Mouse ribonucleotide reductase control: influence of substrate binding upon interactions with allosteric effectors.
Using ribonucleotide reductase encoded by vaccinia virus as a model for the mammalian enzyme, our laboratory developed an assay that allows simultaneous monitoring of the reduction of ADP, CDP, GDP, and UDP. That study found ADP reduction to be specifically inhibited by ADP itself. To learn whether this effect is significant for cellular regulation, we have analyzed recombinant mouse ribonucleotide reductase. We report that allosteric control properties originally described in single-substrate assays operate also under our four-substrate assay conditions. Three distinctions from the vaccinia enzyme were seen: 1) higher sensitivity to allosteric modifiers; 2) higher activity with UDP as substrate; and 3) significant inhibition by ADP of GDP reduction as well as that of ADP itself. Studies of the effects of ADP and other substrates upon binding of effectors indicate that binding of ribonucleoside diphosphates at the catalytic site influences dNTP binding at the specificity site. We also examined the activities of hybrid ribonucleotide reductases, composed of a mouse subunit combined with a vaccinia subunit. As previously reported, a vaccinia R1/mouse R2 hybrid has low but significant activity. Surprisingly, a mouse R1/vaccinia R2 hybrid was more active than either mouse R1/R2 or vaccinia R1/R2, possibly explaining why mutations affecting vaccinia ribonucleotide reductase have only small effects upon viral DNA replication.
Chimploy
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Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331-7305, USA.
Mathews
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D000244:Adenosine Diphosphate / Q000378:metabolism; D000495:Allosteric Site; D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D003565:Cytidine Diphosphate / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006153:Guanosine Diphosphate / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D013997:Time Factors; D014530:Uridine Diphosphate / Q000378:metabolism; D014616:Vaccinia virus / Q000235:genetics
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D006153:Guanosine Diphosphate; D014530:Uridine Diphosphate; D000244:Adenosine Diphosphate; D003565:Cytidine Diphosphate; D012264:Ribonucleotide Reductases