Characterization of the Escherichia coli prsA1-encoded mutant phosphoribosylpyrophosphate synthetase identifies a divalent cation-nucleotide binding site.
The prsA1 allele, specifying a mutant Escherichia coli phosphoribosylpyrophosphate (PRPP) synthetase, has been cloned. The mutation was shown by nucleotide sequence analysis to result from substitution of Asp-128 (GAT) in the wild type by Ala (GCT) in prsA1. This alteration was confirmed by chemical determination of the amino acid sequence of a tryptic peptide derived from the purified mutant enzyme. The mutation lies at the N-terminal end of a 16 residue sequence that is highly conserved in E. coli, Bacillus subtilis, and rat PRPP synthetases and has the following consensus sequence: DLHAXQIQGFFDI/VPI/VD. There was little alteration in the Km for ribose 5-phosphate. The Km for ATP of the mutant enzyme was increased 27-fold when Mg2+ was the activating cation but only 5-fold when Mn2+ was used. Maximal velocities of the wild type and mutant enzymes were the same. The mutant enzyme has a 6-fold lower affinity for Ca2+, as judged by the ability of Ca2+ to inhibit the reaction in the presence of 10 mM Mg2+. Wild type PRPP synthetase is subject to product inhibition by AMP, but AMP inhibition of the prsA1 mutant enzyme could not be detected. It has been previously proposed that a divalent cation binds to PRPP synthetase and serves as a bridge to the alpha-phosphate of ATP and AMP at the active site. The prsA1 mutation appears to alter this divalent cation site.
Bower
S G
SG
Department of Biochemistry, University of Illinois, Urbana 61801.
Harlow
K W
KW
Switzer
R L
RL
Hove-Jensen
B
B
D000483:Alleles; D000595:Amino Acid Sequence; D001665:Binding Sites; D002413:Cations, Divalent; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism; D010770:Phosphotransferases / Q000378:metabolism*; D010957:Plasmids; D011485:Protein Binding; D015183:Restriction Mapping; D012268:Ribose-Phosphate Pyrophosphokinase / Q000235:genetics / Q000378:metabolism*
D002413:Cations, Divalent; D009711:Nucleotides; D010770:Phosphotransferases; D012268:Ribose-Phosphate Pyrophosphokinase