Thermodynamics parameters for binding of substrate-analogue determined by isothermal titration calorimetry.

Substrate-analogue, S-benzyloxycarbonyl-thiosulfonate, was titrated to the variants of acylphosphatases to obtain values of association constant (Ka) and enthalpy (ΔHb) of binding at 298 K. Free energy (ΔGb) and entropy (ΔSb) of binding were calculated by: ΔGb = −RTlnKa and TΔSb = ΔHb − ΔGb, where R is the universal gas constant. ΔΔGb, ΔΔHb, and ΔΔSb represent the changes of these thermodynamic parameters upon the removal of the salt-bridge between the active-site arginine residue and the C-terminal carboxyl group.