Replacement of both the acyl pocket residue Phe295 as well as residue Phe338, adjacent to the catalytic His447 in human acetylcholinesterase (HuAChE), resulted in a 680-fold decline in catalytic activity due to conformational destabilization of the histidine side chain [Barak et al. (2002) Biochemistry 41, 8245]. A possible restriction of this catalytically nonproductive mobility of His447 in a series of F295X/F338A HuAChEs was examined in silico followed by site-directed mutagenesis. Simulations suggested that of the 12 aliphatic residues substituted at position 295, including hydrophobic and polar amino acids, only methionine was capable of maintaining the catalytically viable conformation of His447. Examination of the reactivities of the actual F295X/F338A HuAChEs showed that indeed the F295M/F338A enzyme was only 2-fold less reactive than the F338A mutant toward acetylthiocholine, while enzymes substituted by the similarly bulky residues leucine and isoleucine were catalytically impaired. Furthermore, only the F295M/F338A enzyme exhibited wild-type-like reactivity toward covalent modifiers of the catalytic Ser203 including the methylphosphonate soman and transition state analogue m-(N,N,N-trimethylammonio)trifluoroacetophenone (TMTFA), as well as a facile dealkylation of the F295M/F338A-soman adduct. A different behavior was observed for bulkier ligands which introduce a deformation in the acyl pocket, and therefore their activity seems only marginally affected by the positioning of His447. The findings emphasize the importance of the precise positioning of His447 for catalysis and indicate that, in the absence of aromatic "trapping", restriction of the histidine mobility in F295X/F338A HuAChEs requires a combination of steric interference and a specific polar interaction. The results also underscore the role of the acyl pocket subsite of cholinesterases in maintaining the catalytically viable conformation of the catalytic histidine.