Substrate Specificity of Human Ribonucleotide Reductase from Molt-4F Cells

Abstract

Nucleoside triphosphates were examined as the activator for various nucleoside diphosphate reductions catalyzed by a highly purified ribonucleotide reductase obtained from Molt-4F cultured human cells. It was found that cytidine 5′-diphosphate and uridine diphosphate reductions are activated by adenosine 5′-triphosphate with apparent K_a's of 0.63 ± 0.03 (S.E.) and 1.25 ± 0.10 mm, respectively. Guanosine 5′ diphosphate reduction is activated by deoxythymidine 5′-triphosphate with an apparent K_a of 1.25 ± 0.11 µm, and adenosine 5′-diphosphate reduction is activated by guanosine 5′-triphosphate or deoxyguanosine 5′-triphosphate with an apparent K_a of 1.1 ± 0.09 or 1.1 ± 0.08 mm, respectively. In the presence of saturating amounts of their best activating nucleoside triphosphates, the K_m's of various nucleoside diphosphates for this purified enzyme were studied. Double reciprocal plots of velocity against substrate concentration were found to be linear for all four substrates in the concentration range tested and yielded apparent K_m's of 7 ± 0.3 µm for cytidine 5′-diphosphate, 80 ± 6.5 µm for adenosine 5′-diphosphate, 33 ± 3.1 µm for guanosine 5′-diphosphate, 50 ± 2.0 µm for uridine 5′-diphosphate. The reduction of one ribonucleoside diphosphate could be inhibited by other ribonucleoside diphosphates in a noncompetitive manner.