Reduction of NDPs by murine ribonucleotidc reductase (mRR) requires catalytic (mRl) and free radical-containing (mR2) subunits and is regulated by nucleoside triphosphate allosteric effectors. Here we present a comprehensive and quantitative model forallosteric control of mRR enzymatic activity based on molecular mass, ligand binding and enzyme activity studies. In this model, nucleotide binding to the specificity site (s-site) drives formation of R1_2, ATP or dATP binding to the adenine-specific site (a-site) results in formation of tetramer Rl_(4a) which isomehzes to the more stable R1_(4b), and ATP binding to the newly described hexamerization site (h-site) drives formation of R1_6. The R2_2 complexes of R1_2, R1_(4a), and R1_6 are enzymaticallyactive, whereas the R2_2 complexes of R1_(4b) are not. Thus, a key aspect of the down regulation of RR enzymatic activity is the ability of dATP or ATP binding to the a-site to drive not only Rl letramer formation, but also the conversion of Rl_(4a) toR1_(4b) and it is ATP binding to the h-site which accounts for its activating properties at high (>lmM) concentration. The D57N variant of mRl is not inhibited by dATP because of a block in the formation of R1_(4b), but not of Rl_(4a) or Rl_6. The new model revises and improves upon an earlier phenomenological model (Thelander and Reichard 1979) (the 'RT' model) which ignores aggregation state changes and the h-site, and incorrectly rationalizes ATP activation vs. dATP inhibition as reflecting differentfunctional consequences of ATP vs. dATP binding to the a-site. Our results suggest that the R1_6R2_6 heterohexamer is the major active form of the enzyme in mammalian cell cytoplasm, where [ATP] is the primary modulator of enzyme activity, coupling therate of DNA biosynthesis with the energetic state of the cell. However, it remains possible that the helerodimer formed by complexation of R1_2 with p53R2 is the active form of the enzyme in the nucleus. Using the crystal structure of the Escherichia coli Rl hexamer as a model for the mRl hexamer, a scheme is presented that rationalizes the slow isomerization of the letramer form and suggests an explanation for the low enzymatic activity of tetramers complexed with R2. The similar specific activities of R1_2R2_2 and R1_6R2_6 are inconsistent with a proposed model for R2_2 docking with R1_2 (Uhlin and Eklund. 1994) and an alternative model is proposed.
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