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'-azido-2'-deoxyuridine 5'-diphosphate (N/sub 3/UDP). Incubation of RDPR with (3'-/sup 3/H)N/sub 3/UDP resulted in 0.2 mol of /sup 3/H 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..-/sup 32/P)N/sub 3/UDP resulted in stoichiometric production of inorganic pyrophosphate. One equivalent of uracil was eliminated from N/sub 3/UDP, but no azide release was detected. Analysis of the reaction of RDPR with (/sup 15/N/sub 3/)N/sub 3/UDP 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 N/sub 3/UDP as reported previously, while the specific activity of the B1 subunit was reduced by half. Incubation of (5'-/sup 3/H)N/sub 3/UDP 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.
- Research Organization:
- Univ. of Wisconsin, Madison
- OSTI ID:
- 6375225
- Journal Information:
- Biochemistry; (United States), Vol. 26:12
- Country of Publication:
- United States
- Language:
- English
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AZIDES
BIOCHEMISTRY
LABELLED COMPOUNDS
TRACER TECHNIQUES
OXIDOREDUCTASES
INACTIVATION
ESCHERICHIA COLI
NITROGEN 15
PHOSPHORUS 32
STOICHIOMETRY
TRITIUM COMPOUNDS
URIDINE
AZINES
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CHEMISTRY
DAYS LIVING RADIOISOTOPES
ENZYMES
HETEROCYCLIC COMPOUNDS
HYDROXY COMPOUNDS
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
MICROORGANISMS
NITROGEN COMPOUNDS
NITROGEN ISOTOPES
NUCLEI
NUCLEOSIDES
NUCLEOTIDES
ODD-EVEN NUCLEI
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PHOSPHORUS ISOTOPES
PYRIMIDINES
RADIOISOTOPES
RIBOSIDES
STABLE ISOTOPES
URACILS
550201* - Biochemistry- Tracer Techniques