Thymidine Kinases from Human and Caenorhabditis elegans and their Active Site Mutants

Tine Skovgaard

Studenteropgave: Semesterprojekt

Abstrakt

Thymidine kinase 1 (TK1) is a deoxynucleoside kinase that catalyzes the transfer of γ-phosphate from ATP to the 5`-OH of thymidine (dThd) to form thymidine monophosphate. In humans and other mammalians TK1 is a key enzyme in the salvage of dTTP for DNA synthesis. Deoxynucleoside kinases have several useful applications, but one of the more important ones is the activation of antiviral and anticancer drugs such as the nucleoside analogs AZT (3`-azido-2`-3`-dideoxythymidine) and AraC (1-β-D-arabinofuranosylcytosine) which are used for HIV and cancer treatment, respectively. In humans there are four different deoxynucleoside kinases with overlapping specificities towards the deoxynucleosides, dThd, deoxyadenosine (dAdo), deoxyguanosine (dGuo) and deoxycytidine (dCyd). In C. elegans, however, there only seems to be a single deoxynucleoside kinase-like gene with high sequence similarity to human TK1 (HuTK1). That discovery led to the interesting question regarding the substrate specificity of the C. elegans kinase: Can C. elegans TK1 (CeTK1) phosphorylate other substrates than HuTK1 can since it is apparently the only deoxynucleoside kinase in the cell? In the present characterization of CeTK1 the enzyme is compared with HuTK1 with regard to substrate specificity, native size, activation by ATP, stability and kinetic parameters for dThd. CeTK1 is truncated in the C-terminal (CeTK1-CΔ57) in order to see how the truncation affects stability and activity, and the effects are compared to the effect of a 41 amino acid C-terminal truncation that has previously been performed in HuTK1 (HuTK1-Δ41). The truncations in the two enzymes are performed at the same position after a conserved sequence. Furthermore, a number of mutations have been performed in the active site of both HuTK1-Δ41 (M28I, M28A, L124A, T163S and T163A) and CeTK1 (M42I, L143A, S182T and S182A) in order to elucidate the effect of specific amino acids on substrate specificity and activity of the enzymes. The sequence of CeTK1 is similar to that of human TK1, but besides accepting dThd as a substrate CeTK1 also phosphorylates dGuo. In contrast to HuTK1, the CeTK1 exclusively exists as a dimer with a molecular mass of ~60 kDa even if incubated with ATP. Incubation with ATP induces a transition into a more active enzyme with a higher kcat but unchanged Km. C-terminal deletion of the enzyme results in lower catalytic efficiency and stability. All the mutants showed activity with both dThd and the nucleoside analog AZT, but most of them had lower activities than the enzymes on which the mutation had been performed. Some mutations gave broader substrate specificities, however. Especially interesting is the gain of dCyd activity with CeTK1-M42I and –L143A since dCyd is a substrate that cannot be phosphorylated by WT CeTK1 or any other known TK1. And also very interesting is the highly increased specificity of HuTK1-CΔ41-T163S towards the nucleoside analog AZT compared to the natural substrate dThd.

UddannelserMolekylærbiologi, (Bachelor/kandidatuddannelse) Bachelor el. kandidat
SprogEngelsk
Udgivelsesdato1 jun. 2006
VejledereBirgitte Munch-Petersen

Emneord

  • Caenorhabditis elegans
  • cytosolic
  • thymidine kinase
  • mutants
  • gene therapy
  • TK1