Dissection of the human acetylcholinesterase active center determinants of substrate specificity. Identification of residues constituting the anionic site, the hydrophobic site, and the acyl pocket.
ABSTRACT Substrate specificity determinants of human acetylcholinesterase (HuAChE) were identified by combination of molecular modeling and kinetic studies with enzymes mutated in residues Trp-86, Trp-286, Phe-295, Phe-297, Tyr-337, and Phe-338. The substitution of Trp-86 by alanine resulted in a 660-fold decrease in affinity for acetythiocholine but had no effect on affinity for the isosteric uncharged substrate (3,3-dimethylbutylthioacetate). The results demonstrate that residue Trp-86 is the anionic site which binds, through cation-pi interactions, the quaternary ammonium of choline, and that of active center inhibitors such as edrophonium. The results also suggest that in the non-covalent complex, charged and uncharged substrates with a common acyl moiety (acetyl) bind to different molecular environments. The hydrophobic site for the alcoholic portion of the covalent adduct (tetrahedral intermediate) includes residues Trp-86, Tyr-337, and Phe-338, which operate through nonpolar and/or stacking interactions, depending on the substrate. Substrates containing choline but differing in the acyl moiety (acetyl, propyl, and butyryl) revealed that residues Phe-295 and Phe-297 determine substrate specificity of the acyl pocket for the covalent adducts. Phe-295 also determines substrate specificity in the non-covalent enzyme substrate complex and thus, the HuAChE F295A mutant exhibits over 130-fold increase in the apparent bimolecular rate constant for butyrylthiocholine compared with wild type enzyme. Reactivity toward specific butyrylcholinesterase inhibitors is similarly dependent on the nature of residues at positions 295 and 297. Amino acid Trp-286 at the rim of the active site "gorge" and Trp-86, in the active center, are essential elements in the mechanism of inhibition by propidium, a peripheral anionic site ligand. Molecular modeling and kinetic data suggest that a cross-talk between Trp-286 and Trp-86 can result in reorientation of Trp-86 which may then interfere with stabilization of substrate enzyme complexes. It is proposed that the conformational flexibility of aromatic residues generates a plasticity in the active center that contributes to the high efficiency of AChE and its ability to respond to external stimuli.
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ABSTRACT: The photosensitizer, methylene blue (MB), generates singlet oxygen that irreversibly inhibits Torpedo californica acetylcholinesterase (TcAChE). In the dark, it inhibits reversibly. Binding is accompanied by a bathochromic absorption shift, used to demonstrate displacement by other acetylcholinesterase inhibitors interacting with the catalytic "anionic" subsite (CAS), the peripheral "anionic" subsite (PAS), or bridging them. MB is a noncompetitive inhibitor of TcAChE, competing with reversible inhibitors directed at both "anionic" subsites, but a single site is involved in inhibition. MB also quenches TcAChE's intrinsic fluorescence. It binds to TcAChE covalently inhibited by a small organophosphate (OP), but not an OP containing a bulky pyrene. Differential scanning calorimetry shows an ~8° increase in the denaturation temperature of the MB/TcAChE complex relative to native TcAChE, and a less than twofold increase in cooperativity of the transition. The crystal structure reveals a single MB stacked against Trp279 in the PAS, oriented down the gorge toward the CAS; it is plausible that irreversible inhibition is associated with photooxidation of this residue and others within the active-site gorge. The kinetic and spectroscopic data showing that inhibitors binding at the CAS can impede binding of MB are reconciled by docking studies showing that the conformation adopted by Phe330, midway down the gorge, in the MB/TcAChE crystal structure, precludes simultaneous binding of a second MB at the CAS. Conversely, binding of ligands at the CAS dislodges MB from its preferred locus at the PAS. The data presented demonstrate that TcAChE is a valuable model for understanding the molecular basis of local photooxidative damage.Protein Science 06/2012; 21(8):1138-52. · 2.74 Impact Factor
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ABSTRACT: Enzymes acetylcholinesterase (AChE; E.C. 184.108.40.206) and butyrylcholinesterase (BChE; E.C. 220.127.116.11) have intensively been investigated in biomedicine and toxicology due to important role in organisms. Even if structurally homologous, they differ in catalytic activity, specificity, for substrates, and selectivity in binding to many ligands. This paper compiles the results of research on cholinesterases and their interactions with ligands and inhibitors, and identifies amino acids of active sites involved in these interactions.Archives of Industrial Hygiene and Toxicology 06/2011; 62(2):175-90. · 0.67 Impact Factor
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ABSTRACT: The cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase, are primary targets of organophosphates (OPs). Exposure to OPs can lead to serious cardiovascular complications, respiratory compromise, and death. Current therapy to combat OP poisoning involves an oxime reactivator (2-PAM, obidoxime, TMB4, or HI-6) combined with atropine and on occasion an anticonvulsant. Butyrylcholinesterase, administered in the plasma compartment as a bio-scavenger, has also shown efficacy but is limited by its strict stoichiometric scavenging, slow reactivation, and a propensity for aging. Here, we characterize 10 human (h) AChE mutants that, when coupled with an oxime, give rise to catalytic reactivation and aging resistance of the soman conjugate. With the most efficient human AChE mutant Y337A/F338A, we show enhanced reactivation rates for several OP-hAChE conjugates compared with wild-type hAChE when reactivated with HI-6 (1-(2'-hydroxyiminomethyl-1'-pyridinium)-3-(4'-carbamoyl-1-pyridinium)). In addition, we interrogated an 840-member novel oxime library for reactivation of Y337A/F338A hAChE-OP conjugates to delineate the most efficient oxime-mutant enzyme pairs for catalytic bio-scavenging. Combining the increased accessibility of the Y337A mutation to oximes within the space-impacted active center gorge with the aging resistance of the F338A mutation provides increased substrate diversity in scavenging potential for aging-prone alkyl phosphate inhibitors.Journal of Biological Chemistry 08/2011; 286(34):29718-24. · 4.65 Impact Factor