Products of the inactivation of ribonucleoside diphosphate reductase from Escherichia coli with 2'-azido-2'-deoxyuridine 5'-diphosphate.
Ribonucleoside diphosphate reductase (RDPR) from Escherichia coli was completely inactivated by 1 equiv of the mechanism-based inhibitor 2'-azido-2'-deoxyuridine 5'-diphosphate (N3UDP). Incubation of RDPR with [3'-3H]N3UDP resulted in 0.2 mol of 3H released to solvent per mole of enzyme inactivated, indicating that cleavage of the 3' carbon-hydrogen bond occurred in the reaction. Incubation of RDPR with [beta-32P]N3UDP resulted in stoichiometric production of inorganic pyrophosphate. One equivalent of uracil was eliminated from N3UDP, but no azide release was detected. Analysis of the reaction of RDPR with [15N3]N3UDP by mass spectrometry revealed that the azide moiety was converted to 0.9 mol of nitrogen gas per mole of enzyme inactivated. The tyrosyl radical of the B2 subunit was destroyed during the inactivation by N3UDP as reported previously [Sjöberg, B.-M., Gräslund, A., & Eckstein, F. (1983) J. Biol. Chem. 258, 8060-8067], while the specific activity of the B1 subunit was reduced by half. Incubation of [5'-3H]N3UDP with RDPR resulted in stoichiometric covalent radiolabeling of the enzyme. Separation of the enzyme's subunits by chromatofocusing revealed that the modification was specific for the B1 subunit.
Salowe
S P
SP
Department of Biochemistry, College of Agriculture and Life Sciences, University of Wisconsin-Madison 53706.
Ator
M A
MA
Stubbe
J
J
D000345:Affinity Labels / Q000494:pharmacology*; D001386:Azides / Q000494:pharmacology*; D003856:Deoxyuracil Nucleotides / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology*; D006918:Hydroxyurea / Q000494:pharmacology; D007700:Kinetics; D011485:Protein Binding; D012262:Ribonucleoside Diphosphate Reductase / Q000037:antagonists & inhibitors*; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors*; D014316:Tritium
D000345:Affinity Labels; D001386:Azides; D003856:Deoxyuracil Nucleotides; D014316:Tritium; C053847:2'-azido-2'-deoxyuridine 5'-diphosphate; D012264:Ribonucleotide Reductases; D012262:Ribonucleoside Diphosphate Reductase; D006918:Hydroxyurea