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 α-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 Mg ²⁺, Mn ²⁺, Co ²⁺, or Cd ²⁺, with the most dramatic changes revealed by the D220E and D220F enzymes in the presence of Co ²⁺ and the D221A enzyme in the presence of Mn ²⁺ or Co ²⁺. 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 Co ²⁺. V(app) of the D220E enzyme was similar to that of the wild-type enzyme in the presence of Mg ²⁺, Mn ²⁺, or Cd ²⁺, whereas the V(app) was increased in the presence of Co ²⁺. V(app) values of the D224A and D224S enzymes were lowered to 10-15-fold and 3-4-fold in the presence of Mg ²⁺ or Mn ²⁺, 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 Cd ²⁺. 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.