Human thymidine kinase 2: molecular cloning and characterisation of the enzyme activity with antiviral and cytostatic nucleoside substrates

作者：Liya Wang, Birgitte Munch‐Petersen, Anita Herrström Sjöberg, Ulf Hellman, Tomas Bergman, Hans JÖRNVALL, Staffan Eriksson · 发表于：FEBS Letters · 年份：1999 · DOI：10.1016/s0014-5793(98)01711-6 · 被引用次数：168 · 研究领域：Biochemical and Molecular Research、Chronic Lymphocytic Leukemia Research、HIV/AIDS drug development and treatment

Based on amino acid sequence information from purified mitochondrial thymidine kinase (TK2), a cDNA of 1930 bp was cloned, containing an open reading frame encoding 232 amino acid residues starting with the N-terminal sequence determined from the native human protein preparation. Northern blot analysis with the cDNA coding region demonstrated several TK2 mRNAs, with 2 and 4 kb forms present in many tissues. We also characterised N-terminally truncated (starting at position 18) human TK2 with pharmacologically important antiviral and cytostatic nucleoside analogues. Results were highly similar to those with the native TK2 preparation. The anti-leukaemic drug arabinosyl cytosine is phosphorylated. The antitumour drug difluorodeoxycytidine and its metabolite difluorodeoxyuridine are good substrates, with K(m) values of 66 and 29 microM, respectively, and a relative Vmax of 0.6 compared to that of thymidine. Negative cooperativity was found with thymidine and the anti-HIV drug 3'-azidothymidine, but the reaction followed Michaelis-Menten kinetics with deoxycytidine, arabinosyl cytosine, and arabinosyl thymine. The results demonstrate a broad substrate specificity and complex kinetics, and suggest a role for TK2 in the activation of chemotherapeutic nucleoside analogues.