Reversible ATP-dependent transition between two forms of human cytosolic thymidine kinase with different enzymatic properties.

Human cytosolic thymidine kinase, subunit molecular mass about 24 kDa, is a tetramer in the presence of ATP but a dimer in the presence of thymidine or without substrates. The pure, substrate-free enzyme showed complex, non-hyperbolic thymidine substrate kinetics with an apparent Km of 15 microM. Incubation with ATP at 4 degrees C induced a time-dependent transition to an enzyme form with hyperbolic kinetics and a 20-fold lower Km value for thymidine (0.7 microM) but the same maximal velocity as for cytosolic thymidine kinase (TK1) without ATP. Removal of the ATP by carboxymethyl chromatography reestablished the non-hyperbolic kinetics with the low affinity for thymidine (Km(app) = 12 microM), and this enzyme form could be reversed once more by ATP incubation to the high affinity enzyme form. Similar shifts could not be induced by thymidine. The activating effect of ATP depended on the concentration of enzyme protein in a linear manner. These results indicate that ATP is a positive effector of cytosolic thymidine kinase, controlling a kinetically slow transition between two molecular forms of the enzyme. A hypothetical reaction mechanism is presented to explain the complex kinetic behavior.