Ribonucleotide reductases (RNRs) catalyze the de novo reduction of NDPs (CDP, UDP, ADP, GDP) to deoxynucleotides in all organisms, thus controlling their relative ratios and amounts and, consequently, the fidelity of DNA replication and repair. The class Ia RNRs are composed of alpha (α) and beta (β) subunits which form an active and dynamic α2β2 complex. Studies over the last 20 years with mechanism-based inhibitors such a 2’-azido-2’-deoxynucleotides and gemcitabine diphosphate exhibited half-sites reactivity with only one equivalent of inhibitor required per α2β2 for rapid enzyme inactivation. Recent studies with unnatural amino acids site-specifically replacing the Y122 radical in β2, which is essential for initiating the reduction process in α2 by long range radical transfer (RT, (Y122-Y356 )-β to (Y730-Y731-C439)-α) over 35 Å, also revealed half-sites reactivity. With 2,3,5-F3Y122 radical , half of this radical was lost on incubation with α2, substrate and effector, concomitant with formation of 0.5 equivalents of Y356radical in the RT pathway. We have recently shown that generating a radical within the RT pathway causes formation of a “tight” α2β2 complex. Structural analysis of this stalled complex reveals an asymmetric interaction between α2 and β2, consistent with all the biochemical data.

Half-Sites reactivity of ribonucleotide reductase: biochemical and EM structural evidenceα

256th National Meeting (2018)

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