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

Ribonucleoticle reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. The class I RNRs are composed of two subunits, alpha and beta, with proposed quaternary structures of alpha 2 beta 2, alpha 6 beta 2, or alpha 6 beta 6, depending on the organism. The a subunits bind the nucleoside diphosphate substrates and the dNTP/ATP allosteric effectors that govern specificity and turnover. The beta 2 subunit houses the diferric Y-center dot (1 radical per beta 2) cofactor that is required to initiate nucleotide reduction. 2',2'-Difluoro-2'deoxycytidine (F2C is presently used clinically in a variety of cancer treatments and the 5'-diphosphorylated F2C (F2CDP) is a potent inhibitor of RNRs. The studies with [1'-3H]-F2CDP and [5-H-3]-F2CDP have established that F2CDP is a substoichiometric mechanism based inhibitor (0.5 eq F2CDP/alpha) of both the Escherichia coli and the human MRS in the presence of reductant. Inactivation is caused by covalent labeling of RNR by the sugar of F2CDP (0.5 eq/alpha) and is accompanied by release of 0.5 eq cytosine/alpha. Inactivation also results in loss of 40% of beta 2 activity. Studies using size exclusion chromatography reveal that in the E. coli RNR, an alpha 2 beta 2 tight complex is generated subsequent to enzyme inactivation by F2CDP, whereas in the human RNR, an alpha 6 beta 6 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 F2CDP and ATP. This information and the proposed asymmetry between the interactions of alpha n beta n provide an explanation for complete inactivation of RNR with substoichiometric amounts of F2CDP.