Direct photoaffinity labeling of an allosteric site on subunit protein M1 of mouse ribonucleotide reductase by dTTP.
The protein M1 subunit of ribonucleotide reductase contains at least two allosteric nucleotide binding sites that control the capacity of the enzyme to reduce ribonucleotides to the deoxyribonucleotides required for DNA synthesis. Direct photoaffinity labeling of partially purified protein M1 from mouse T-lymphoma (S49) cells was observed after UV irradiation in the presence of dTTP at 0 degrees C. The relative molar incorporation of nucleotide per subunit was 4-8%. Competition experiments showed that the dTTP was bound to an allosteric domain genetically and kinetically defined as the substrate specificity site of the enzyme. An altered protein M1 isolated from a thymidine-resistant mutant cell line showed significantly decreased photoincorporation of dTTP, consistent with the fact that its CDP reductase activity is resistant to feedback inhibition by dTTP. Specific photolabeling of several other proteins with pyrimidine and purine nucleotides was also found, indicating the general usefulness of direct photoaffinity labeling in the study of enzymes involved in nucleotide and nucleic acid metabolism.
D000345:Affinity Labels / Q000494:pharmacology*; D000494:Allosteric Regulation; D000495:Allosteric Site; D000818:Animals; D002460:Cell Line; D007700:Kinetics; D008223:Lymphoma / Q000201:enzymology; D051379:Mice; D009154:Mutation; D009374:Neoplasms, Experimental / Q000201:enzymology; D009705:Nucleosides / Q000494:pharmacology; D009711:Nucleotides / Q000494:pharmacology; D012264:Ribonucleotide Reductases / Q000378:metabolism*; D013942:Thymine Nucleotides / Q000494:pharmacology*
D000345:Affinity Labels; D009705:Nucleosides; D009711:Nucleotides; D013942:Thymine Nucleotides; D012264:Ribonucleotide Reductases; C024157:thymidine 5'-triphosphate