Abstract
Thymidine kinase (ATP : thymidine 5′-phosphotransferase, EC 2.7.1.21), purified to apparent homogeneity from human liver, was found to have Michaelis constants for thymidine and ATP of 5 and 90 μM, respectively. Based on studies of initial velocity and product inhibition, the enzyme kinetic mechanism is compatible with an ordered sequential reaction with thymidine binding first and thymidine monophosphate released last. The activity of various triphosphate nucleosides as phosphate donors for human liver thymidine kinase showed little specificity with ATP > CTP > UTP > GTP and the respective Michaelis constants ranged from 0.10 to 0.30 mM. Among various purine and pyrimidine compounds, only TTP and dCTP were effective inhibitors of the enzyme. Inhibition with TTP was competitive with respect to both thymidine and ATP with K i values of 13.5 and 8.5 μM, respectively, while the inhibition produced by dCTP was complex. Deoxycytidine was found to be an effective nucleoside substrate for human liver thymidine kinase with a Michaelis constant of 6 μM. This finding suggests that human mitochondrial deoxycytidine and thymidine kinase activity is a single protein.
Published Version
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