Many
nucleoside analog drugs, such as
ribavirin and
viramidine, are activated or metabolized in vivo through 5'-phosphorylation. In this report, we determined the steady-state kinetic parameters for 5'-monophosphorylation of
ribavirin and
viramidine by
adenosine kinase. The apparent Km for
ribavirin is 540 microM, and k(cat) is 1.8 min-1. Its catalytic efficiency of 3.3 x 10(-3) min-1 . microM-1 is 1,200-fold lower than that of
adenosine. In contrast to the common belief that
ribavirin is exclusively phosphorylated by
adenosine kinase, cytosolic
5'-nucleotidase II was found to catalyze
ribavirin phosphorylation in vitro. The reaction is optimally stimulated by the physiological concentration of
ATP or
2,3-bisphosphoglycerate. In
phosphate-buffered saline plus
ATP and
2,3-bisphosphoglycerate, the apparent Km for
ribavirin is 88 microM, and k(cat) is 4.0 min-1. These findings suggest that cytosolic
5'-nucleotidase II may be involved in
ribavirin phosphorylation in vivo. Like
ribavirin,
viramidine was found to be phosphorylated by either
adenosine kinase or cytosolic
5'-nucleotidase II, albeit with a much lower activity. The catalytic efficiency for
viramidine phosphorylation is 10- to 330-fold lower than that of
ribavirin, suggesting that other
nucleoside kinase(s) may be involved in
viramidine phosphorylation in vivo. Both
ribavirin and
viramidine are not phosphorylated by
deoxycytidine kinase and
uridine-cytidine kinase. The coincidence of presence of high concentrated
2,3-bisphosphoglycerate in erythrocytes suggests that cytosolic
5'-nucleotidase II could play an important role in phosphorylating
ribavirin and contribute to anabolism of
ribavirin triphosphate in erythrocytes. Elucidation of
ribavirin and
viramidine phosphorylation mechanism should shed light on their in vivo metabolism, especially the
ribavirin-induced
hemolytic anemia in erythrocytes.