Subunit Interaction Dynamics of Class Ia Ribonucleotide Reductases: In Search of a Robust Assay.

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides (NDP) to deoxynucleotides (dNDP), in part, by controlling the ratios and quantities of dNTPs available for DNA replication and repair. The active form of class Ia RNR is an asymmetric αβ complex in which α contains the active site and β contains the stable diferric-tyrosyl radical cofactor responsible for initiating the reduction chemistry. Each dNDP is accompanied by disulfide bond formation. We now report that, under conditions, β can initiate turnover in α catalytically under both "one" turnover (no external reductant, though producing two dCDPs) and multiple turnover (with an external reductant) assay conditions. In the absence of reductant, rapid chemical quench analysis of a reaction of α, substrate, and effector with variable amounts of β (1-, 10-, and 100-fold less than α) yields 3 dCDP/α at all ratios of α:β with a rate constant of 8-9 s, associated with a rate-limiting conformational change. Stopped-flow fluorescence spectroscopy with a fluorophore-labeled β reveals that the rate constants for subunit association (163 ± 7 μM s) and dissociation (75 ± 10 s) are fast relative to turnover, consistent with catalytic β. When assaying in the presence of an external reducing system, the turnover number is dictated by the ratio of α:β, their concentrations, and the concentration and nature of the reducing system; the rate-limiting step can change from the conformational gating to a step or steps involving disulfide rereduction, dissociation of the inhibited αβ state, or both. The issues encountered with RNR are likely of importance in all class I RNRs and are central to understanding the development of screening assays for inhibitors of these enzymes.