We have studied the effect of pH on the interactions between
thymidine kinase,
thymidine triphosphate, and
5'-amino-2',5'-dideoxythymidine (5'-AdThd) in purified preparations of the
enzyme and in intact 647V cells, a human
bladder cancer cell line.
Thymidine kinase is competitively inhibited by 5'-AdThd.
dTTP feedback inhibits in a noncompetitive fashion. However, 5'-AdThd partially reverses the inhibition produced by
dTTP resulting in enhanced
enzyme activity. We have found that
dTTP (pKa = 7.5) is a much more potent inhibitor of purified preparations of
thymidine kinase activity at low pH conditions. For example, 2.5 microM
dTTP inhibited
thymidine kinase activity by 50, 85, and 95% at pH values of 8.0, 7.5, and 6.5, respectively. The interaction of 5'-AdThd (pKa = 8.5) at either the active (competitive) or the regulatory (deinhibition) site is not altered significantly over a pH range of 6.5 to 9.5. To extend these findings to intact cells, we studied the perturbation of the uptake of
thymidine by 5'-AdThd in 647V cells incubated in media buffered at various pH values. In cells exposed to media buffered at pH 8.5 or 7.5, 5'-AdThd maximally stimulated
thymidine uptake about 250 and 300%
at 10 and 30 microM, respectively. However, at pH 6.5, 300 microM 5'AdThd was required to produce maximal stimulation of about 500%. These observations are consistent with the greater sensitivity of
thymidine kinase (in situ) to feedback inhibition by
dTTP at the lower pH conditions. Intracellular
dTTP pool sizes were not affected by variations in pH during the short time course of our experiments. However, after 1 h, the intracellular concentration of 5'-AdThd was twice that of the extracellular medium in conditions at pH 7.5 and 8.5 but was equimolar across the membrane at pH 6.5. This does not account for the differences in the perturbation of
thymidine uptake by 5'-AdThd at various pH values. In general, our results indicate that regulation of
thymidine kinase by
dTTP is pH dependent, while its modulation by 5'-AdThd is not, and that regulation of
thymidine kinase in situ is sensitive to alterations in pH.