THERMODYNAMIC CHARACTERIZATION OF THE BINDING OF DCMP TO THE ASN229ASP MUTANT OF THYMIDYLATE SYNTHASE

Isothermal titration microcalorimetry and equilibrium dialysis have be en used to characterize the binding of 2'-deoxycytidine 5'-monophospha te (dCMP) to the Asn229Asp mutant of Lactobacillus casei recombinant t hymidylate 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 syn thase, A concomitant net uptake of protons with binding of dCMP to bot h enzymes, was detected carrying out calorimetric experiments In vario us buffer systems with different heats of ionization, The change in pr otonation for binding of dCMP to wild-type enzyme is lower than that o btained for binding of this nucleotide to TS N229D, which suggests tha t 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 an d N229D TS is favoured by both enthalpy and entropy changes, the entha lpy change is more negative for the mutant protein, Thus, the substitu tion of Asn 229 for Asp results in a higher affinity of TS for dCMP du e to a more favourable enthalpic contribution, The Gibbs energy change of binding of dCMP to the mutant enzyme is weakly temperature-depende nt, because of the enthalpy-entropy compensation arising from a negati ve heat capacity change of binding equal to -0.83 +/- 0.02 kJ K-1 per mol of dCMP bound. (C) 1997 Federation of European Biochemical Societi es.