Characterization of a substrate-derived radical detected during the inactivation of ribonucleotide reductase from Escherichia coli by 2'- fluoromethylene-2'-deoxycytidine 5'-diphosphate

Gary J. Gerfen, Wilfred A. Van Der Donk, Guixue Yu, James R. McCarthy, Esa T. Jarvi, Donald P. Matthews, Christian Farrar, Robert G. Griffin, JoAnne Stubbe

Research output: Contribution to journalArticle

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Abstract

Ribonucleotide reductase (RDPR) from E. coli catalyzes the conversion of nucleotides to deoxynucleotides and contains an unusual tyrosyl radical- diferric cluster cofactor (E)-2'-Fluoromethylene-2'-deoxycytidine 5'- diphosphate [(E)-1] obtained by phosphorylation of the clinically promising antitumor agent MDL 101,731, is a potent time-dependent inactivator of this protein. Electron paramagnetic resonance (EPR) spectroscopy reveals that inactivation is accompanied by loss of the essential tyrosyl radical cofactor and formation of a new radical species. The 9.4-GHz EPR spectrum of this new radical reveals two hyperfine splittings of approximately 1.5 mT producing a triplet-like signal. Incubation of the enzyme with [6'-2H]-(E)-1 alters this EPR spectrum, providing the first evidence for a nucleotide-based radical species generated by RDPR. The observed spectrum is a 1:1 composite of a doublet and a triple signal, the latter being identical to that obtained with unlabeled (E)-1. Studies with (E)-1 in 2H2O also produce a 1:1 mixture of these two radical signals. The results of these isotope labeling experiments suggest wash-out or wash-in of ~0.5 equiv of deuterium at the 6' position, respectively. Incubation of the enzyme with [6'-2H]-(E)-1 in 2H2O produced only the doublet as would be expected on the basis of this hypothesis. EPR (139.5 GHz) spectra established the principal g values of the new radical species. Simulation of the 9.4- and 139.5-GHz EPR spectra yield a self- consistent set of principal hyperfine values. A structure is proposed for the radical intermediate that is consistent with the EPR data and kinetic data on release of the fluoride ion that accompanies inactivation. The proposed structure and a postulated mechanism for its formation provide further support for the hypothesis that catalysis is initiated by 3'-hydrogen atom abstraction from the nucleotide substrate.

Original languageEnglish (US)
Pages (from-to)3823-3835
Number of pages13
JournalJournal of the American Chemical Society
Volume120
Issue number16
DOIs
StatePublished - Apr 29 1998

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Ribonucleotide Reductases
Electron Spin Resonance Spectroscopy
Escherichia coli
Paramagnetic resonance
Substrates
Nucleotides
Enzymes
Isotope Labeling
Phosphorylation
Deuterium
Catalysis
Fluorides
Catalyst supports
Isotopes
Antineoplastic Agents
Labeling
2'-fluoromethylene-2'-deoxycytidine 5'-diphosphate
Oxidoreductases
Hydrogen
Spectrum Analysis

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

Characterization of a substrate-derived radical detected during the inactivation of ribonucleotide reductase from Escherichia coli by 2'- fluoromethylene-2'-deoxycytidine 5'-diphosphate. / Gerfen, Gary J.; Van Der Donk, Wilfred A.; Yu, Guixue; McCarthy, James R.; Jarvi, Esa T.; Matthews, Donald P.; Farrar, Christian; Griffin, Robert G.; Stubbe, JoAnne.

In: Journal of the American Chemical Society, Vol. 120, No. 16, 29.04.1998, p. 3823-3835.

Research output: Contribution to journalArticle

Gerfen, Gary J. ; Van Der Donk, Wilfred A. ; Yu, Guixue ; McCarthy, James R. ; Jarvi, Esa T. ; Matthews, Donald P. ; Farrar, Christian ; Griffin, Robert G. ; Stubbe, JoAnne. / Characterization of a substrate-derived radical detected during the inactivation of ribonucleotide reductase from Escherichia coli by 2'- fluoromethylene-2'-deoxycytidine 5'-diphosphate. In: Journal of the American Chemical Society. 1998 ; Vol. 120, No. 16. pp. 3823-3835.
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abstract = "Ribonucleotide reductase (RDPR) from E. coli catalyzes the conversion of nucleotides to deoxynucleotides and contains an unusual tyrosyl radical- diferric cluster cofactor (E)-2'-Fluoromethylene-2'-deoxycytidine 5'- diphosphate [(E)-1] obtained by phosphorylation of the clinically promising antitumor agent MDL 101,731, is a potent time-dependent inactivator of this protein. Electron paramagnetic resonance (EPR) spectroscopy reveals that inactivation is accompanied by loss of the essential tyrosyl radical cofactor and formation of a new radical species. The 9.4-GHz EPR spectrum of this new radical reveals two hyperfine splittings of approximately 1.5 mT producing a triplet-like signal. Incubation of the enzyme with [6'-2H]-(E)-1 alters this EPR spectrum, providing the first evidence for a nucleotide-based radical species generated by RDPR. The observed spectrum is a 1:1 composite of a doublet and a triple signal, the latter being identical to that obtained with unlabeled (E)-1. Studies with (E)-1 in 2H2O also produce a 1:1 mixture of these two radical signals. The results of these isotope labeling experiments suggest wash-out or wash-in of ~0.5 equiv of deuterium at the 6' position, respectively. Incubation of the enzyme with [6'-2H]-(E)-1 in 2H2O produced only the doublet as would be expected on the basis of this hypothesis. EPR (139.5 GHz) spectra established the principal g values of the new radical species. Simulation of the 9.4- and 139.5-GHz EPR spectra yield a self- consistent set of principal hyperfine values. A structure is proposed for the radical intermediate that is consistent with the EPR data and kinetic data on release of the fluoride ion that accompanies inactivation. The proposed structure and a postulated mechanism for its formation provide further support for the hypothesis that catalysis is initiated by 3'-hydrogen atom abstraction from the nucleotide substrate.",
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AU - Yu, Guixue

AU - McCarthy, James R.

AU - Jarvi, Esa T.

AU - Matthews, Donald P.

AU - Farrar, Christian

AU - Griffin, Robert G.

AU - Stubbe, JoAnne

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