EFFECTS OF MUTAGENESIS OF ASPARTIC-ACID RESIDUES IN THE PUTATIVE PHOSPHORIBOSYL DIPHOSPHATE BINDING-SITE OF ESCHERICHIA-COLI PHOSPHORIBOSYLDIPHOSPHATE SYNTHETASE ON METAL-ION SPECIFICITY AND RIBOSE 5-PHOSPHATE BINDING

The three conserved aspartic acid residues of the 5-phospho-D-ribosyl alpha-1-diphosphate binding site (213-GRDCVLVDDMIDTGGT-228) of Escheri chia coli phosphoribosyl diphosphate synthetase were studied by analys is of the mutant enzymes D220E, D220F, D221A, D224A, and D224S. The mu tant enzymes showed an increase in K-M for ribose 5-phosphate in the p resence 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 o f Mn2+ or Co2+. The D220F and D221A enzymes both showed large decrease s in V-app in the presence of the various divalent metal ions, except for the D221A enzyme in the presence of Mg2+. V-app of the D220E enzym e 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-f old and 3-4-fold in the presence of Mg2+ or Mn2+, respectively, wherea s V-app was similar to that of the wild-type and K-M for Rib-5-P was i ncreased 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 me tal ion present, suggesting a function of the investigated aspartic ac id residues both in the binding of ribose 5-phosphate, possibly via a divalent metal ion, and in the interaction with a divalent metal ion d uring catalysis.