Role of an invariant lysine residue in folate binding on Escherichia coli thymidylate synthase: Calorimetric and crystallographic analysis of the K48Q mutant
Por:
Arvizu-Flores A.A., Sugich-Miranda R., Arreola R., Garcia-Orozco K.D., Velazquez-Contreras E.F., Montfort W.R., Maley F., Sotelo-Mundo R.R.
Publicada:
1 ene 2008
Resumen:
Thymidylate synthase (TS) catalyzes the reductive methylation of deoxyuridine monophosphate (BUMP) 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(cal) of the K48Q mutant was 430-fold lower than wild-type TS in activity, while the Km for the (R)-stereoisomer of CH(2)THF was 300 mu M, about 30-fold larger than Km 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
Filiaciones:
Arvizu-Flores A.A.:
Aquatic Molecular Biology Laboratory, Centro de Investigación en Alimentación y Desarrollo, A.C. Hermosillo, Sonora 83000, Mexico
Sugich-Miranda R.:
Univ Sonora, Dept Invest Polimeros & Mat, Hermosillo 83000, Sonora, Mexico
Arreola R.:
Univ Nacl Autonoma Mexico, Inst Fisiol Celular, Dept Bioquim, Mexico City 04510, DF, Mexico
Garcia-Orozco K.D.:
Aquatic Molecular Biology Laboratory, Centro de Investigación en Alimentación y Desarrollo, A.C. Hermosillo, Sonora 83000, Mexico
Velazquez-Contreras E.F.:
Univ Sonora, Dept Invest Polimeros & Mat, Hermosillo 83000, Sonora, Mexico
Montfort W.R.:
Department of Biochemistry and Molecular Biophysics, The University of Arizona, Tucson, AZ 85721, United States
Maley F.:
Wadsworth Center, New York State Department of Health, Albany, NY 12201, United States
Sotelo-Mundo R.R.:
Aquatic Molecular Biology Laboratory, Centro de Investigación en Alimentación y Desarrollo, A.C. Hermosillo, Sonora 83000, Mexico
All Open Access; Green
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