Enhanced Sampling and Conformation-Dependent pKa of Histidine Side Chains: A Case Study in Human Carbonic Anhydrase II

An extensive constant pH replica-exchange molecular dynamics (CpH-REMD) simulation study on the enzyme human carbonic anhydrase (HCA) II has been carried out to compute the pK_a of the side chains of eight histidine residues with varying degrees of solvent exposure and relevance in the catalytic function. First, the estimates of total pK_a of four noncatalytic His side chains accurately reproduce their experimental values [Fisher, S. Z.; Raum, H. N.; Weininger, U. ChemBioChem 2025, 26, e202400930]. The observed deviations in the case of three noncatalytic His side chains are attributed to the preferential sampling of one of the possible protonation states dynamically stabilized by π–π stacking along with cation–π, salt bridge, and hydrogen bonding interactions. We also report the emergent concept of conformation-dependent side chain pK_a of both catalytically important and noncatalytic His residues. The estimated pK_as of two different conformations of the catalytically important His-64 side chain quantitatively match their values measured in recent NMR studies [Raum, H. N.; Fisher, S. Z.; Weininger, U. Cell. Mol. Life Sci. 2023, 80, 286]. The noncatalytic histidine residues also exhibit multiple conformations associated with significantly different values of pK_a. Our results indicate the importance of devising new methods to enhance the sampling of long time scales introduced by local noncovalent interactions and long-range cooperative fluctuations of ionizable side chain conformations.