Title: Three distinct domains in the cholinesterase molecule confer selectivity for acetyl- and butyrylcholinesterase inhibitors Radic Z, Pickering NA, Vellom DC, Camp S, Taylor P Ref: Biochemistry, 32:12074, 1993 : PubMed
By examining inhibitor interactions with single and multiple site-specific mutants of mouse acetylcholinesterase, we have identified three distinct domains in the cholinesterase structure that are responsible for conferring selectivity for acetyl- and butyrylcholinesterase inhibitors. The first domain is the most obvious; it defines the constraints on the acyl pocket dimensions where the side chains of F295 and F297 primarily outline this region in acetylcholinesterase. Replacement of these phenylalanine side chains with the aliphatic residues found in butyrylcholinesterase allows for the catalysis of larger substrates and accommodates butyrylcholinesterase-selective alkyl phosphates such as isoOMPA. Also, elements of substrate activation characteristic of butyrylcholinesterase are evident in the F297I mutant. Substitution of tyrosines for F295 and F297 further alters the catalytic constants. The second domain is found near the lip of the active center gorge defined by two tyrosines, Y72 and Y124, and by W286; this region appears to be critical for the selectivity of bisquaternary inhibitors, such as BW284C51. The third domain defines the site of choline binding. Herein, in addition to conserved E202 and W86, a critical tyrosine, Y337, found only in the acetylcholinesterases is responsible for sterically occluding the binding site for substituted tricyclic inhibitors such as ethopropazine. Analysis of a series of substituted acridines and phenothiazines defines the groups on the ligand and amino acid side chains in this site governing binding selectivity. Each of the three domains is defined by a cluster of aromatic residues. The two domains stabilizing the quaternary ammonium moieties each contain a negative charge, which contributes to the stabilization energy of the respective complexes.
Acetyl- and butyrylcholinesterase have 51-54% sequence identity in mammalian species; they exhibit distinct substrate and inhibitor specificities. The crystal structure of acetylcholinesterase enables one to predict folding of related esterases as well as assign residues responsible for differences in substrate specificity. These predictions were tested by expression of esterase chimeras and site-specific mutants using mouse acetylcholinesterase as a template. Chimeras of acetylcholinesterase in which the amino-terminal 174 and the carboxyl-terminal 88 amino acids have been converted to the butyrylcholinesterase sequences still exhibit acetyl-like substrate specificity. Four nonconserved amino acids which are within the central sequence and appear to surround the acyl pocket, F295, R296, F297, and V300, have been mutated alone and in combination to the corresponding residues found in butyrylcholinesterase, L286, S287, I288, and G291. The V300 and R296 mutants slightly enhance butyrylthiocholine hydrolysis while the F295 and F297 mutants, alone and in combination, confer butyrylcholinesterase character by enhancing activity to butyrylthiocholine, and diminishing activity to acetylthiocholine. The F297 mutation eliminates substrate inhibition. F295 and F297 may form a clamp around the acetoxy methyl group. They have distinctive roles in affecting catalysis of the two acylcholines and precisely control acyl ester specificity. Comparison of the susceptibilities of the chimeras and site-specific mutants to cholinesterase-specific inhibitors isoOMPA, ethopropazine, and BW284c51 suggests that inhibitor selectivity for isoOMPA is attributable to residues limiting the size of the acyl pocket, while residues in the amino-terminal domain presumably near the lip of the gorge affect BW284c51 selectivity.
