DETECTION OF A NEW SUBSTRATE-DERIVED RADICAL DURING INACTIVATION OF RIBONUCLEOTIDE REDUCTASE FROM ESCHERICHIA-COLI BY GEMCITABINE 5'-DIPHOSPHATE

Ribonucleotide reductases (RNRs) play a central role in replication an d repair by catalyzing the conversion of nucleotides to deoxynucleotid es, Gemcitabine 5'-diphosphate (F2CDP), the nucleoside of which was re cently approved by the FDA for treatment of pancreatic cancer, is a po tent mechanism-based inhibitor of class I and II RNRs. Inactivation of the Eschericia coli class I RNR is accompanied by loss of two fluorid es and one cytosine, This RNR is composed of two homodimeric subunits: R1 and R2. R1 is the site of nucleotide reduction, and R2 contains th e essential diferric-tyrosyl radical cofactor. The mechanism of inacti vation depends on the availability of reductant, In the presence of re ductant [thioredoxin (TR)/thioredoxin reductase (TRR)/NADPH or dithiot hreitol], inhibition results from R1 inactivation. In the absence of r eductant with prereduced R1 and R2, inhibition results from loss of th e essential tyrosyl radical in R2, The same result is obtained with C7 54S/C759S-R1 in the presence of TR/TRR/NADPH. In both cases, tyrosyl r adical loss is accompanied by formation of a new stable radical (0.15- 0.25 equiv/RNR). EPR studies in (H2O)-H-2, With [U-H-2]R1, and examina tion of the microwave power saturation of the observed signal, indicat e by process of elimination that this new radical is nucleotide-based, In contrast to all previously investigated 2'-substituted nucleotide inhibitors of RNR, inactivation is not accompanied by formation of a n ew protein-associated chromophore under any conditions. The requiremen t for reductant in the R1 inactivation pathway, the lack of chromophor e on the protein, the loss of two fluoride ions, and the stoichiometry of the inactivation all suggest a unique mechanism of RNR inactivatio n not previously observed with other 2'-substituted nucleotide inhibit ors of RNR. This unique mode of inactivation is proposed to be respons ible for its observed clinical efficacy.