Extensive constant pH molecular dynamics (CpHMD) simulation studies have been carried out to probe pH-dependent structure and dynamics of the most efficient isoenzyme II of human carbonic anhydrase (HCA) that controls the pH in the human body. Analysis of unbiased CpHMD trajectories for pH 5-8 up to 1 µs reveals no significant deviation of the equilibrium structure and hydration at and near the active site, in close agreement with known experimental results. Fluctuations in the protonation states of several hydrophilic amino acids at the active site in the pH range studied facilitate the formation of a well-ordered water network (W1, W2, W3a, and W3b) for the rate determining proton transfer between the Zn-bound water molecule and His-64 sidechain. The latter is located at the edge of the active site cavity and is found to exhibit distinct values of pK_a in two catalytically important sidechain conformations. Enhanced sampling of the fluctuating conformational and protonation states of His-64 sidechain through replica exchange CpHMD simulations leads to a quantitative agreement of its estimated conformation dependent pK_a values to those measured in recent NMR based studies. We also present a new method to promote sampling of relatively rare fluctuations in the protonation states of residues such as Tyr that, when present in the enzyme, are known to have large shifts in their known high values of solution pK_a.

Full titration curves of His-64 sidechain in its inward and outward orientations computed from replica exchange constant pH MD simulation of HCA II in water

Dulal Mondal

Appearances