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

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Abstract
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 μM, respectively, and a relative V max 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.