INACTIVATION OF RIBONUCLEOTIDE REDUCTASE BY (E)2'-FLUOROMETHYLENE-2'-DEOXYCYTIDINE 5'-DIPHOSPHATE - A PARADIGM FOR NUCLEOTIDE MECHANISM-BASED INHIBITORS

Ribonucleotide reductase (RDPR) from Escherichia coli catalyzes the co nversion of nucleotides to deoxynucleotides and is composed of two hom odimeric subunits: R1 and R2. (E)- and (Z)-2'-fluoromethylene-2'-deoxy cytidine 5'-diphosphate (FMCDP) are time dependent inactivators of thi s protein, with similar to 1.5 equiv being sufficient for complete los s of catalytic activity, Inactivation results from loss of the essenti al tyrosyl radical on R2 and alkylation of R1. Studies using electron spin resonance spectroscopy reveal that tyrosyl radical loss is accomp anied by formation of a new, substrate-based radical, Experiments usin g [6'-C-14]-(E)-FMCDP and [5-H-3]-(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, simila r to 1 equiv of label per R1 is observed only in studies carried out w ith [C-14]FMCDP. Under these same conditions with [H-3]FMCDP, 1.5 equi v of radiolabel is detected as cytosine. Inactivation of R1 thus resul ts from alkylation by the sugar moiety of FMCDP, While studies to isol ate 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 resid ue, Inactivation is accompanied by rapid formation of a new chromophor e with a lambda(max) at 334 nm, Dithiothreitol does not protect the en zyme against inactivation by FMCDP, although it does prevent chromopho re formation. Two possible mechanisms are proposed to accommodate thes e experimental observations.