Direct photoaffinity labeling of an allosteric site of subunit protein M1 of mouse ribonucleotide reductase by dATP. Evidence for two independent binding interactions within the allosteric specificity site.
The M1 subunit of ribonucleotide reductase contains two kinds of allosteric sites, the activity site and the specificity site, which regulate the overall catalytic activity and the substrate specificity of the enzyme, respectively. The effector nucleotides, dGTP and dTTP, bind only to the specificity site; dATP and ATP bind to both sites. Partially purified protein M1 was photolabeled specifically after UV irradiation in the presence of [32P]dATP. The labeling occurred exclusively at the allosteric specificity site as evidenced by 1) total inhibition of the labeling by dGTP and dTTP, 2) normal photoincorporation of [32P]dATP by mutant protein M1 molecules that lack a functional activity site, and 3) coidentity of one-dimensional peptide maps of protein M1 labeled with either [32P]dATP or [32P]dTTP. A mutant protein M1 that is resistant to normal regulation by dGTP and dTTP (indicating an alteration in the allosteric specificity site) showed normal photoincorporation of [32P]dATP (but not [32P]dTTP). This labeling was not inhibited by dGTP or dTTP. Our data suggest that this mutation has altered the binding of dGTP and dTTP but not dATP (or ATP) at the specificity site. Thus, by the combination of genetic and photolabeling techniques, two independent nucleotide binding interactions occurring within this one complex regulatory domain can be distinguished.
D000345:Affinity Labels; D000495:Allosteric Site / Q000187:drug effects; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D001667:Binding, Competitive; D002460:Cell Line; D003838:Deoxyadenine Nucleotides / Q000378:metabolism / Q000528:radiation effects; D003848:Deoxyguanine Nucleotides / Q000494:pharmacology; D008228:Lymphoma, Non-Hodgkin; D051379:Mice; D009154:Mutation; D009374:Neoplasms, Experimental; D012264:Ribonucleotide Reductases / Q000378:metabolism*; D013942:Thymine Nucleotides / Q000494:pharmacology; D014466:Ultraviolet Rays
D000345:Affinity Labels; D003838:Deoxyadenine Nucleotides; D003848:Deoxyguanine Nucleotides; D013942:Thymine Nucleotides; C029603:deoxyguanosine triphosphate; D012264:Ribonucleotide Reductases; C026600:2'-deoxyadenosine triphosphate; C024157:thymidine 5'-triphosphate