Abstract
The two thymidine (dThd) kinases in human cells, the cytosolic, S-phase-specific TK1 and the mitochondrial, constitutively expressed TK2 were purified to homogeneity as judged from sodium dodecyl sulfate-gel electrophoresis. The substrate specificity of TK1 and TK2 toward natural substrates and important nucleoside analogues was compared. With TK1, the Km values for 5-fluorodeoxyuridine (FdUrd), 3'-azido-2',3'-dideoxythymidine (AZT), and 3'-fluoro-2',3'-dideoxythymidine (FLT) were 2.2, 0.6, and 2.1 microM as compared to 0.5 microM for dThd and 9 microM for deoxyuridine (dUrd). With TK2, dUrd, deoxycytidine (dCyd), and 5-fluorodeoxyuridine (FdUrd) were efficiently phosphorylated, but with distinctly different kinetics: Michaelis-Menten kinetics with dCyd, dUrd, and FdUrd; negative cooperativity with dThd. Negative cooperativity was also observed with AZT, although this drug was a very poor substrate for TK2 with a Vmax of 5-6% of that with dThd. FLT, 2',3'-dideoxycytidine (ddCyd), and arabinofuranosylcytosine (araC) were not substrates for TK2, and 2',3'-didehydrodideoxy-thymidine (D4T) was not a substrate for TK1 or TK2. On the other hand, AZT, FLT, and D4T were competitive inhibitors with Ki values of 0.6, 6, and 2073 microM for TK1, and 2, 10, and 78 microM for TK2, respectively. The much lower tolerance for modifications of the deoxyribose moiety of TK2 as compared to TK1 is important for the design of new antiviral nucleoside analogues intended for use in cells with different expression of TK1 and TK2.
Highlights
The two thymidine kinases in human cells, dThd to dTMP, but theexact role of the enzyme activity in the cytosolic, S-phase-specific TK1 and themitochon- cell division and DNA synthesis is far from clear
Fluorodeoxyuridine(FdUrd) were efficiently phosfairly constant level of dThd kinase activity in resting cells is due to the presence of TK2 which has beenshown to be predominantly localized in mitochondria (3, 9-14)
Both cellular fractionation experiments (9, 13) and the well known fact that [3H]dThd is incorporated into nonproliferative cells with radiation or chemical damaged DNA (15) have phorylated, but with distinctly different kinetics:Mi- shown that TK2 is present in the cytosol and mitochondria chaelis-Menten kinetics with dCyd, dUrd, and FdUrd; in resting and proliferating cells
Summary
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