Effects of Nucleoside Triphosphates on Human Ribonucleotide Reductase from Molt-4F Cells¹

The effects of nucleoside triphosphates on various nucleoside diphosphate reductions catalyzed by a highly purified ribonucleotide reductase from Molt-4F cultured human cells were examined. It was found that deoxyadenosine 5′-triphosphate strongly inhibited all four reductions. The reduction of pyrimidine nucleoside diphosphate in the presence of an activator [adenosine 5′-triphosphate (ATP)] was inhibited in a noncompetitive manner with respect to ATP by deoxyguanosine 5′-triphosphate (dGTP) and deoxythymidine 5′-triphosphate (dTTP). For cytidine 5′-diphosphate reduction, the value of the K_i intercept for dGTP was 47 µm and for dTTP, it was 270 µm; the K_i slope was 25 µm for dGTP, 100 µm for dTTP. Similarly, for uridine diphosphate reduction, the K_i intercept was 4.3 µm for dGTP, 25 µm for dTTP. The K_i slope was 1.5 µm for dGTP and 9 µm for dTTP. The reduction of ADP in the presence of its activator (dGTP) was inhibited noncompetitively by dTTP. The values of K_i intercept and slope of dTTP for adenosine 5′-diphosphate (ADP) reduction were 1.8 and 0.9 mm, respectively. Although guanosine 5′-triphosphate (GTP) and dGTP were found to serve equally well as activators for ADP reductions with the same apparent K_a and V_max, the inhibition pattern of GTP and dGTP on the enzyme activity for cytidine 5′-diphosphate reduction was different. ATP was found to be an accessory activator for ADP reduction due to the fact that ATP at 1.0 or 0.3 mm concentration decreased the apparent K_a of GTP for ADP reduction from 1.1 to 0.14 or 0.08 mm, respectively. In the absence of ATP, the V_max for ADP reduction was increased 2-fold. ATP at a concentration of 1.0 or 0.3 mm also changed the apparent K_a of dTTP for guanosine 5′-diphosphate reduction from 1.25 to 0.9 or 0.6 µm, respectively, but V_max for guanosine 5′-diphosphate reduction was increased.

GTP at a concentration of 0.3 or 0.5 mm decreased the apparent K_a's of ATP for pyrimidine nucleoside reduction. At these concentrations of GTP, the V_max for cytidine 5′-diphosphate reduction was decreased 7 and 12%, and the V_max for uridine 5′-diphosphate reduction was decreased 33 and 45% when they were compared with those in the absence of GTP. Therefore, a change in any of these nucleotide concentrations could lead to changes in ribonucleotide reductase activity. ATP emerges as a most important factor in controlling the reduction of all four ribonucleoside diphosphates.