Direct photoaffinity labeling of the catalytic site of mouse ribonucleotide reductase by CDP.
Ribonucleotide reductase reduces all four ribonucleoside diphosphates to the deoxyribonucleotides required for DNA synthesis. The enzyme is composed of two nonidentical subunits, M1 and M2. The 89-kilodalton M1 subunit contains at least two allosteric sites which, by binding nucleotide effectors, regulate the catalytic activity and substrate specificity of the enzyme. We now show that in addition, protein M1 contains a substrate-binding (catalytic) site which is specifically photolabeled after UV irradiation in the presence of the natural substrate, [32P]CDP. The photolabeling of protein M1 by [32P]CDP required the presence of the second subunit, protein M2, and ATP, the positive allosteric effector for CDP reduction. The negative effectors, dATP, dGTP, and dTTP, inhibited the photolabeling of wild type protein M1. Deoxy-ATP did not inhibit the labeling of a mutant protein M1 that is resistant to feedback inhibition by dATP. In addition, hydroxyurea and 4-methyl-5-aminoisoquinoline thiosemicarbazone, two inhibitors of ribonucleotide reductase which affect protein M2, also inhibited the [32P]CDP labeling of protein M1. These data provide new insights into the role and interaction of the two ribonucleotide reductase subunits, proteins M1 and M2, and the mechanism of action of the allosteric effectors.
D000345:Affinity Labels; D000818:Animals; D003565:Cytidine Diphosphate / Q000494:pharmacology*; D003597:Cytosine Nucleotides / Q000494:pharmacology*; D003854:Deoxyribonucleotides / Q000494:pharmacology; D006918:Hydroxyurea / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D008223:Lymphoma / Q000201:enzymology; D046911:Macromolecular Substances; D051379:Mice; D008970:Molecular Weight; D009374:Neoplasms, Experimental / Q000201:enzymology; D012264:Ribonucleotide Reductases / Q000378:metabolism*; D012265:Ribonucleotides / Q000494:pharmacology
D000345:Affinity Labels; D003597:Cytosine Nucleotides; D003854:Deoxyribonucleotides; D007546:Isoquinolines; D046911:Macromolecular Substances; D012265:Ribonucleotides; C013118:4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone; D003565:Cytidine Diphosphate; D012264:Ribonucleotide Reductases; D006918:Hydroxyurea