CHIMERIC SMALL-SUBUNIT INHIBITORS OF MAMMALIAN RIBONUCLEOTIDE REDUCTASE - A DUAL FUNCTION FOR THE R2 C-TERMINUS

Here we report on the formation and activity of complexes between the large submit (mR1) dimer of mouse ribonucleotide reductase (mRR) and s mall subunit chimeric dimers (cR2) derived from Escherichia coli and m ouse small subunits, cR2 submits were constructed by substituting mous e C-terminal gene sequences, coding for either 7 or 33 amino acid resi dues, for the corresponding E.coli R2 (eR2) sequences, with the remain der of the gene corresponding to eR2, The purified cR2s contained the mu-oxo bridged diferric center and tyrosine radical necessary for redu ctase activity, although the radical signal was broadened compared wit h wild-type eR2, Neither chimera formed an active complex with mR1, bu t each was a competitive inhibitor, with respect to mR2, of mRR activi ty, The inhibition constants for both chimeras were similar, and were sevenfold higher than the dissociation constant of mR2 dimer to mR1 di mer (0.24 +/- 0.02 mu M). Analysis of inhibition data showed that chim eric R2 subunits bind to mammalian R1 with a 1:1 (R1:R2) stoichiometry and permit the inference that both C-termini in a cR2 dimer bind to t he two sites per mR1 dimer. The lack of enzymatic activity in the mR1- cR2 complex is attributed to perturbation or elimination of interactio ns linking the tyrosine radical/dinuclear iron center and the C-termin us within R2.