Studies on the partial reaction of thymine 7-hydroxylase in the presence of 5-fluorouracil.

The uncoupling of 2-oxoglutarate decarboxylation from hydroxylation in the reaction catalyzed by thymine 7-hydroxylase (thymine, 2-oxoglutarate:oxygen oxidoreductase (7-hydroxylating), EC 1.14.11.6) in the presence of 5-fluorouracil has been studied. In the complete reaction no external reductant is formally needed. The uncoupled reaction is almost negligible in the absence of ascorbate and the optimal ascorbate concentration is 5-times higher than in the presence of a hydroxylatable substrate. This indicates that ascorbate acts as the external reductant that is formally needed in the catalytic cycle. The complete reaction follows the steady-state kinetics of an ordered ter reactant mechanism where 2-oxoglutarate and thymine have to be bound to the enzyme before oxygen (E. Holme (1975) Biochemistry 14, 4999-5003). The uncoupled reaction follows the same kinetic pattern as the complete reaction, and in accordance with this no decarboxylation of 2-oxoglutarate occurs in the absence of a substrate analogue even at elevated oxygen tension. There is a good agreement between Kia values for 2-oxoglutarate of the two reactions, but there is at least a 6-fold increase in KO2 where a minimum value of 25% O2 in the gas phase was found for the partial reaction. The high KO2 found means that the reaction rate could increase considerably at elevated oxygen tension.