title: Comparative active-site mutation study of human and Caenorhabditis elegans thymidine kinase 1.
abstract: The first step for the intracellular retention of several anticancer or antiviral nucleoside analogues is the addition of a phosphate group catalysed by a deoxyribonucleoside kinase such as thymidine kinase 1 (TK1). Recently, human TK1 (HuTK1) has been crystallized and characterized using different ligands. To improve our understanding of TK1 substrate specificity, we performed a detailed, mutation-based comparative structure-function study of the active sites of two thymidine kinases: HuTK1 and Caenorhabditis elegans TK1 (CeTK1). Specifically, mutations were introduced into the hydrophobic pocket surrounding the substrate base. In CeTK1, some of these mutations led to increased activity with deoxycytidine and deoxyguanosine, two unusual substrates for TK1-like kinases. In HuTK1, mutation of T163 to S resulted in a kinase with a 140-fold lower K(m) for the antiviral nucleoside analogue 3'-azido-3'-deoxythymidine (AZT) compared with the natural substrate thymidine. The crystal structure of the T163S-mutated HuTK1 reveals a less ordered conformation of the ligand thymidine triphosphate compared with the wild-type structure but the cause of the changed specificity towards AZT is not obvious. Based on its highly increased AZT activity relative to thymidine activity this TK1 mutant could be suitable for suicide gene therapy.
authors.affiliation: Department of Science, Systems and Models, Roskilde University, Denmark. tine.skovgaard@cpr.ku.dk
authors.forename: Tine
authors.initials: T
authors.lastname: Skovgaard
authors.forename: Ulla
authors.initials: U
authors.lastname: Uhlin
authors.forename: Birgitte
authors.initials: B
authors.lastname: Munch-Petersen
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000737:chemistry / Q000378:metabolism; D017173:Caenorhabditis elegans / Q000201:enzymology* / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D006801:Humans; D007700:Kinetics; D009154:Mutation; D013379:Substrate Specificity; D013937:Thymidine Kinase / Q000737:chemistry* / Q000235:genetics*; D015215:Zidovudine / Q000737:chemistry / Q000378:metabolism
chemical_list: D000998:Antiviral Agents; D029742:Caenorhabditis elegans Proteins; D015215:Zidovudine; D013937:Thymidine Kinase; C092717:thymidine kinase 1