Ribonucleotide Reductase Induced by Herpes Simplex Type

The ribonucleotide reductase induced by herpes simplex virus type 1 (HSV-1) was purified in high yield from serum-starved baby hamster kidney (BHK-21) cells infected with HSV-1 (strain H-29). The enzyme preparation was essentially free of both eucaryotic ribonucleotide reductase and contaminating enzymes that could cause significant depletion f substrates. The HSV-l-induced enzyme was assayed in 0.2 M 4-(2hydroxyethy1)1-piperazineethanesulfonic acid-Na at the pH optimum of 8.1 and the optimal dithiothreitol concentration of 10 mM. Nucleoside diphosphates were the substrates of this enzyme. The HSVl-induced ribonucleotide reductase was inhibited by anions. EDTA also inhibited the enzyme and this inhibition was not reversed by the addition of FeCl,. Hydroxyurea acted as a noncompetitive inhibitor versus CDP reduction (Kii = 1.3 mM Ki. = 2.4 mM). The enzyme was inhibited by either free Mg” or free ATP. However, it was neither inhibited nor activated by the ATP. Mg complex. Reduction of either CDP or ADP was only weakly inhibited by dATP, dTTP, dGTP, and dCTP. No activation of this enzyme by these compounds was observed. The VL values for reduction of CDP, UDP, and ADP were similar, while the GDP VL was 2-fold greater. The KL values were 80, 12, 1.2, and 0.65 PM for UDP, ADP, GDP, and CDP, respectively. The KL values for CDP, GDP, and ADP were the lowest values observed for any ribonucleotide reductase. Each ribonucleoside diphosphate substrate competitively inhibited the reduction of each other substrate. The KL values obtained for inhibition by a given ribonucleoside diphosphate were similar to the KL value obtained for that compound as a substrate. Kinetic analysis of the combined rate of product formation when both CDP and ADP were simultaneously present as substrates produced patterns that were consistent with reduction at a common catalytic site. The 2‘-deoxynucleoside diphosphate products were also competitive inhibitors versus the substrates. The KI values versus CDP reduction were 310, 140, 9, and 5 PM for dUDP, dADP, dGDP, and dCDP, respectively. Similar KL values were obtained when ADP was the substrate. All of these data are most consistent with the hypothesis that the HSVl-induced ribonucleotide reductase catalyzes the reduction of all substrates at a common site. The apparent lack of significant allosteric modulation of HSV-1induced ribonucleotide reductase, its kinetic behavior, and its low KL for CDP, GDP, and ADP clearly differentiate this enzyme from other ribonucleotide reductases.