A photoaffinity-labeled allosteric site in Escherichia coli ribonucleotide reductase.
The B1 subunit of Escherichia coli ribonucleotide reductase is coded for by the nrdA gene, of determined structure. Protein B1 contains two types of allosteric binding sites. One type (h-sites) determines the substrate specificity while the other type (l sites) governs the overall activity. The effectors dGTP and dTTP bind only to the h-sites while dATP and ATP bind to both the h- and the l-sites. Protein B1 has been photoaffinity-labeled with radioactive dTTP and dATP using direct UV irradiation. Following tryptic digestion of labeled protein B1 only one peptide labeled with dTTP was found, while several peptides were labeled with dATP. One of the dATP-labeled peptides had chromatographic properties very similar to that labeled with dTTP and this peptide most likely forms part of the h-site of protein B1. Labeling of the l-site could not be conclusively shown since substantial non-specific labeling occurred with dATP. CNBr fragments of dTTP-labeled protein B1 were used to localize the region of nucleotide binding in the deduced primary structure of the nrdA gene. The dTTP label was further localized to a tryptic octapeptide with the sequence Ser-X-Ser-Gln-Gly-Gly-Val-Arg. The labeled amino acid was found at position 2, but the residue itself could not be directly identified. Unexpectedly, this sequence was not found in the earlier reported primary structure of the nrdA gene. However, a recent revised structure of the gene identifies the labeled residue as Cys-289 and fully confirms the rest of the peptide sequence. Thus the present result clearly defines one of the allosteric binding sites in ribonucleotide reductase.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000345:Affinity Labels; D000494:Allosteric Regulation; D000495:Allosteric Site; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D002855:Chromatography, Thin Layer; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D010455:Peptides / Q000032:analysis; D012264:Ribonucleotide Reductases / Q000032:analysis*; D013942:Thymine Nucleotides / Q000378:metabolism
D000345:Affinity Labels; D001426:Bacterial Proteins; D003838:Deoxyadenine Nucleotides; D003848:Deoxyguanine Nucleotides; D010455:Peptides; D013942:Thymine Nucleotides; C029603:deoxyguanosine triphosphate; D000255:Adenosine Triphosphate; D012264:Ribonucleotide Reductases; C026600:2'-deoxyadenosine triphosphate; C024157:thymidine 5'-triphosphate