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J Biol Chem, Vol. 273, Issue 45, 29519-29523, November 6, 1998

Differential Effects of Hydroxyurea upon Deoxyribonucleoside Triphosphate Pools, Analyzed with Vaccinia Virus Ribonucleotide Reductase

Stephen P. Hendricks and Christopher K. Mathews

From the Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331-7305

Hydroxyurea inhibits DNA synthesis by destroying the catalytically essential free radical of class I ribonucleoside diphosphate (rNDP) reductase, thereby blocking the de novo synthesis of deoxyribonucleotides. In mammalian cells, including those infected by vaccinia virus, hydroxyurea treatment causes a differential depletion of the four deoxyribonucleoside triphosphate pools, suggesting that the activities of rNDP reductase are differentially sensitive to hydroxyurea. In the presence of different substrates and allosteric modifiers, we measured rates of free radical destruction in the vaccinia virus-coded rNDP reductase, by following absorbance at 417 nm as a function of time after hydroxyurea addition. Also, we followed enzyme activity directly, by using a recently developed assay that allows simultaneous monitoring of the four activities, in the presence of substrates and effectors at concentrations that approximate the intracellular environment. We found the primary determinant of radical loss to be not the ensemble of allosteric ligands bound but the activity of the enzyme. Nucleoside triphosphate effectors accelerated radical decay, compared with rates seen with the free enzyme. Adding substrate to the holoenzyme, under conditions where the enzymatic reaction is proceeding, further accelerated radical decay. Alternative models are discussed, to account for selective depletion of purine nucleotide pools by hydroxyurea.

Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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S. P. Hendricks and C. K. Mathews
Allosteric Regulation of Vaccinia Virus Ribonucleotide Reductase, Analyzed by Simultaneous Monitoring of Its Four Activities
J. Biol. Chem., November 6, 1998; 273(45): 29512 - 29518.
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Copyright © 1998 by the American Society for Biochemistry and Molecular Biology.