Spectroscopic analysis of folate binding to thymidylate synthase active site

Aldo A. Arvizu-Flores, Enrique F. Velazquez-Contreras, Lorena Machi, Rodrigo Arreola, Frank Maley, Rogerio R. Sotelo-Mundo

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The fluorescence properties of folate binding to thymidylate synthase (TS) were analyzed. Two antifolates with different binding modes to the TS active site were the ligands. Intrinsic tryptophan fluorescence was used to evaluate the binding of both antifolates to the wild-type TS and a mutant Escherichia coli TS (K48Q) that is impaired in folate binding. During titration of wild-type TS with PDDF, tryptophan fluorescence was quenched at 330 nm, which was accompanied by an increase in emission at 379 nm, suggesting an energy transfer process from a tryptophan in the TS active site to the folate analogue. Energy transfer was not observed with the mutant TS, as expected. Tryptophan emission is a very useful tool to test for substrate-like inhibitors with biological activity.

Original languageEnglish
Pages (from-to)142-146
Number of pages5
JournalSpectroscopy Letters
Issue number3
StatePublished - Apr 2009

Bibliographical note

Funding Information:
This work was supported by Consejo Nacional de Ciencia y Tecnología (CONACYT) grant 48991-Z (R.R.S.). A. Arvizu-Flores received a graduate scholarship from CONACYT. We thank Drs. Gloria Yepiz-Plascencia and Maria Islas-Osuna for critical reading and suggestions.


  • Antifolate binding
  • Mutant K48Q
  • Protein fluorescence
  • Thymidylate synthase


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