Probing the Peripheral Site of Human Butyrylcholinesterase

Abstract

Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) catalyze the hydrolysis of the neurotransmitter acetylcholine and, thereby, function as coregulators of cholinergic neurotransmission. For both enzymes, hydrolysis takes place near the bottom of a 20 Å deep active site gorge. A number of amino acid residues within the gorge have been identified as important in facilitating efficient catalysis and inhibitor binding. Of particular interest is the catalytic triad, consisting of serine, histidine, and glutamate residues, that mediates hydrolysis. Another site influencing the catalytic process is located above the catalytic triad toward the periphery of the active site gorge. This peripheral site (P-site) contains a number of aromatic amino acid residues as well as an aspartate residue that is able to interact with cationic substrates and guide them down the gorge to the catalytic triad. In human AChE, certain aryl residues in the vicinity of the anionic aspartate residue (D74), such as W286, have been implicated in ligand binding and have therefore been considered part of the P-site of the enzyme. The present study was undertaken to explore the P-site of human BuChE and determine whether, like AChE, aromatic side chains near the peripheral aspartate (D70) of this enzyme contribute to ligand binding. Results obtained, utilizing inhibitor competition studies and BuChE mutant species, indicate the participation of aryl residues (F329 and Y332) in the E-helix component of the BuChE active site gorge, along with the anionic aspartate residue (D70), in binding ligands to the P-site of the enzyme.

Comparison of the Binding of Reversible Inhibitors to Human Butyrylcholinesterase and Acetylcholinesterase: A Crystallographic, Kinetic and Calorimetric Study

“…To further compare the binding sites of inhibitors with AChE and BChE, we employed an inhibitor competition assay. This assay is designed to detect any ternary complex formed when two inhibitors are added to AChE or BChE simultaneously [ 15 , 23 , 34 , 41 , 42 , 43 ]. The assay is conducted with a substrate like acetylthiocholine whose second-order hydrolysis at low substrate concentrations is diffusion controlled and thus is inhibited by ligands that bind to either the A-site or the P-site [ 15 ] (see Figure 7 A below).…”

“…Since inhibitor binding site competition analysis and mutant studies were successful in mapping ligand binding to the AChE P-site, a similar approach was made to probe the BChE active site gorge [ 23 ]. Wild-type and mutant BChE species and the enzyme inhibitors ThT, propidium, edrophonium and two synthetic phenothiazine derivatives were examined, and the results indicated the participation of aryl residues (Phe329 and Tyr332) in the alpha helix (E-helix; residues 326–332) bordering the BChE active site gorge, along with the anionic aspartate residue (Asp70), in the binding of ligands to the P-site of the enzyme.…”

Novel Enzymatic Potentiometric Approaches for Surfactant Analysis

“…According to the reported results, Hyamine 1622 significantly inhibits AChE activities more than other tested surfactants in the concentration range from 0.1 to 1 mg L −1 . Cholinesterase inhibitors can bind into esteratic part of active site, anionic part of active site (α anionic site), aromatic gorge, and peripheral (or β in some sources) anionic site . The action of surfactant on ChE can be mediated through binding of the inhibitor to the catalytic site or peripheral anionic site of the enzyme .…”

Highly sensitive enzymatic potentiometric approaches for anticholinesterase drugs rugs

“…Hence, AChE is widely used as a potent recognition element for the construction of biosensors for pesticide detection [9,10]. Biosensors based on AChE as well as butyrylcholinesterase were first ELMORSY KHALED et al and peripheral (or β in some sources) anionic site [17,18]. Inhibitors binding into aromatic gorge are quite rare.…”