Thermodynamic analysis of the binding of 5-fluoro-2'-deoxyuridine 5'-monophosphate to thymidylate synthase over a range of temperatures.
The binding of 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) to Lactobacillus casei recombinant thymidylate synthase has been studied by isothermal titration microcalorimetry at pH 7.1 over the temperature range 16-35 degrees C. Calorimetric measurements in various buffer systems with different heats of ionization suggest that a proton uptake is involved in the binding process of the nucleotide. In the temperature range investigated, the mol protons bound/mol nucleotide increases as the temperature decreases. A model of two equal and independent sites fits well with the binding isotherms for thymidylate synthase. The binding constants, the changes in Gibbs energy, enthalpy, and entropy/site for FdUMP binding were calculated at each temperature. The results show that the binding is driven by both enthalpy and entropy contributions in the range 16-35 degrees C. The enthalpy changes become more negative as the temperature increases, with delta Cp = -170 +/- 20 J.K-1.(mol FdUMP bound)-1. The behavior of the system supports the observation that FdUMP binds to thymidylate synthase without producing profound conformational changes in the protein dimer.
García-Fuentes
L
L
Departamento de Química Física, Bioquímica y Química Inorgánica, Facultad de Ciencias Experimentales, Universidad de Almería, Spain.
Reche
P
P
López-Mayorga
O
O
Santi
D V
DV
González-Pacanowska
D
D
Barón
C
C
D001665:Binding Sites; D002151:Calorimetry; D004926:Escherichia coli / Q000235:genetics; D005468:Fluorodeoxyuridylate / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007780:Lacticaseibacillus casei / Q000201:enzymology / Q000235:genetics; D008956:Models, Chemical; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D013940:Thymidylate Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D005468:Fluorodeoxyuridylate; D013940:Thymidylate Synthase