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

Based on amino acid sequence information from purified mitochondrial thymid ine kinase (TK2), a cDNA of 1930 bp was cloned, containing an open reading frame encoding 232 amino acid residues starting with the N-terminal sequenc e determined from the native human protein preparation, Northern blot analy sis with the cDNA coding region demonstrated several TK2 mRNAs, with 2 and 4 kb forms present in many tissues. We also characterised N-terminally trun cated (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 arabinosy l cytosine is phosphorylated. The antitumour drug difluorodeoxycytidine and its metabolite difluorodeoxyuridine are good substrates, with K-m values o f 66 and 29 mu M, respectively, and a relative V-max of 0.6 compared to tha t of thymidine. Negative cooperativity was found with thymidine and the ant i-HIV drug 3'-azidothymidine, but the reaction followed Michaelis-Menten ki netics with deoxycytidine, arabinosyl cytosine, and arabinosyl thymine. The results demonstrate a broad substrate specificity and complex kinetics, an d suggest a role for TK2 in the activation of chemotherapeutic nucleoside a nalogues. (C) 1999 Federation of European Biochemical Societies.