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 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.
Wang
L
L
Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Biomedical Center, Uppsala.
Munch-Petersen
B
B
Herrström Sjöberg
A
A
Hellman
U
U
Bergman
T
T
Jörnvall
H
H
Eriksson
S
S
D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000378:metabolism*; D000998:Antiviral Agents / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006801:Humans; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D013937:Thymidine Kinase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism