p.W286A Trp286Ala (p.W317A Trp317Ala in primary sequence with 31 amino-acids signal peptide) Peripheral Anionic Site Substrate inhibition;10 fold increase of inhibition K for PAS ligands; Acyl specificity;replacement of aromatic active center residues in human-ACHE by the corresponding residues in human-BCHE
The antidotal property of oximes is attributed to their ability to reactivate acetylcholinesterase (AChE) inhibited by organophosphorus compounds (OP) such as pesticides and nerve warfare agents. Understanding their interactions within the active site of phosphylated AChE is of great significance for the search for more efficient reactivators, especially in the case of the most resistant OP to reactivation, tabun. Therefore, herein we studied the interactions and reactivation of tabun-inhibited AChE by site-directed mutagenesis and a series of bispyridinium oximes. Our results indicated that the replacement of aromatic residues with aliphatic ones at the acyl pocket and choline binding site mostly interfered with the stabilisation of the oxime's pyridinium ring(s) within the active site gorge needed to obtain the proper orientation of the oxime group toward the phosphorylated active site serine. However, in the case of W286A, the mutation in the peripheral binding site by preventing a pi-pi interaction with one of the oxime's pyridinium rings allowed a more favourable position of the oxime for a nucleophilic attack on the phosphorylated catalytic serine. The mutation resulted in a 2-5 fold increase in the reactivation rates when compared to the AChE wild type. Therefore, it seems that aromatic amino acids at the peripheral binding site presented a limitation in bispyridinium oxime reactivation efficiency of tabun-phosphorylated AChE. Moreover, this is further corroborated by the reactivation by mono-pyridinium oxime 2-PAM, in which mutations at the peripheral site did not influence either the affinity or reactivation of tabun-inhibited AChE.
Title: Does butyrylization of acetylcholinesterase through substitution of the six divergent aromatic amino acids in the active center gorge generate an enzyme mimic of butyrylcholinesterase? Kaplan D, Ordentlich A, Barak D, Ariel N, Kronman C, Velan B, Shafferman A Ref: Biochemistry, 40:7433, 2001 : PubMed
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.
Title: The 'aromatic patch' of three proximal residues in the human acetylcholinesterase active centre allows for versatile interaction modes with inhibitors Ariel N, Ordentlich A, Barak D, Bino T, Velan B, Shafferman A Ref: Biochemical Journal, 335:95, 1998 : PubMed
The role of the functional architecture of the human acetylcholinesterase (HuAChE) active centre in accommodating the non-covalent inhibitors tacrine and huperzine A, or the carbamates pyridostigmine and physostigmine, was analysed using 16 mutants of residues lining the active-centre gorge. Despite the structural diversity of the ligands, certain common properties of the complexes could be observed: (a) replacement of aromatic residues Tyr133, Tyr337 and especially Trp86, resulted in pronounced changes in stability of all the complexes examined; (b) effects due to replacements of the five other aromatic residues along the active-centre gorge, such as the acyl pocket (Phe295, Phe297) or at the peripheral anionic site (Tyr124, Trp286, Tyr341) were relatively small; (c) effects due to substitution of the carboxylic residues in the gorge (Glu202, Glu450) were moderate. These results and molecular modelling indicate that the aromatic side chains of residues Trp86, Tyr133 and Tyr337 form together a continuous 'aromatic patch' lining the wall of the active-centre gorge, allowing for the accommodation of the different ligands via multiple modes of interaction. Studies with HuAChE mutants carrying replacements at positions 86, 133 and 337 indicate that the orientations of huperzine A and tacrine in the HuAChE complexes in solution are significantly different from those observed in X-ray structures of the corresponding complexes with Torpedo californica AChE (TcAChE). These discrepancies may be explained in terms of structural differences between the complexes of HuAChE and TcAChE or, more likely, by the enhanced flexibility of the AChE active-centre gorge in solution as compared with the crystalline state.
Title: Role of tyrosine 337 in the binding of huperzine A to the active site of human acetylcholinesterase Ashani Y, Grunwald J, Kronman C, Velan B, Shafferman A Ref: Molecular Pharmacology, 45:555, 1994 : PubMed
Huperzine A (HUP), a natural, potent, 'slow,' reversible inhibitor of antiacetylcholinesterase (AChE), has been suggested to be superior to antiacetylcholinesterase drugs now being used for management of Alzheimer's disease. To delineate the binding site of human AChE (HuAChE) for HUP, the biochemical constants kon, koff, and Ki were determined for complexes formed between HUP and single-site (Y337F, Y337A, F295A, W286A, and E202Q) or double-site (F295L/F297V) mutants of recombinant HuAChE (rHuAChE). The kinetic and dissociation constants were compared with those obtained for wild-type rHuAChE and AChE from Torpedo californica. Results demonstrate that the inhibition of AChE by HUP occurs through association with residues located inside the active site 'gorge,' rather than at the rim of the gorge. Tyrosine at position 337 (Y337) is essential for inhibition of rHuAChE by HUP (Ki = 26 nM). An aromatic array constituted from residues Y337, F295, and probably W86 is likely to offer a multicontact subsite that interacts with the ammonium group and with both the exo-and endocyclic double bond moieties of HUP. Lack of the aromatic side chain in the position homologous to Y337 explains the poor inhibitory potency of HUP toward human butyrylcholinesterase (Ki > 20,000 nM). Replacement of the carboxylate-containing E202 by glutamine had only marginal effect on the stability of the complex formed between HUP and rHuAChE. The pH-rate profiles suggest that destabilization of the complex after proton gain cannot be attributed solely to protonation of E202. These findings are expected to establish HUP as a lead compound for the design of new anti-AChE drugs.
Several of the residues constituting the peripheral anionic site (PAS) in human acetylcholinesterase (HuAChE) were identified by a combination of kinetic studies with 19 single and multiple HuAChE mutants, fluorescence binding studies with the Trp-286 mutant, and by molecular modeling. Mutants were analyzed with three structurally distinct positively charged PAS ligands, propidium, decamethonium, and di(p-allyl-N-dimethylaminophenyl)pentane-3-one (BW284C51), as well as with selective active center inhibitors, hexamethonium and edrophonium. Single mutations of residues Tyr-72, Tyr-124, Glu-285, Trp-286, and Tyr-341 resulted in up to 10-fold increase in inhibition constants for PAS ligands, whereas for multiple mutants up to 400-fold increase was observed. The 6th PAS element residue Asp-74 is unique in its ability to affect conformation of both the active site and the PAS (Shafferman, A., Velan, B., Ordentlich, A., Kronman, C., Grosfeld, H., Leitner, M., Flashner, Y., Cohen, S., Barak, D., and Ariel, N. (1992) EMBO J. 11, 3561-3568) as demonstrated by the several hundred-fold increase in Ki for D74N inhibition by the bisquaternary ligands decamethonium and BW284C51. Based on these studies, singular molecular models for the various HuAChE inhibitor complexes were defined. Yet, for the decamethonium complex two distinct conformations were generated, accommodating the quaternary ammonium group by interactions with either Trp-286 or with Tyr-341. We propose that the PAS consists of a number of binding sites, close to the entrance of the active site gorge, sharing residues Asp-74 and Trp-286 as a common core. Binding of ligands to these residues may be the key to the allosteric modulation of HuAChE catalytic activity. This functional degeneracy is a result of the ability of the Trp-286 indole moiety to interact either via stacking, aromatic-aromatic, or via pi-cation attractions and the involvement of the carboxylate of Asp-74 in charge-charge or H-bond interactions.
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.
Amino acids located within and around the 'active site gorge' of human acetylcholinesterase (AChE) were substituted. Replacement of W86 yielded inactive enzyme molecules, consistent with its proposed involvement in binding of the choline moiety in the active center. A decrease in affinity to propidium and a concomitant loss of substrate inhibition was observed in D74G, D74N, D74K and W286A mutants, supporting the idea that the site for substrate inhibition and the peripheral anionic site overlap. Mutations of amino acids neighboring the active center (E202, Y337 and F338) resulted in a decrease in the catalytic and the apparent bimolecular rate constants. A decrease in affinity to edrophonium was observed in D74, E202, Y337 and to a lesser extent in F338 and Y341 mutants. E202, Y337 and Y341 mutants were not inhibited efficiently by high substrate concentrations. We propose that binding of acetylcholine, on the surface of AChE, may trigger sequence of conformational changes extending from the peripheral anionic site through W286 to D74, at the entrance of the 'gorge', and down to the catalytic center (through Y341 to F338 and Y337). These changes, especially in Y337, could block the entrance/exit of the catalytic center and reduce the catalytic efficiency of AChE.
Title: Acetylcholinesterase Catalysis - Protein Engineering Studies Shafferman A, Velan B Ref: In Multidisciplinary approaches to cholinesterase functions - Proceedings of Fourth International Meeting on Cholinesterases, (Shafferman, A. and Velan, B., Eds) Plenum Press, New York:165, 1992 : PubMed
