TY - JOUR
T1 - Spectroscopic analysis of folate binding to thymidylate synthase active site
AU - Arvizu-Flores, Aldo A.
AU - Velazquez-Contreras, Enrique F.
AU - Machi, Lorena
AU - Arreola, Rodrigo
AU - Maley, Frank
AU - Sotelo-Mundo, Rogerio R.
N1 - 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.
PY - 2009/4
Y1 - 2009/4
N2 - 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.
AB - 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.
KW - Antifolate binding
KW - Mutant K48Q
KW - Protein fluorescence
KW - Thymidylate synthase
UR - http://www.scopus.com/inward/record.url?scp=67651065730&partnerID=8YFLogxK
U2 - 10.1080/00387010902768763
DO - 10.1080/00387010902768763
M3 - Artículo
SN - 0038-7010
VL - 42
SP - 142
EP - 146
JO - Spectroscopy Letters
JF - Spectroscopy Letters
IS - 3
ER -