pH Regulates Ligand Binding to an Enzyme Active Site by Modulating Intermediate Populations.

Understanding the mechanism of ligands binding to their protein targets and the influence of various factors governing the binding thermodynamics is essential for rational drug design. The solution pH is one of the critical factors that can influence ligand binding to a protein cavity, especially in enzymes whose function is sensitive to the pH. Using computer simulations, we studied the pH effect on the binding of a guanidinium ion (Gdm) to the active site of hen egg-white lysozyme (HEWL). HEWL serves as a model system for enzymes with two acidic residues in the active site and ligands with Gdm moieties, which can bind to the active sites of such enzymes and are present in several approved drugs treating various disorders. The computed free energy surface (FES) shows that Gdm binds to the HEWL active site using two dominant binding pathways populating multiple intermediates. We show that the residues close to the active site that can anchor the ligand could play a critical role in ligand binding. Using a Markov state model, we quantified the lifetimes and kinetic pathways connecting the different states in the FES. The protonation and deprotonation of the acidic residues in the active site in response to the pH change strongly influence the Gdm binding. There is a sharp jump in the ligand-binding rate constant when the pH approaches the largest p of the acidic residue present in the active site. The simulations reveal that, at most, three Gdm can bind at the active site, with the Gdm bound in the cavity of the active site acting as a scaffold for the other two Gdm ions binding. These results can aid in providing greater insights into designing novel molecules containing Gdm moieties that can have high binding affinities to inhibit the function of enzymes with acidic residues in their active site.