Does “Butyrylization” of Acetylcholinesterase through Substitution of the Six Divergent Aromatic Amino Acids in the Active Center Gorge Generate an Enzyme Mimic of Butyrylcholinesterase?

• Published in: Biochemistry (Easton) Vol. 40; no. 25; pp. 7433 - 7445
• Main Authors: Kaplan, Dana, Ordentlich, Arie, Barak, Dov, Ariel, Naomi, Kronman, Chanoch, Velan, Baruch, Shafferman, Avigdor
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 26.06.2001
• Subjects: Acetylcholinesterase - chemistry; Acetylcholinesterase - genetics; Acetylcholinesterase - metabolism; Amino Acid Substitution - genetics; Benzenaminium, 4,4'-(3-oxo-1,5-pentanediyl)bis(N,N-dimethyl-N-2-propenyl-), Dibromide - chemistry; Benzenaminium, 4,4'-(3-oxo-1,5-pentanediyl)bis(N,N-dimethyl-N-2-propenyl-), Dibromide - metabolism; Binding Sites - genetics; Butyrates - metabolism; Butyrylcholinesterase - chemistry; Butyrylcholinesterase - metabolism; Cholinesterase Inhibitors - chemistry; Cholinesterase Inhibitors - metabolism; Decamethonium Compounds - chemistry; Decamethonium Compounds - metabolism; Edrophonium - chemistry; Edrophonium - metabolism; Humans; Hydrolysis; Kinetics; Ligands; Molecular Mimicry - genetics; Mutagenesis, Site-Directed; Organophosphorus Compounds - chemistry; Organophosphorus Compounds - metabolism; Substrate Specificity - genetics; Tacrine - chemistry; Tacrine - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi010181x

The active center gorge of human acetylcholinesterase (HuAChE) is lined by 14 aromatic residues, whereas in the closely related human butyrylcholinesterase (HuBChE) 3 of the aromatic active center residues (Phe295, Phe297, Tyr337) as well as 3 of the residues at the gorge entrance (Tyr72, Tyr124, Trp286) are replaced by aliphatic amino acids. To investigate whether this structural variability can account for the reactivity differences between the two enzymes, gradual replacement of up to all of the 6 aromatic residues in HuAChE by the corresponding residues in HuBChE was carried out. The affinities 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-type HuAChE. For most of these prototypical noncovalent active center and peripheral site ligands, the hexamutant HuAChE displayed a reactivity phenotype closely resembling that of HuBChE. These results support the accepted view that the active center architectures of AChE and BChE differ mainly by the presence of a larger void space in BChE. Nevertheless, reactivity of the hexamutant HuAChE toward the substrates acetylthiocholine and butyrylthiocholine, or covalent ligands such as phosphonates and the transition state analogue m-(N,N,N-trimethylammonio)trifluoroacetophenone (TMTFA), is about 45−170-fold lower than that of HuBChE. Most of this reduction in reactivity can be related to the combined replacements of the three aromatic residues at the active center, Phe295, Phe297, and Tyr337. We propose that the hexamutant HuAChE, unlike BChE, is impaired in its capacity to accommodate certain tetrahedral species in the active center. This impairment may be related to the enhanced mobility of the catalytic histidine His447, which is observed in molecular dynamics simulations of the hexamutant and the F295L/F297V/Y337A HuAChE enzymes but not in the wild-type HuAChE.