Modification of Escherichia coli Thymidylate Synthase at Tyrosine-94 by 5-Imidazolylpropynyl-2‘-deoxyuridine 5‘-Monophosphate
| Autor: | James T. Reston, Zhe Nie, Ruth L. Saxl, Thomas I. Kalman, Frank Maley |
|---|---|
| Rok vydání: | 2003 |
| Předmět: |
chemistry.chemical_classification
Spectrometry Mass Electrospray Ionization biology Stereochemistry Proteolytic enzymes Active site Peptide Thymidylate Synthase Trypsin Biochemistry Thymidylate synthase Cofactor Kinetics chemistry Escherichia coli biology.protein medicine Nucleic acid Tyrosine Nucleotide Deoxyuracil Nucleotides Chromatography High Pressure Liquid medicine.drug |
| Zdroj: | Biochemistry. 42:4544-4551 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi0268089 |
| Popis: | Evidence is presented that 5-imidazolylpropynyl-2'-deoxyuridine 5'-monophosphate (IP-dUMP) is a mechanism-based, irreversible inactivator of Escherichia coli thymidylate synthase (TS), which covalently modifies Tyr94 at the active site of the enzyme. The inactivation of TS was time and concentration dependent and did not require the folate cofactor. Due to the rapidity of the inactivation process, accurate kinetic parameters could be determined only in the presence of saturating concentrations (1000K(M)) of the competing substrate, dUMP. Under these conditions, a K(I) of 0.36 +/- 0.09 microM and an inactivation rate constant (k(inact)) of 0.53 +/- 0.15 min(-1) were obtained from Kitz-Wilson plots. Electrospray ionization-mass spectrometry (ESI-MS) determined a 412 amu mass increase of TS after inhibition by IP-dUMP with no mass difference being detected for the TS mutants Tyr94Phe or Cys146Ala, thus indicating the importance of these residues for complex formation. The change in WT-TS mass was consistent with covalent modification by IP-dUMP, which was confirmed by proteolytic digestion of the modified protein followed by ESI-MS. By these means, a 43-residue trypsin peptide (residues 54-96), a 16-residue endoAspN peptide (residues 89-104), and an 8-residue endoAspN/endoLysC peptide (residues 89-96), each containing the IP-dUMP adduct, were observed. MS/MS analysis of the IP-dUMP-endoAspN peptide identified a modified 3-residue daughter ion, YGK (residues 94-96). A mechanistic scheme requiring the participation of Cys146 is proposed for the covalent modification of IP-dUMP by Tyr94, which, unlike an earlier proposal [Kalman, T. I., Nie, Z., and Kamat, A. (2001) Nucleosides Nucleotides Nucleic Acids 20, 869-871], does not require the release of imidazole for the activation of the inhibitor. |
| Databáze: | OpenAIRE |
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