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

doi:10.1080/00387010902768763

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

Departamento de Ciencias Químico-Biológicas

Research output: Contribution to journal › Article › peer-review

1 Scopus citations

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.

Original language	English
Pages (from-to)	142-146
Number of pages	5
Journal	Spectroscopy Letters
Volume	42
Issue number	3
DOIs	https://doi.org/10.1080/00387010902768763
State	Published - 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.

Keywords

Antifolate binding
Mutant K48Q
Protein fluorescence
Thymidylate synthase

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10.1080/00387010902768763

Cite this

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title = "Spectroscopic analysis of folate binding to thymidylate synthase active site",

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",

author = "Arvizu-Flores, {Aldo A.} and Velazquez-Contreras, {Enrique F.} and Lorena Machi and Rodrigo Arreola and Frank Maley and Sotelo-Mundo, {Rogerio R.}",

note = "Funding Information: This work was supported by Consejo Nacional de Ciencia y Tecnolog{\'i}a (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.",

year = "2009",

month = apr,

doi = "10.1080/00387010902768763",

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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

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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 -

Spectroscopic analysis of folate binding to thymidylate synthase active site

Abstract

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Keywords

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