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.

Tomato Thymidine Kinase Is Subject to Inefficient TTP Feedback Regulation

“…Plant nucleoside kinases have so far been only poorly characterized [8,9] but here, we focused on one of them, toTK1, and characterized its feedback regulation. huTK1 is known to exist in two forms, dependent on the enzyme concentration and the presence of ATP.…”

Terapia génica citotóxica para el tratamiento del cáncer cerebral

“…La toTK tiene alta afinidad y especificidad por el azidotimidina (AZT) y muestra un efecto citotóxico potente en células de GBM humanas in vitro 43 . Las ventajas de este sistema son que AZT penetra fácilmente la barrera hematoencefálica y que toTK puede eficientemente fosforilar tanto AZT como AZT-monofosfato 44 .…”

Thymidine kinase 1 regulatory fine‐tuning through tetramer formation

“…The conversion between the active tetramer and the less‐active dimer is suggested to serve as a fine‐tuner of the TK1 activity in human cells. So far, only a couple of other eukaryotic TK1s have been examined for their oligomerization properties and not a single bacterial TK1 has been studied for a possible ATP‐dependent shift in oligomerization and kinetic properties. It is therefore unclear when during the evolutionary history such a fine‐regulatory mechanism originated.…”