Comprehensive Model for Allosteric Regulation of Mammalian Ribonucleotide Reductase:  Refinements and Consequences

Reduction of NDPs by murine ribonucleotide reductase (mRR) requires catalytic (mR1) and free radical-containing (mR2) subunits and is regulated by nucleoside triphosphate allosteric effectors. Here we present the results of several studies that refine the recently presented comprehensive model for the allosteric control of mRR enzymatic activity [Kashlan, O. B., et al. (2002) Biochemistry 41, 462−474], in which nucleotide binding to the specificity site (s-site) drives formation of an active R12R22 dimer, ATP or dATP binding to the adenine site (a-site) drives formation of a tetramer, mR14a, which isomerizes to an inactive form, mR14b, and ATP binding to the hexamerization site (h-site) drives formation of an active R16R26 hexamer. Analysis of the a-site D57N variant of mR1, which differs from wild-type mR1 (wt-mR1) in that its RR activity is activated by both ATP and dATP, demonstrates that dATP activation of the D57N variant RR arises from a blockage in the formation of mR14b from mR14a, and provides strong evidence that mR14a forms active complexes with mR22. We further demonstrate that (a) differences in the effects of ATP versus dATP binding to the a-site of wt-mR1 provide specific mechanisms by which the dATP/ATP ratio in mammalian cells could modulate in vivo RR enzymatic activity, (b) the comprehensive model is valid over a range of Mg2+ concentrations that include in vivo concentrations, and (c) equilibrium constants derived for the comprehensive model can be used to simulate the distribution of R1 among dimer, tetramer, and hexamer forms in vivo. Such simulations indicate that mR16 predominates over mR12 in the cytoplasm of normal mammalian cells, where the great majority of RR activity is located, but that mR12 may be important for nuclear RR activity and for RR activity in cells in which the level of ATP is depleted.