Enzyme regulatory site-directed drugs: study of the interactions of 5'-amino-2', 5'-dideoxythymidine (5'-AdThd) and thymidine triphosphate with thymidine kinase and the relationship to the stimulation of thymidine uptake by 5'-AdThd in 647V cells.
5'-Amino-2',5'-dideoxythymidine (5'-AdThd) is a nontoxic thymidine (dThd) analogue capable of antagonizing the feedback inhibition exerted by thymidine triphosphate (dTTP) on thymidine kinase (EC 2.7.1.21). In intact cells, this results in stimulation of thymidine uptake by 5'-AdThd. We have studied the interaction between 5'-AdThd and thymidine kinase purified from 647V cells. We found that 5'-AdThd inhibited competitively thymidine kinase activity (Ki of 0.5 microM) in the absence of dTTP whereas dTTP inhibited thymidine kinase activity in a noncompetitive manner. However, in the presence of dTTP, 5'-AdThd was able to stimulate enzyme activity in a mode that suggests competition with dTTP for the regulatory site. Altered interactions were observed at high substrate (dThd) concentrations, with dThd showing competitive kinetics with dTTP. In intact cells, we evaluated the hypothesis that antagonism of feedback inhibition could account for stimulation of dThd uptake by 5'-AdThd. If inhibition of thymidine kinase activity by dTTP is critical, then depletion of cellular dTTP by methotrexate should reduce the ability of 5'-AdThd to stimulate dThd uptake. Indeed, this was the case. If the dTTP pools were repleted by the addition of higher concentrations of dThd, the ability of 5'-AdThd to stimulate dThd uptake was restored. Furthermore, effects of 5'-AdThd on nucleoside phosphorylase or cytoplasmic 5'-nucleotidase activity (dTMP breakdown) could not account for the stimulation of dThd uptake in 647V cells. In summary, our results indicate that 5'-AdThd interacts with thymidine kinase at the dTTP-binding site, resulting in stimulation of enzyme activity and stimulation of dThd uptake in intact cells.
Vazquez-Padua
M A
MA
Department of Human Oncology, University of Wisconsin Clinical Cancer Center, University of Wisconsin School of Medicine, Madison 53792.
Kunugi
K
K
Fischer
P H
PH
D015720:5'-Nucleotidase; D015224:Dideoxynucleosides / Q000494:pharmacology*; D005246:Feedback; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology; D009708:Nucleotidases / Q000032:analysis; D013936:Thymidine / Q000378:metabolism*; D013937:Thymidine Kinase / Q000037:antagonists & inhibitors*; D013939:Thymidine Phosphorylase / Q000032:analysis; D013942:Thymine Nucleotides / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D015224:Dideoxynucleosides; D013942:Thymine Nucleotides; C024082:5'-amino-5'-deoxythymidine; D013939:Thymidine Phosphorylase; D013937:Thymidine Kinase; D009708:Nucleotidases; D015720:5'-Nucleotidase; C024157:thymidine 5'-triphosphate; D013936:Thymidine; D008727:Methotrexate