Inactivation of ribonucleotide reductase by (E)-2'-fluoromethylene-2'- deoxycytidine 5'-diphosphate: A paradigm for nucleotide mechanism-based inhibitors

Wilfred A. Van der Donk, Guixue Yu, Domingos J. Silva, Joanne Stubbe, James R. McCarthy, Esa T. Jarvi, Donald P. Matthews, Robert J. Resvick, Eugene Wagner

Research output: Contribution to journalArticlepeer-review


Ribonucleotide reductase (RDPR) from Escherichia coli catalyzes the conversion of nucleotides to deoxynucleotides and is composed of two homodimeric subunits: R1 and R2. (E)- and (Z)-2'-fluoromethylene-2'- deoxycytidine 5'-diphosphate (FMCDP) are time dependent inactivators of this protein, with ~1.5 equiv being sufficient for complete loss of catalytic activity. Inactivation results from loss of the essential tyrosyl radical on R2 and alkylation of R1. Studies using electron spin resonance spectroscopy reveal that tyrosyl radical loss is accompanied by formation of a new, substrate-based radical. Experiments using [6'-14C]-(E)-FMCDP and [5-3H]- (E)-FMCDP reveal that alkylation of R1 is accompanied by release of 0.5 equiv of cytosine and 1.4 equiv of fluoride ion. When R1 is denatured subsequent to inactivation, ~1 equiv of label per R1 is observed only in studies carried out with [14C]FMCDP. Under these same conditions with [13H]FMCDP, 1.5 equiv of radiolabel is detected as cytosine. Inactivation of R1 thus results from alkylation by the sugar moiety of FMCDP. While studies to isolate the alkylated amino acid on R1 were unsuccessful, studies using a variety of site-directed mutants of R1 (C462S, C225S, C754/759S, C439S, and E441Q) indicate that E441 or possibly C439 is the modified residue. Inactivation is accompanied by rapid formation of a new chromophore with a λ(max) at 334 nm. Dithiothreitol does not protect the enzyme against inactivation by FMCDP, although it does prevent chromophore formation. Two possible mechanisms are proposed to accommodate these experimental observations.

Original languageEnglish (US)
Pages (from-to)8381-8391
Number of pages11
Issue number25
StatePublished - 1996
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry


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