Chemistry of the binuclear iron/tyrosyl radical cofactor of ribonucleotide reductase from E. coli
Ribonucleoside diphosphate reductase (RDPR) of E. coli contains a unique binuclear iron/tyrosyl radical cofactor which is believed to participate in the catalysis of 2'-deoxyribonucleotide formation. To facilitate these studies, the genes for the subunits were individually cloned into multicopy plasmids. The reaction of RDPR with the mechanisms-based inactivator 2'-azido-2'-deoxyuridine 5'-diphosphate (N/sub 3/UDP) has been investigated in detail with isotopically labeled compounds. Complete inactivation occurred in one turnover with destruction of the tyrosyl radical and stoichiometric production of uracil, pyrophosphate, and dinitrogen. An isotope effect upon inactivation and tyrosyl radical loss demonstrated cleavage of the 3' carbon-hydrogen bond. The specific activity of B1 was reduced by half and stoichiometric covalent modification of this subunit was observed during extended reactions. The enzyme was not modified when denatured after short reaction times although inactivation was nearly complete, indicating that the alkylating agent was formed in a slower second process. The transient new radical special formed during the reaction was studied by EPR and electron spin echo envelope modulation (ESEEM) spectroscopies. Direct evidence for localization of spin density on an azido nitrogen atom of the nucleotide was obtained by labeling N/sub 3/UDP with /sup 15/N.
- Research Organization:
- Wisconsin Univ., Madison (USA)
- OSTI ID:
- 7164291
- Resource Relation:
- Other Information: Thesis (Ph. D.)
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
NUCLEOTIDES
BIOCHEMICAL REACTION KINETICS
OXIDOREDUCTASES
BIOCHEMISTRY
CLONING
ELECTRON SPIN RESONANCE
ESCHERICHIA COLI
GENES
ISOTOPE EFFECTS
NITROGEN 15
PLASMIDS
RADICALS
STOICHIOMETRY
TRACER TECHNIQUES
BACTERIA
CELL CONSTITUENTS
CHEMISTRY
ENZYMES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIGHT NUCLEI
MAGNETIC RESONANCE
MICROORGANISMS
NITROGEN ISOTOPES
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
REACTION KINETICS
RESONANCE
STABLE ISOTOPES
550201* - Biochemistry- Tracer Techniques