Thermodynamic characterization of the binding of dCMP to the Asn229Asp mutant of thymidylate synthase.
Isothermal titration microcalorimetry and equilibrium dialysis have been used to characterize the binding of 2'-deoxycytidine 5'-monophosphate (dCMP) to the Asn229Asp mutant of Lactobacillus casei recombinant thymidylate synthase at pH 7.4 over a temperature range of 15 degrees C to 35 degrees C. Equilibrium dialysis analysis shows that dCMP binds to two sites in the dimer of both wild-type and mutant thymidylate synthase. A concomitant net uptake of protons with binding of dCMP to both enzymes, was detected carrying out calorimetric experiments in various buffer systems with different heats of ionization. The change in protonation for binding of dCMP to wild-type enzyme is lower than that obtained for binding of this nucleotide to TS N229D, which suggests that the pK value of Asp-229 is increased upon dCMP binding to the mutant enzyme. At 25 degrees C, although the binding of dCMP to wild-type and N229D TS is favoured by both enthalpy and entropy changes, the enthalpy change is more negative for the mutant protein. Thus, the substitution of Asn 229 for Asp results in a higher affinity of TS for dCMP due to a more favourable enthalpic contribution. The Gibbs energy change of binding of dCMP to the mutant enzyme is weakly temperature-dependent, because of the enthalpy-entropy compensation arising from a negative heat capacity change of binding equal to -0.83 +/- 0.02 kJ K(-1) per mol of dCMP bound.
Téllez-Sanz
R
R
Departamento de Química Fisica, Bioquímica y Química Inorgánica, Facultad de Ciencias Experimentales, Universidad de Almería, La Cañada de San Urbano, Spain.
Bernier-Villamor
V
V
García-Fuentes
L
L
González-Pacanowska
D
D
Barón
C
C
D001216:Asparagine / Q000737:chemistry / Q000235:genetics; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics; D002151:Calorimetry; D003843:Deoxycytidine Monophosphate / Q000737:chemistry*; D003956:Dialysis; D009154:Mutation; D011485:Protein Binding / Q000235:genetics; D013696:Temperature; D013816:Thermodynamics; D013940:Thymidylate Synthase / Q000737:chemistry / Q000235:genetics*
D003843:Deoxycytidine Monophosphate; D001224:Aspartic Acid; D001216:Asparagine; D013940:Thymidylate Synthase