Other factors may also be at play here, for instance the overall electrostatic field or subtle differences in the architecture. hBChE is slightly more similar to AChE from Torpedo californica than to hAChE (72% vs 69% sequence similarity), and tcAChE also harbors Asn in the position in question (Fig ). To quantify the role of position 331/322, we extended the analysis onto tcAChE and performed in silico mutagenesis to the opposite amino acid for all three cholinesterases. We found a consistent trend of the N>V substitution lowering the basicity of the internal Glu, and the V>N substitution raising it (Fig 5B ).