MECHANISM-BASED INACTIVATION OF THYMINE HYDROXYLASE, AN ALPHA-KETOGLUTARATE-DEPENDENT DIOXYGENASE, BY 5-ETHYNYLURACIL

5-Ethynyluracil was shown to be a mechanism-based inactivator of thymi ne 7-hydroxylase, with K(i) = 22 muM and a k2 = 2.6 min-1. Inactivatio n resulted in covalent modification of the enzyme with a stoichiometry of approximately 1 adduct/enzyme molecule. The reaction of thymine 7- hydroxylase with 5-ethynyluracil also generated two products: 5-carbox yuracil and uracil-5-acetylglycine. The enzyme adduct was stable at pH 2, 8, and 10 and stable to treatment with hydroxylamine. Following tr ypsin digestion of labeled enzyme, two labeled peptides corresponding to 45% of the adduct were isolated and sequenced. The results demonstr ated the presence of a single modified amino acid. Tandem mass spectro metry suggested that the modified amino acid is tyrosine, which is lin ked to the inhibitor in an unprecedented fashion.