Figure 1. The key hydrogen bond network in (A) the crystal structure of BChE (PDB ID 4BDS), and in (B) the crystal structure of AChE (PDB ID 4EY4). The oxygen and nitrogen atoms are rendered with red and blue colors, respectively. The carbon atoms in BChE and AChE are rendered with cyan and green colors, respectively. The black dashed lines indicate hydrogen bonds, and the red spheres at the center are the water molecules.

In the crystal structure of AChE, as we can see from Figure 1B, such a key hydrogen bond network also exists, and it is highly similar to that in BChE. Therefore, just as that in BChE, the Glu202 in AChE is very likely to be protonated in order to join and stabilize the key hydrogen bond network. Interestingly, this hydrogen bond network has been pointed out to be critical in stabilizing the active center, but the protonation state of Glu202 was not mentioned.¹⁵ Thus, it is of great interest to know what protonation state is adopted by Glu202 in stabilizing the key hydrogen bond network, and what consequence is if Glu202 adopts a different protonation state. In the present work, we carried out a series of molecular dynamics simulations to investigate the dynamical behaviors of the key hydrogen bond network with the Glu202 adopting different protonation states. Their effects on the stability of catalytic His447 are also examined.