Regulation of thymidine kinase activity by 5-iodo-2'-deoxyuridine 5'-triphosphate and deoxythymidine 5'-triphosphate
Prusoff, W.H.; Chang, P.K.
Chemico-Biological Interactions 1(3): 285-299
ISSN/ISBN: 0009-2797 PMID: 5524800 DOI: 10.1016/0009-2797(70)90015-3
A comparison was made of the inhibition of deoxythymidine kinase derived froin Ehrlich ascites carcinoma cells by 5-iodo-2'-deoxyuridine 5'-triphosphate and deoxythymidine 5'-triphosphate. The inhibition produced by the halogenated triphosphate derivative was observed to be qualitatively similar to deoxythymidine 5'-triphosphate over a rangs of p H, temperature and substrate concentrations; however, 5-iodo-2'-deoxyuridine 5'-triphosphate was quantitatively more inhibitory. Although the apparent Km for thymidine was not significantly different at p H 5.0, 6.5 and 7.5, the inhibitory effect of deoxythymidine 5'-triphosphate and 5-iodo-2'-deoxyuridine 5'-triphosphate was 43- and 26-fold larger respectively at p H 5.0 compared to p H 6.5; whereas the inhibitory effect for both compounds at p H 6.5 was about 4-fold greater than that at p H 7.5. In the presence or absence of the inhibitors, analysis indicated "allosteric" kinetics prevailed at p H 5.0 and 6.5, whereas Michaelis competitive kinetics occurred at p H 7.5 for both. These triphosphate derivatives protected thymidine kinase froin thermal inactivation (65°, 30 min); however, the halogenated derivative was effective at lower concentrations. Thus the halogenated triphosphate not only exerted a greater inhibitory effect than deoxythymidine triphosphate, but also afforded greater protection to thermal inactivation. A Hill plot indicated cooperative interactions at p H 5.0 and 6.5 but not at p H 7.5, and the presence of either triphosphate derivative increased the Hill coefficient (n) at p H 5.0 and 6.5 but not at p H 7.5. The order of addition of the substrate (thymidine) or of the inhibitory effectors had no significant effect on the amount of inhibition observed.