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 thymine 7-hydroxylase, with Ki = 22 microM and a k2 = 2.6 min-1l Inactivation 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-carboxyuracil and uracil-5-acetylglycine. The enzyme adduct was stable at pH 2, 8, and 10 and stable to treatment with hydroxylamine. Following trypsin digestion of labeled enzyme, two labeled peptides corresponding to 45% of the adduct were isolated and sequenced. The results demonstrated the presence of a single modified amino acid. Tandem mass spectrometry suggested that the modified amino acid is tyrosine, which is linked to the inhibitor in an unprecedented fashion.
Thornburg
L D
LD
Department of Biochemistry, University of Wisconsin-Madison 53706.
Stubbe
J
J
D000595:Amino Acid Sequence; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D010449:Peptide Mapping; D014357:Trypsin; D014498:Uracil / Q000031:analogs & derivatives* / Q000494:pharmacology
D007656:Ketoglutaric Acids; D010446:Peptide Fragments; C073482:eniluracil; D014498:Uracil; D006899:Mixed Function Oxygenases; C023164:thymine, 2-oxoglutarate dioxygenase; D014357:Trypsin