title: Kinetic properties of mutant human thymidine kinase 2 suggest a mechanism for mitochondrial DNA depletion myopathy.
abstract: 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.
authors.affiliation: Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, The Biomedical Centre, P. O. Box 575, SE-751 23 Uppsala, Sweden. liya.wang@vmk.slu.se
authors.forename: Liya
authors.initials: L
authors.lastname: Wang
authors.forename: Ann
authors.initials: A
authors.lastname: Saada
authors.forename: Staffan
authors.initials: S
authors.lastname: Eriksson
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry; D001667:Binding, Competitive; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007532:Isoleucine / Q000737:chemistry; D007700:Kinetics; D017240:Mitochondrial Myopathies / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids; D017354:Point Mutation; D017386:Sequence Homology, Amino Acid; D013937:Thymidine Kinase / Q000737:chemistry / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; D004272:DNA, Mitochondrial; D007532:Isoleucine; D006639:Histidine; D001216:Asparagine; D000255:Adenosine Triphosphate; C105339:thymidine kinase 2; D013937:Thymidine Kinase