Journal article
Mg2+ binds to the surface of thymidylate synthase and affects hydride transfer at the interior active site
Journal of the American Chemical Society, Vol.135(20), pp.7583-7592
05/22/2013
DOI: 10.1021/ja400761x
PMCID: PMC3674108
PMID: 23611499
Abstract
Thymidylate synthase (TSase) produces the sole intracellular
de novo
source of thymidine (i.e. the DNA base T) and thus is a common target for antibiotic and anticancer drugs. Mg
2+
has been reported to affect TSase activity, but the mechanism of this interaction has not been investigated. Here we show that Mg
2+
binds to the surface of
Escherichia coli
TSase and affects the kinetics of hydride transfer at the interior active site (16 Å away). Examination of the crystal structures identifies a Mg
2+
near the glutamyl moiety of the folate cofactor, providing the first structural evidence for Mg
2+
binding to TSase. The kinetics and NMR relaxation experiments suggest that the weak binding of Mg
2+
to the protein surface stabilizes the closed conformation of the ternary enzyme complex and reduces the entropy of activation on the hydride transfer step. Mg
2+
accelerates the hydride transfer by
ca
. 7-fold but does not affect the magnitude or temperature-dependence of the intrinsic kinetic isotope effect. These results suggest that Mg
2+
facilitates the protein motions that bring the hydride donor and acceptor together, but it does not change the tunneling ready state of the hydride transfer. These findings highlight how variations in cellular Mg
2+
concentration can modulate enzyme activity through long-range interactions in the protein, rather than binding at the active site. The interaction of Mg
2+
with the glutamyl-tail of the folate cofactor and nonconserved residues of bacterial TSase may assist in designing antifolates with poly-glutamyl substitutes as species-specific antibiotic drugs.
Details
- Title: Subtitle
- Mg2+ binds to the surface of thymidylate synthase and affects hydride transfer at the interior active site
- Creators
- Zhen Wang - University of IowaPaul J Sapienza - University of North Carolina at Chapel HillThelma Abeysinghe - University of IowaCalvin Luzum - University of IowaAndrew L Lee - University of North Carolina at Chapel HillJanet S Finer-Moore - University of California, San FranciscoRobert M Stroud - University of California, BerkeleyAmnon Kohen - University of Iowa
- Resource Type
- Journal article
- Publication Details
- Journal of the American Chemical Society, Vol.135(20), pp.7583-7592
- DOI
- 10.1021/ja400761x
- PMID
- 23611499
- PMCID
- PMC3674108
- NLM abbreviation
- J Am Chem Soc
- ISSN
- 0002-7863
- eISSN
- 1520-5126
- Grant note
- R01 GM065368 || GM / National Institute of General Medical Sciences : NIGMS
- Language
- English
- Date published
- 05/22/2013
- Academic Unit
- Chemistry
- Record Identifier
- 9984201427202771
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