Binding Thermodynamics of Phosphorylated Inhibitors to Triosephosphate Isomerase and the Contribution of Electrostatic Interactions

Electrostatic interactions have a central role in some biological processes, such as recognition of charged ligands by proteins. We characterized the binding energetics of yeast triosephosphate isomerase (TIM) with phosphorylated inhibitors 2-phosphoglycollate (2PG) and phosphoglycolohydroxamate (PGH). We determined the thermodynamic parameters of the binding process (Kb, ΔGb, ΔHb, ΔSb and ΔCp) with different concentrations of NaCl, using fluorimetric and calorimetric titrations in the conventional mode of ITC and a novel method, multithermal titration calorimetry (MTC), which enabled us to measure ΔCp in a single experiment. We ruled out specific interactions of Na+ and Cl– with the native enzyme and did not detect significant linked protonation effects upon the binding of inhibitors. Increasing ionic strength (I) caused Kb, ΔGb and ΔHb to become less favorable, while ΔSb became less unfavorable. From the variation of Kb with I, we determined the electrostatic contribution of TIM−2PG and TIM−PGH to ΔGb at I = 0.06 M and 25 °C to be 36% and 26%, respectively. The greater affinity of PGH for TIM is due to a more favorable ΔHb compared to 2PG (by 19–24 kJ mol-1 at 25 °C). This difference is compatible with PGH establishing up to five more hydrogen bonds with TIM. Both binding ΔCps were negative, and less negative with increasing ionic strength. ΔCps at I = 0.06 M were much more negative than predicted by surface area models. Water molecules trapped in the interface when ligands bind to protein could explain the highly negative ΔCps. Thermodynamic binding functions for TIM−2PG changed more with ionic strength than those for TIM−PGH. This greater dependence is consistent with linked, but compensated, protonation equilibriums yielding the dianionic species of 2PG that binds to TIM, process that is not required for PGH.