Catalytic Site pK_a Values of Aspartic, Cysteine, and Serine Proteases: Constant pH MD Simulations

Enzymatic function and activity of proteases is closely controlled by the pH value. The protonation states of titratable residues in the active site react to changes in the pH value, according to their pK_a, and thereby determine the functionality of the enzyme. Knowledge of the titration behavior of these residues is crucial for the development of drugs targeting the active site residues. However, experimental pK_a data are scarce, since the systems' size and complexity make determination of these pK_a values inherently difficult. In this study, we use single pH constant pH MD simulations as a fast and robust tool to estimate the active site pK_a values of a set of aspartic, cysteine, and serine proteases. We capture characteristic pK_a shifts of the active site residues, which dictate the experimentally determined activity profiles of the respective protease family. We find clear differences of active site pK_a values within the respective families, which closely match the experimentally determined pH preferences of the respective proteases. These shifts are caused by a distinct network of electrostatic interactions characteristic for each protease family. While we find convincing agreement with experimental data for serine and aspartic proteases, we observe clear deficiencies in the description of the titration behavior of cysteines within the constant pH MD framework and highlight opportunities for improvement. Consequently, with this work, we provide a concise set of active site pK_a values of aspartic and serine proteases, which could serve as reference for future theoretical as well as experimental studies.