Characterization of the Escherichia coli prsA1-encoded mutant phosphoribosylpyrophosphate synthetase identifies a divalent cation-nucleotide binding site

• Published in: The Journal of biological chemistry 1989-06, Vol.264 (17), p.10287-10291
• Main Authors: Bower, S G, Harlow, K W, Switzer, R L, Hove-Jensen, B
• Format: Article
• Language: English
• Subjects: Alleles; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Binding Sites; Biological and medical sciences; Cations, Divalent; Cloning, Molecular; Enzyme Activation; Enzymes and enzyme inhibitors; Escherichia coli - enzymology; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Genes; Genes, Bacterial; Kinetics; Miscellaneous; Molecular Sequence Data; Mutation; Nucleotides - metabolism; Phosphotransferases - metabolism; Plasmids; Protein Binding; Restriction Mapping; Ribose-Phosphate Pyrophosphokinase - genetics; Ribose-Phosphate Pyrophosphokinase - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)81798-7

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 α-phosphate of ATP and AMP at the active site. The prsA1 mutation appears to alter this divalent cation site.