Characterization of Oligomeric and Kinetic Properties of Tomato Thymidine Kinase 1

Abstract

The gene encoding thymidine kinase 1 from tomato (toTK1) has in combination with azidothymidine (AZT) recently been proposed as a powerful suicide gene for anticancer gene therapy. The toTK1/AZT combination has been demonstrated to have several advantages for the treatment of glioblastomas because AZT can easily penetrate the blood–brain barrier and toTK1 can efficiently phosphorylate AZT and also AZT-monophosphate. In a pursuit to further understand the properties of toTK1, we examined the oligomerization properties of recombinant toTK1 and its effect on enzyme kinetics. Previously, it has been shown that human TK1 is a dimer in the absence of ATP and a tetramer if preincubated with ATP. However, we show here that ATP preincubation did not result in a structural shift from dimer to tetramer in toTK1. For human TK1 pretreated with ATP, the K_m value decreased 20-fold, but toTK1's K_m value did not show a dependence on the presence or absence of ATP. Furthermore, toTK1 was always found in a highly active form.

Keywords:

• Thymidine kinase 1
• tomato (Solanum lycopersicum)
• oligomerization
• ATP activation
• enzyme kinetics

Notes

^a Kinetics values for hTK1 are from ref. [3].