Functional Characteristics of the Oxyanion Hole in Human Acetylcholinesterase

Abstract

The contribution of the oxyanion hole to the functional architecture and to the hydrolytic efficiency of human acetylcholinesterase (HuAChE) was investigated through single replacements of its elements, residues Gly-121, Gly-122 and the adjacent residue Gly-120, by alanine. All three substitutions resulted in about 100-fold decrease of the bimolecular rate constants for hydrolysis of acetylthiocholine; however, whereas replacements of Gly-120 and Gly-121 affected only the turnover number, mutation of residue Gly-122 had an effect also on the Michaelis constant. The differential behavior of the G121A and G122A enzymes was manifested also toward the transition state analog m-(N,N,N-trimethylammonio)trifluoroacetophenone (TMTFA), organophosphorous inhibitors, carbamates, and toward selected noncovalent active center ligands. Reactivity of both mutants toward TMTFA was 2000 -11,000-fold lower than that of the wild type HuAChE; however, the G121A enzyme exhibited a rapid inhibition pattern, as opposed to the slow binding kinetics shown by the G122A enzyme. For both phosphates (diethyl phosphorofluoridate, diisopropyl phosphorofluoridate, and paraoxon) and phosphonates (sarin and soman), the decrease in inhibitory activity toward the G121A enzyme was very substantial (2000 -6700-fold), irrespective of size of the alkoxy substituents on the phosphorus atom. On the other hand, for the G122A HuAChE the relative decline in reactivity toward phosphonates (500 -460-fold) differed from that toward the phosphates (12-95-fold). Although formation of Michaelis complexes with substrates does not seem to involve significant interaction with the oxyanion hole, interactions with this motif are a major stabilizing element in accommodation of covalent inhibitors like organophosphates or carbamates. These observations and molecular modeling suggest that replacements of residues Gly-120 or Gly-121 by alanine alter the structure of the oxyanion hole motif, abolishing the H-bonding capacity of residue at position 121. These mutations weaken the interaction between HuAChE and the various ligands by 2.7-5.0 kcal/mol. In contrast, variations in reactivity due to replacement of residue Gly-122 seem to result from steric hindrance at the active center acyl pocket.

A Molecular Mechanism of Enantiorecognition of Tertiary Alcohols by Carboxylesterases

“…[27] This binding pocket stabilizes the oxyanion in the tetrahedral intermediate formed during the catalytic cycle of ester hydrolysis. [28] . GGG(A)X-type hydrolases are mostly carboxylesterases and often from eukaryotic origin.…”

Design of acetylcholinesterases for biosensor applications

“…More sensitive AChE variants and sophisticated multianalyte detection systems are critical issues when it comes to turning AChE biosensors into marketable products. (Ordentlich et al, 1996) Hu F295A 1800 65 830 (Ordentlich et al, 1996) Hu F295L 800 264 940 (Ordentlich et al, 1996) Hu F295L/ F297V 117 340 310 (Ordentlich et al, 1996) Hu E405A 1.9 0.09 1.5 (Ordentlich et al, 1996) Hu G121A 0.085 0.007 (Ordentlich et al, 1998) (Villatte et al, 1998) Dm B08 220 (Montesinos et al, 2000) WT Rb 80 52 0.07 (Pleiss et al, 1999) Rb F295L 550 120 5.6 (Pleiss et al, 1999) WT: wild type; Hu: human AChE, amino acid numbering corresponds to human AChE; HuBChE: human butyrylcholinesterase; Dm: Drosophila melanogaster AChE, amino acid numbering corresponds to DmAChE; Rb: rat brain AChE, amino acid numbering corresponds to RbAChE.…”

“…Amino acids were identified which are important for maintaining the structural integrity of the enzyme (Velan et al, 1991a;Shafferman et al, 1992a) as well as residues that are involved in catalysis and interaction with various substrates and reversible inhibitors (Shafferman et al, 1992b;Ordentlich et al, 1993;Ordentlich et al, 1996). The group also analysed the functional characteristics of the oxyanion hole in HuAChE (Ordentlich et al, 1998). Substitutions in the acyl pocket led to an increase in sensitivity of human AChE towards three tested organophosphate inhibitors (Ordentlich et al, 1996).…”

“…Modifications in the oxyanion hole (G122A and G121A) led to a decrease in sensitivity to DFP, DEFP, and paraoxon by up to three orders of magnitude (Ordentlich et al, 1998). The substitution of six aromatic amino acids in the active (Aldridge, 1950); E = enzyme, CX = carbamate or organophosphate compound, X = leaving group, C = remaining group, k 3 = decarbamylation rate constant and ki = bimolecular rate constant.…”

Synthesis and in silico evaluation of 1N-methyl-1S-methyl-2-nitroethylene (NMSM) derivatives against Alzheimer disease: to understand their interacting mechanism with acetylcholinesterase

“…Moreover, the N3 (P pKa 07.76) atom of this ligand is placed towards the conserved Ser125 so as in Lig_1 and Lig_2-hAChE complexes with bond distance of about 2.95 Å. This ligand also makes VdW interactions with the residues of PAS site (Tyr72, Asp74, and Tyr124), CAS site (Trp86 and Tyr337), oxyanion hole (Gly121) [56], and with other residues (Val 73, Asn87, Pro88, and Ser125) of hAChE. The C-H-π interaction was formed by the position of Lig_3 methylsulfanyl group towards the benzyl ring of Trp86 indole with a distance of 2.79 Å, which is mainly focused for their contribution in the gorge, similar to cation-π interaction of ACh-hAChE complex.…”