Acetyl- and butyrylcholinesterases (AChE, BuChE) from various species differ in their substrate specificities and sensitivities to a wide range of inhibitors, yet display conserved sequence, structure and catalytic properties. To determine features that confer these selective properties, residues 58 through 133 of recombinant human BuChE were replaced with the corresponding sequence from human AChE. The replaced region (> 60% identity) spans the Asp70 residue, important for ligand interactions, and the choline binding site, and introduces differences of charge and hydrophobicity in the outer rim and on the surface of the active site gorge. Expressed in microinjected Xenopus laevis oocytes, the resultant chimera retained the catalytic activity, substrate specificity and the Km value toward butyrylthiocholine characteristic of BuChE. Further, it did not acquire substrate inhibition, which is unique to AChE, although it lost the property of substrate activation, characteristic of BuChE. Moreover, the chimera resembled BuChE in its sensitivity to succinylcholine and physostigmine, but acquired the AChE-like sensitivity to echothiophate and iso-OMPA, and displayed an intermediate pattern of inhibition, more similar to that of AChE than of BuChE, toward bambuterol, dibucaine and BW284C51. These findings demonstrate that the exchanged residues are involved in inhibitor recognition, but not in substrate distinction and in direct catalysis. Furthermore, substrate interaction with the exchanged domain may mediate structural changes leading to substrate activation in BuChE and inhibition in AChE. The two AChE-specific aromatic tyrosine residues positioned near Asp70 within this region are hence implicated in the peripheral anionic site of cholinesterases, which is involved in the recognition of various ligands.
Chimeric human cholinesterase. Identification of interaction sites responsible for recognition of acetyl- or butyrylcholinesterase-specific ligands
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