excess of Mg 2ϩ (56,57).…”

Steady State Kinetic Model for the Binding of Substrates and Allosteric Effectors to Escherichia coliPhosphoribosyl-diphosphate Synthase

“…Mg 2ϩ ions are required to form the actual substrate MgATP and as an activator of the enzyme (8 -13). PRPP synthases from Salmonella typhimurium (8,14,15), Escherichia coli (11,16), Bacillus subtilis (10), human (17), and rat (18) possess an absolute requirement for P i as an activator and are subject to inhibition by ADP and for B. subtilis and mammalian enzymes also by GDP, which binds to a specific allosteric site. In addition, ADP competes with MgATP for binding to the active site.…”