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 structurefunction 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 Km 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.

Keywords

crystal structure; enzyme kinetics; mutagenesis; nucleoside analogue; thymidine kinase 1

Published in

FEBS Journal
2012, Volume: 279, number: 10, pages: 1777-1787
Publisher: WILEY-BLACKWELL

SLU Authors

• Uhlin, Ulla

  • Department of Molecular Biology, Swedish University of Agricultural Sciences
    

Sustainable Development Goals

Ensure healthy lives and promote well-being for all at all ages


UKÄ Subject classification

Biochemistry and Molecular Biology


Publication identifier

DOI: https://doi.org/10.1111/j.1742-4658.2012.08554.x

Permanent link to this page (URI)

https://res.slu.se/id/publ/45615