Mechanism-based inactivation of thymine hydroxylase, an alpha-ketoglutarate-dependent dioxygenase, by 5-ethynyluracil.

doi:10.1021/BI00213A037

Paper

Mechanism-based inactivation of thymine hydroxylase, an alpha-ketoglutarate-dependent dioxygenase, by 5-ethynyluracil.

Published Dec 21, 1993 · Lora D. Thornburg, JoAnne Stubbe

Biochemistry

Q1 SJR score

23

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0

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Abstract

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.

In Vitro Study

Study Snapshot

5-ethynyluracil effectively inactivates thymine 7-hydroxylase, resulting in a stable covalent modification of the enzyme and a single modified amino acid, tyrosine, linked to the inhibitor in an unprecedented manner.

PopulationOlder adults (50-71 years)

Sample size24

MethodsObservational

OutcomesBody Mass Index projections

ResultsSocial networks mitigate obesity in older groups.

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Mechanism-based inactivation of thymine hydroxylase, an alpha-ketoglutarate-dependent dioxygenase, by 5-ethynyluracil.

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