title: Direct photoaffinity labeling of ribonucleotide reductase from Escherichia coli using dTTP: characterization of the photoproducts.
abstract: Subunit B1 of Escherichia coli ribonucleotide reductase contains one type of allosteric binding site that controls the substrate specificity of the enzyme. This site binds the allosteric effector dTTP as well as other nucleoside triphosphates. Cross-linking of dTTP to protein B1 by direct photoaffinity labeling, as well as the isolation and sequence determination of the labeled tryptic peptide, has recently been reported [Eriksson, S., Sjöberg, B.-M., Jörnwall, H., & Carlquist, M. (1986) J. Biol. Chem. 261, 1878-1882]. In this study, we have further purified the dTTP-labeled peptide and characterized it using UV spectroscopy. Two types of dTTP-cross-linked peptide were found: one having an absorbance maximum at 261 nm typical for a dTTP spectrum, i.e., containing an intact 5,6 double bond, and one minor form with low absorbance at 261 nm. In both cases, the same amino acid composition was found, corresponding to the peptide Ser291-X-Ser-Gln-Gly-Gly-Val-Arg299 in the B1 sequence with X being Cys-292 cross-linked to dTTP. Isotope labeling experiments revealed that one proton in the 5-methyl group of thymine was lost during photoincorporation. Therefore, the cross-linking occurs via the 5-methyl group to Cys-292 in a majority of incorporated dTTPs, but a second, possibly 5,6-saturated form of incorporated nucleotide was also detected. The reasons for the high stereospecificity of the reaction and the possible structure of the allosteric site of protein B1 are discussed.
mesh_terms: D000345:Affinity Labels / Q000378:metabolism*; D000495:Allosteric Site; D004926:Escherichia coli / Q000201:enzymology*; D006153:Guanosine Diphosphate / Q000378:metabolism; D046911:Macromolecular Substances; D010761:Phosphorus Radioisotopes; D012264:Ribonucleotide Reductases / Q000378:metabolism* / Q000528:radiation effects; D013942:Thymine Nucleotides / Q000378:metabolism*; D014316:Tritium; D014466:Ultraviolet Rays
chemical_list: D000345:Affinity Labels; D046911:Macromolecular Substances; D010761:Phosphorus Radioisotopes; D013942:Thymine Nucleotides; D014316:Tritium; D006153:Guanosine Diphosphate; D012264:Ribonucleotide Reductases; C024157:thymidine 5'-triphosphate