Enhanced subunit interactions with gemcitabine-5′-diphosphate inhibit ribonucleotide reductases

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. The class I RNRs are composed of two subunits, α and β, with proposed quaternary structures of α2β2, α6β2, or α6β6, depending on the organism. The α subunits bind the nucleoside diphosphate substrates and the dNTP/ATP allosteric effectors that govern specificity and turnover. The β2 subunit houses the diferric Y • (1 radical per β2) cofactor that is required to initiate nucleotide reduction. 2′,2′-Difluoro-2′-deoxycytidine (F 2 C) is presently used clinically in a variety of cancer treatments and the 5′-diphosphorylated F 2 C (F 2 CDP) is a potent inhibitor of RNRs. The studies with [1′- 3 H]-F 2 CDP and [5- 3 H]-F 2 CDP have established that F 2 CDP is a substoichiometric mechanism based inhibitor (0.5 eq F 2 CDP/α) of both the Escherichia coli and the human RNRs in the presence of reductant. Inactivation is caused by covalent labeling of RNR by the sugar of F 2 CDP (0.5 eq/α) and is accompanied by release of 0.5 eq cytosine/α. Inactivation also results in loss of 40% of β2 activity. Studies using size exclusion chromatography reveal that in the E. coli RNR, an α2β2 tight complex is generated subsequent to enzyme inactivation by F 2 CDP, whereas in the human RNR, an α6β6 tight complex is generated. Isolation of these complexes establishes that the weak interactions of the subunits in the absence of nucleotides are substantially increased in the presence of F 2 CDP and ATP. This information and the proposed asymmetry between the interactions of αnβn provide an explanation for complete inactivation of RNR with substoichiometric amounts of F 2 CDP.