Role of an invariant lysine residue in folate binding on Escherichia coli thymidylate synthase: calorimetric and crystallographic analysis of the K48Q mutant.

The International Journal of Biochemistry & Cell Biology
Aldo A Arvizu-FloresRogerio R Sotelo-Mundo

Abstract

Thymidylate synthase (TS) catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) using methylene tetrahydrofolate (CH(2)THF) as cofactor, the glutamate tail of which forms a water-mediated hydrogen bond with an invariant lysine residue of this enzyme. To understand the role of this interaction, we studied the K48Q mutant of Escherichia coli TS using structural and biophysical methods. The k(cat) of the K48Q mutant was 430-fold lower than wild-type TS in activity, while the K(m) for the (R)-stereoisomer of CH(2)THF was 300 microM, about 30-fold larger than K(m) from the wild-type TS. Affinity constants were determined using isothermal titration calorimetry, which showed that binding was reduced by one order of magnitude for folate-like TS inhibitors, such as propargyl-dideazafolate (PDDF) or compounds that distort the TS active site like BW1843U89 (U89). The crystal structure of the K48Q-dUMP complex revealed that dUMP binding is not impaired in the mutant, and that U89 in a ternary complex of K48Q-nucleotide-U89 was bound in the active site with subtle differences relative to comparable wild-type complexes. PDDF failed to form ternary complexes with K48Q and dUMP. Thermodynamic data correlated with the structu...Continue Reading

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Citations

Jun 30, 2009·Comparative Biochemistry and Physiology. Toxicology & Pharmacology : CBP·Aldo A Arvizu-FloresRogerio R Sotelo-Mundo
Oct 31, 2015·Journal of the American Chemical Society·Paul J SapienzaAndrew L Lee
Mar 10, 2010·Journal of Molecular Recognition : JMR·Robert J FalconerBrett M Collins
Apr 28, 2019·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Cecilia PozziStefano Mangani

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