Effects of mutagenesis of aspartic acid residues in the putative phosphoribosyl diphosphate binding site of Escherichia coli phosphoribosyl diphosphate synthetase on metal ion specificity and ribose-5-phosphate binding

The three conserved aspartic acid residues of the 5-phospho-d-ribosyl a-1-diphosphate binding site (213-GRDCVLVDDMIDTGGT-228) of Escherichia coli phosphoribosyl diphosphate synthetase were studied by analysis of the mutant enzymes D220E, D220F, D221A, D224A, and D224S. The mutant enzymes showed an increase in K_M for ribose 5-phosphate in the presence of at least one of the divalent metal ions Mg²⁺, Mn²⁺, Co²⁺, or Cd²⁺, with the most dramatic changes revealed by the D220E and D220F enzymes in the presence of Co²⁺ and the D221A enzyme in the presence of Mn²⁺ or Co²⁺. The D220F and D221A enzymes both showed large decreases in V_app in the presence of the various divalent metal ions, except for the D221A enzyme in the presence of Co²⁺. V_app of the D220E enzyme was similar to that of the wild-type enzyme in the presence of Mg²⁺, Mn²⁺, or Cd²⁺, whereas the V_app was increased in the presence of Co²⁺. V_app values of the D224A and D224S enzymes were lowered to 10-15-fold and 3-4-fold in the presence of Mg²⁺ or Mn²⁺, respectively, whereas V_app was similar to that of the wild-type and K_M for Rib-5-P was increased 4-fold in the presence of Cd²⁺. The changes in K_M for ribose 5-phosphate and V_app of the mutant enzymes were dependent on the metal ion present, suggesting a function of the investigated aspartic acid residues both in the binding of ribose 5-phosphate, possibly via a divalent metal ion, and in the interaction with a divalent metal ion during catalysis.