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

FEBS Letters

Herrström Sjöberg, Anita¹  Hellman, Ulf²  Bergman, Tomas⁴  Eriksson, Staffan¹  Munch-Petersen, Birgitte³  Wang, Liya¹  Jörnvall, Hans⁴ 

[1] Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Biomedical Center, Box 575, S-751 23 Uppsala, Sweden;Ludwig Institute of Cancer Research, Biomedical Center, Box 595, S-751 24 Uppsala, Sweden;Department of Life Sciences and Chemistry, Roskilde University Center, Box 260, DK-4000 Roskilde, Denmark;Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden

关键词: Cloning;    Thymidine kinase;    Antiviral nucleoside;    Cytostatic nucleoside;    Metabolic conversion;    Mitochondrial enzyme;   

DOI  :  10.1016/S0014-5793(98)01711-6

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown