Enhanced Sampling and Conformation-Dependent p<i>K</i>_a 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 p<i>K</i>_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 p<i>K</i>_a of four noncatalytic His side chains accurately reproduce their experimental values [Fisher, S. Z.; Raum, H. N.; Weininger, U. <i>ChemBioChem</i> <b>2025,</b> <i>26,</i> 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 p<i>K</i>_a of both catalytically important and noncatalytic His residues. The estimated p<i>K</i>_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. <i>Cell. Mol. Life Sci.</i> <b>2023,</b> <i>80,</i> 286]. The noncatalytic histidine residues also exhibit multiple conformations associated with significantly different values of p<i>K</i>_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.