Temperature-Dependent Enzyme-Ligand Recognition Emerges from the Coupling of Protein Dynamics and Hydration

Protein-ligand interactions are governed by a subtle interplay between binding thermodynamics, protein dynamics, and hydration. However, how these factors are coupled remains poorly understood, particularly for enzymes whose function depends on coordinated water rearrangements. Here, we investigate the binding of the fluorinated sulfonamide inhibitors 4-fluorobenzenesulfonamide (4FBS) and pentafluorobenzenesulfonamide (PFBS) to the zinc metalloenzyme bovine carbonic anhydrase (BCA) at 298 and 308 K. Binding thermodynamics were characterized by isothermal titration calorimetry (ITC), while quasi-elastic neutron scattering (QENS) was employed to probe ligand-induced changes in protein dynamics. ITC measurements in D₂O were complemented by measurements in H₂O over 288-318 K to assess temperature and solvent isotope effects. PFBS binds more strongly to BCA than 4FBS over the entire temperature range, whereas the thermodynamic parameters are similar in H₂O and D₂O. QENS-derived estimates indicate that binding of either ligand increases the conformational entropy of BCA. In contrast to the thermodynamic trends, however, the dynamic response depends strongly on the ligand: the diffusion coefficient associated with localized internal motions decreases with increasing temperature for the BCA-PFBS complex but increases for BCA-4FBS, indicating that the two ligands modulate the protein energy landscape in distinct ways. Despite their closely related structures, PFBS and 4FBS exhibit markedly different temperature-dependent effects on BCA dynamics. Our results show that subtle changes in ligand chemistry can reshape the balance between protein flexibility, hydration, and binding thermodynamics.