What question did this study set out to answer?

To determine the biochemical, chemical, and structural mechanism by which 5-(trifluoromethyl)-2'-deoxyuridine 5'-monophosphate (CF3dUMP) inhibits Lactobacillus casei thymidylate synthase.

December 1, 1994Biochemistry

Mechanism-Based Inhibition of Thymidylate Synthase by 5-(Trifluoromethyl)-2'-deoxyuridine 5'-Monophosphate

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Authors

JEJens W. EcksteinEnanta Pharmaceuticals (United States)PFPaul FosterResearch Triangle Park Foundation JFJanet Finer-MooreUniversity of California, San Francisco

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Implication

Biochemical and structural analysis demonstrates covalent trapping of thymidylate synthase by CF3dUMP in Lactobacillus casei, revealing a mechanism-based pathway for enzyme inactivation.

Key Points

To determine the biochemical, chemical, and structural mechanism by which 5-(trifluoromethyl)-2'-deoxyuridine 5'-monophosphate (CF3dUMP) inhibits Lactobacillus casei thymidylate synthase.
Treated purified L. casei thymidylate synthase with radiolabeled [2'-3H]CF3dUMP without folate cofactor, followed by proteolytic digestion and peptide sequencing.
Evaluated the chemical stability and breakdown products of the covalent enzyme-inhibitor complex in the presence of reducing agents dithiothreitol and beta-mercaptoethanol.
Solved the three-dimensional atomic structure of the enzyme-inhibitor complex using X-ray crystallography.
Catalytic nucleophilic attack by cysteine 198 at the pyrimidine C6 triggered fluoride release, inducing covalent adduct formation involving both cysteine 198 and tyrosine 146.
X-ray crystallography confirmed the direct, stable covalent attachment of the modified nucleotide inhibitor to tyrosine 146 in the active site.
Incubation with thiol-reducing agents caused decomposition of the covalent complex, allowing structural characterization of the resulting breakdown products.

Cite This Study

Eckstein et al. (1994) studied this question.

synapsesocial.com/papers/6a8e63a390e8ec90ff28dace https://doi.org/10.1021/bi00254a018

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Implication

Biochemical and structural analysis demonstrates covalent trapping of thymidylate synthase by CF3dUMP in Lactobacillus casei, revealing a mechanism-based pathway for enzyme inactivation.

Mechanism-Based Inhibition of Thymidylate Synthase by 5-(Trifluoromethyl)-2'-deoxyuridine 5'-Monophosphate

Discussion

Member takes

Implication

Key Points

Cite This Study

Mechanism-Based Inhibition of Thymidylate Synthase by 5-(Trifluoromethyl)-2'-deoxyuridine 5'-Monophosphate

Discussion

Member takes

Implication

Key Points

Cite This Study

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