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 subunit (mR1) dimer of mouse ribonucleotide reductase (mRR) and small subunit chimeric dimers (cR2) derived from Escherichia coli and mouse small subunits. cR2 subunits were constructed by substituting mouse C-terminal gene sequences, coding for either 7 or 33 amino acid residues, for the corresponding E.coli R2 (eR2) sequences, with the remainder of the gene corresponding to eR2. The purified cR2s contained the micro-oxo bridged diferric center and tyrosine radical necessary for reductase activity, although the radical signal was broadened compared with wild-type eR2. Neither chimera formed an active complex with mR1, but each was a competitive inhibitor, with respect to mR2, of mRR activity. The inhibition constants for both chimeras were similar, and were sevenfold higher than the dissociation constant of mR2 dimer to mR1 dimer (0.24 +/- 0.02 microM). Analysis of inhibition data showed that chimeric 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 the two sites per mR1 dimer. The lack of enzymatic activity in the mR1-cR2 complex is attributed to perturbation or elimination of interactions linking the tyrosine radical/dinuclear iron center and the C-terminus within R2.
Hamann
C S
CS
Department of Chemistry, School of Medicine, University of Pennsylvania, Philadelphia 19104, USA.
Lentainge
S
S
Li
L S
LS
Salem
J S
JS
Yang
F D
FD
Cooperman
B S
BS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D012262:Ribonucleoside Diphosphate Reductase / Q000187:drug effects / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C019857:CDP reductase; D012264:Ribonucleotide Reductases; D012262:Ribonucleoside Diphosphate Reductase