A kinetic study of thymine 7-hydroxylase from neurospora crassa

Abstract

The steady-state kinetics of thymine 7-hydroxylase (thymine, 2-oxoglutarate dioxygenase, EC 1.14.11.6) has been investigated. Initial velocity plots were all found to be linear and intersecting. Variation in concentration of two of the substrates, when the third substrate was at a constant high or low concentration, gave initial velocity plots that conform to an ordered sequential mechanism, where thymine is the second substrate to add. With 5-carboxyuracil, which is the end product in the sequential oxygenation of thymine, a competitive inhibition pattern was observed when 2-ketoglutarate was the variable substrate. When either thymine or oxygen was the variable substrate a noncompetitive inhibition pattern was obtained. When either 2-ketoglutarate or thymine was the variable substrate the inhibition patterns observed with bicarbonate were noncompetitive. With succinate noncompetitive inhibition patterns with hyperbolic intercept replots were obtained. These results are consistent with an ordered sequential kinetic mechanism, where 2-ketoglutarate is added first, followed by thymine and oxygen, and the products are released in the order: bicarbonate, succinate, and 5-hydroxymethyluracil. The order of the two last mentioned products, however, is changed in the presence of succinate.