Thermodynamics–Structure Correlations of Sulfonamide Inhibitor Binding to Carbonic Anhydrase

Microcalorimetry is an excellent method of determining the thermodynamics of ligand–protein binding in solution, because the signal being measured is the observed binding enthalpy (Db-obsH [Editors’ note: this term can also be written DHobs or DbHobs as seen elsewhere in this book]), which is free of any model assumptions. The affinity (DbG) can be computed from the shape of a binding curve, and the binding entropy (DbS) is obtained from the difference between the two. The calorimetrically observed binding enthalpy, however, can be the sum of several partial reactions, including changes in the protonation of ligand, protein, and buffer, each of which may have its own contribution to the observed enthalpy of binding. Which enthalpy is the ‘true’ enthalpy of a reaction? In order to obtain an enthalpy of binding that can be correlated with structure, one must elucidate the thermodynamics for all reactions that occur simultaneously upon ligand binding. It is fairly straightforward to distinguish net protonation effects by measuring Db-obsH in buffers with varying ionization enthalpies,1 and extrapolating the value to zero Dbuffer-protonationH. This process simultaneously gives the net protonation effect for all remaining linked reactions. However, a thorough deconvolution of the energetics of the remaining linked reactions remains complex.