Thymidine kinase 2 (TK2) is a mitochondrial (mt)
pyrimidine deoxynucleoside salvage
enzyme involved in
mtDNA precursor synthesis. The full-length human TK2
cDNA was cloned and sequenced. A discrepancy at
amino acid 37 within the mt leader sequence in the
DNA compared with the determined
peptide sequence was found. Two mutations in the human TK2 gene, His-121 to Asn and Ile-212 to Asn, were recently described in patients with severe
mtDNA depletion
myopathy (Saada, A., Shaag, A., Mandel, H., Nevo, Y., Eriksson, S., and Elpeleg, O. (2001)
Nat. Genet. 29, 342-344). The same mutations in TK2 were introduced, and the mutant
enzymes, prepared in recombinant form, were shown to have similar subunit structure to wild type TK2. The I212N mutant showed less than 1% activity as compared with wild type TK2 with all deoxynucleosides. The H121N mutant
enzyme had normal K(m) values for
thymidine (dThd) and
deoxycytidine (dCyd), 6 and 11 microm, respectively, but 2- and 3-fold lower V(max) values as compared with wild type TK2 and markedly increased K(m) values for
ATP, leading to decreased
enzyme efficiency. Competition experiments revealed that dCyd and dThd interacted differently with the H121N mutant as compared with the wild type
enzyme. The consequences of the two point mutations of TK2 and the role of TK2 in mt disorders are discussed.