Enzymes are biopolymers and their consisting monomers are amino acids. Amino acid residues can have different protonation states, depending on the residue neighbors inside enzymes. Assigning right protonation states for amino acid residues is controversial in some cases. The assignment is possible by visual hydrogen bond analysis and using programs like PROPKA [1]. However, the assigned protonation state might not be exact by these methods. Molecular dynamics (MD) simulation is a powerful method to assign protonation sates of enzyme residues. This method has been used to assign protonation states of Histidines for three enzymes [2] and protonation states of Homocitrate and nearby residues in nitrogenase [3]. The philosophy is that if an incorrect protonation state is employed, the atoms in that residue or in nearby residues will move to release steric or electrostatic clashes or to form new favorable interactions. Therefore, the root mean square deviation (RMSD) will be higher for incorrect protonation states. In this study, we performed four MD simulations to assign protonation states of the active site glutamate residues (Glu-150 and Glu-307) of β-galactosidase (PDB ID 3TTS) [4]. Glutamates inside enzymes can either be protonated (GLU) or deprotonated (GLH). Our results show that the lowest RMSD values of Glu-150 and Glu-307 belongs to the GLH protonation state.
