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 alpha-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 KM 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 Vapp in the presence of the various divalent metal ions, except for the D221A enzyme in the presence of Co2+. Vapp of the D220E enzyme was similar to that of the wild-type enzyme in the presence of Mg2+, Mn2+, or Cd2+, whereas the Vapp was increased in the presence of Co2+. Vapp 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 Vapp was similar to that of the wild-type and KM for Rib-5-P was increased 4-fold in the presence of Cd2+. The changes in KM for ribose 5-phosphate and Vapp 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.
Willemoës
M
M
Center for Enzyme Research, University of Copenhagen, Denmark.
Nilsson
D
D
Hove-Jensen
B
B
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002413:Cations, Divalent / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010754:Phosphoribosyl Pyrophosphate / Q000378:metabolism; D012268:Ribose-Phosphate Pyrophosphokinase / Q000235:genetics* / Q000378:metabolism*; D012267:Ribosemonophosphates / Q000378:metabolism
D002413:Cations, Divalent; D012267:Ribosemonophosphates; D001224:Aspartic Acid; C031626:ribose-5-phosphate; D010754:Phosphoribosyl Pyrophosphate; D012268:Ribose-Phosphate Pyrophosphokinase