Spectroscopic Analysis of Folate Binding to Thymidylate Synthase Active Site |
| |
| Authors: | Aldo A. Arvizu-Flores Enrique F. Velázquez-Contreras Lorena Machi Rodrigo Arreola Frank Maley |
| |
| Affiliation: | 1. Aquatic Molecular Biology Laboratory, Centro de Investigación en Alimentación y Desarrollo, A.C. , Hermosillo, Sonora, Mexico;2. Departamento de Investigación en Polímeros y Materiales , Universidad de Sonora , Hermosillo, Sonora, Mexico;3. Department of Biotechnology and Bioengineering , Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN) , Mexico City, Mexico;4. Wadsworth Center, New York State Department of Health, Albany , New York, USA |
| |
| Abstract: | 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. |
| |
| Keywords: | antifolate binding mutant K48Q protein fluorescence thymidylate synthase |
|
|
