Properties of most enzymes strongly depend on electrostatic interactions, both within the active site and between the groups of the active site and the surrounding protein. Such interactions crucially depend on the protonation state of the various residues, i.e., whether they are charged or not. Protonation or deprotonation of titratable groups can cause changes in binding affinities, enzymatic activities, and structural properties. Very often, protonation or deprotonation are the key events in enzymatic reactions. In this work, we have studied the protonation states of several groups in Escherichia Coli Glyoxalase I (GlxI). There are five residues in which the protonation-state assignment was not fully conclusive, two histidines (His-5, and -74), two glutamates (Glu-56, and -122), and one aspartate (Asp-115). We have performed molecular dynamics (MD) simulations to determine the most stable protonation states of these (besides the standard procedure of studying the surroundings, the solvent accessibility, and the H-bond network around the residue). MD simulations have been run on a 1.50 Å crystal structure of GlxI (Protein Data Bank entry 1F9Z). The entire enzyme was included in the calculations. The protein is a dimer, and the two subunits were treated the same way. The enzyme was solvated in a periodic truncated octahedral box of TIP3P water molecules, extending at least 10 Å from the solute using the leap program in the Amber suite. The final system contained ~30 000 atoms. After the solvation, we minimized the structure followed by a 500 ns production simulation, during which coordinates were sampled every 10 ps. The root-mean-square deviation (RMSD) from the starting crystal structure as has been done before for His residues in three proteins, titratable residues in the active site of myrosinase, and homocitrate and nearby residues in nitrogenase. It is possible to determine which protonation state is more realistic based on the RMSD from the crystal structure. It is believed that if an incorrect protonation state is used, the atoms in that residue or nearby residues will move to reduce steric or electrostatic clashes or to form new favorable interactions. Therefore, the RMSD will be higher in the case of incorrect protonation states (assuming the reference structure is representative of the studied state). The results show that His-5 and -74 are protonated on the ND1 atom, not on the NE2 atom, Glu-56 and -122 and Asp-115 are deprotonated.
