Allosteric regulation of vaccinia virus ribonucleotide reductase, analyzed by simultaneous monitoring of its four activities.
As determined by simultaneous monitoring of its four activities, vaccinia virus-coded ribonucleoside diphosphate (rNDP) reductase shows responses to individual nucleoside triphosphate effectors-ATP, dATP, dGTP, and dTTP-similar to those previously reported for rNDP reductase of mouse, which the viral enzyme closely resembles. This investigation uses the vaccinia enzyme as a readily available and convenient model for understanding the cellular enzyme. As previously reported for T4 phage aerobic rNDP reductase, we found the relative activities of ADP, CDP, GDP, and UDP reduction to be reasonably close to the proportions of the four deoxyribonucleotides in the vaccinia virus genome, but only when the four substrates and the four allosteric effectors were all provided at their approximate intracellular concentrations. GDP reductase levels were somewhat higher, proportionately, than the representation of dGMP in vaccinia virus DNA. To understand this behavior and also to evaluate possible relationships between ribonucleotide reductase control and the very low dGTP pools seen in eukaryotic cells, we carried out substrate saturation experiments with a "bioproportional" mixture containing the four rNDP substrates at their relative in vivo concentrations as determined from rNDP pool measurements. Reduction of the two purine substrates was inhibited at high concentrations of this mixture, and data suggest that ADP acts as a specific inhibitor of its own reduction and that of GDP. Use of the four-substrate assay revealed also that a mixture of vaccinia virus R1 protein and mouse R2 protein is catalytically active, making this the first reported chimeric rNDP reductase to show biological activity.
Hendricks
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SP
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331-7305, USA.
Mathews
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CK
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000494:Allosteric Regulation; D000818:Animals; D002460:Cell Line; D000882:Haplorhini; D051379:Mice; D011994:Recombinant Proteins / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000378:metabolism*; D014616:Vaccinia virus / Q000201:enzymology*
D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D012264:Ribonucleotide Reductases