Does "butyrylization" of acetylcholinesterase through substitution of the six divergent aromatic amino acids in the active center gorge generate an enzyme mimic of butyrylcholinesterase?

The active center gorge of human acetylcholinesterase (HuAChE) is lined by 14 aromatic residues, whereas in the closely related human butyrylcholinest erase (HuBChE) 3 of the aromatic active center residues (Phe295, Phe297, Ty r337) as well as 3 of the residues at the gorge entrance (Tyr72, Tyr124, Tr p286) are replaced by aliphatic amino acids. To investigate whether this st ructural variability can account for the reactivity differences between the two enzymes, gradual replacement of up to all of the 6 aromatic residues i n HuAChE by the corresponding residues in HuBChE was carried out. The affin ities of the hexamutant (Y72N/Y124Q/W286A/F295L/F297V/Y337A) toward tacrine , decamethonium, edrophonium, huperzine A, or BW284C51 differed by about 5- , 80-, 170-, 25000-, and 17000-fold, respectively, from those of the wild-t ype HuAChE. For most of these prototypical noncovalent active center and pe ripheral site ligands, the hexamutant HuAChE displayed a reactivity phenoty pe closely resembling that of HuBChE. These results support the accepted vi ew that the active center architectures of AChE and BChE differ mainly by t he presence of a larger void space in BChE. Nevertheless, reactivity of the hexamutant HuAChE toward the substrates acetylthiocholine and butyrylthioc holine, or covalent ligands such as phosphonates and the transition state a nalogue m-(N,N,N-trimethylammonio)trifluoro-acetophenone (TMTFA), is about 45 - 170-fold lower than that of HuBChE. Most of this reduction in reactivi ty can be related to the combined replacements of the three aromatic residu es at the active center, Phe295, Phe297, and Tyr337. We propose that the he xamutant HuAChE, unlike BChE, is impaired in its capacity to accommodate ce rtain tetrahedral species in the active center. This impairment may be rela ted to the enhanced mobility of the catalytic histidine His447, which is ob served in molecular dynamics simulations of the hexamutant and the F295L/F2 97V/Y337A HuAChE enzymes but not in the wild-type HuAChE.