Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.1.7 (acetylcholinesterase)
28,390 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The three-dimensional solution structure of the MTX2 toxin (65 amino acids and 4 disulfides) from the green mamba venom (Dendroaspis angusticeps), a toxin that activates the pharmacological M1 muscarinic acetylcholine receptors, has been determined by nuclear magnetic resonance and molecular modeling. Seventeen structures were calculated from 810 distance and 68 dihedral angle restraints using DIANA and X-PLOR. The average rms deviation between the 17 refined structures and the energy-minimized average structure is 0.95 A for the backbone atoms. The overall folding of MTX2 consists of three loops stabilized by the four disulfides and forming a two- and a three-stranded beta-sheet. This structure appears to be very similar to that of other snake toxins, such as neurotoxins, fasciculins, and cardiotoxins, that also possess the same three-finger fold. For instance, the RMSd for the backbone atoms between MTX2 and the curaremimetic toxin alpha (from Naja nigricollis), the acetylcholinesterase inhibitor fasciculin 1 (from Dendroaspis angusticeps), and the cardiotoxic toxin gamma (from Naja nigricollis) are 1.86, 1.87, and 2.04 A, respectively. Local differences are observed between this toxin and the other structurally related toxins. Some of these differences could be relevant for the functional specificity of MTX2. In particular, this toxin presents a large twist at the tip of loop II due to a bulge (V31, T32; N35) that accommodates an inserted amino acid in the loop. This spatial arrangement brings the side chain of K34 in the beta-turn of the loop to be aligned with the beta-sheet. Hypotheses about a possible functional role of this lysine are described. Other characteristics in the side-chain distribution that could be related to the MTX2 function are presented.
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PMID:Solution structure of a green mamba toxin that activates muscarinic acetylcholine receptors, as studied by nuclear magnetic resonance and molecular modeling. 782 75

The hydrophilic, salt-soluble (SS) form of acetylcholinesterase (AChE) from bovine brain caudate nucleus exists mainly as a tetramer sedimenting at 10.3S (approximately 40%), and a monomer sedimenting at 3.4S (approximately 60%). The enzyme is N-glycosylated and contains similar HNK-1 carbohydrates as detergent-soluble (DS) AChE. No O-linked carbohydrates could be detected. Amino acid sequencing showed that the N terminus of SS-AChE is identical to that of DS-AChE. In tetrameric SS-AChE, two pairs of disulfide-linked dimers are associated by hydrophobic forces located in the C terminus. Antibodies were raised against a peptide identical to the last 10 amino acid residues of bovine brain DS-AChE. The peptide included the sequence of residues 574-583 (H-Tyr-Ser-Lys-Gln-Asp-Arg-Cys-Ser- Asp-Leu-OH) of the enzyme. The antibodies cross-reacted with tetrameric, but not with monomeric, SS-AChE, showing that in the latter form, the C terminus is truncated. Limited proteolysis of tetrameric SS-AChE at the C terminus led to the formation of an enzymatically active monomer, which did not react with anti-C-terminal antibody. Although the DS form of AChE contains a structural subunit that serves as membrane anchor, no anchor was detected in SS-AChE. Enzyme antigen immunoassays showed that SS-AChE reacted with all monoclonal antibodies directed against the catalytic subunit of DS-AChE, but not with monoclonal antibodies targeting the membrane-anchored subunits. From our results, we conclude that SS-AChE utilizes the same alternative splicing pattern as DS-AChE, leading to tetrameric SS-AChE devoid of the membrane anchor.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of salt-soluble forms of acetylcholinesterase from bovine brain. 793 Dec 96

To characterize the structure of the active site of acetylcholinesterase (AChE) from the electric organ of E. electricus, we identified sites of incorporation of two active-site affinity labels, [3H]diisopropyl fluorophosphate ([3H]DFP), and 1-bromo-2-[14C]pinacolone ([14C]BrPin). AChE was isolated, purified, inactivated and digested with trypsin, and peptides containing 3H or 14C were purified by reverse-phase HPLC and characterized by N-terminal sequence analysis. [3H]DFP, labelling Ser-200, was found in a single peptide, QVTIFGESAGAASVGMHLLSPDSR, 83% identical with the sequence from Thr-193 to Arg-216 deduced for AChE of T. californica, with Gln, Ala, Leu, and Asp in place of Thr-193, Gly-203, Ile-210 and Gly-214, respectively, and 87% identical with that from bovine and human brain AChEs. Inactivation by [14C]BrPin led to two radioactive peptides. One, ASNLVWPEWMGVIHGYEIEFVFGLPLEK, was 96% identical with that extending from Ala-427 to Lys-454 of T. californica. Release of 14C in cycle 14 established reaction of [14C]BrPin with active-site His-440, protected by 5-trimethylammonio-2-pentanone (TAP). The other peptide, LLXVTENIDDAER, 77% homologous with that of T. californica extending from Leu-531 to Arg-543, had label associated with the third cycle, not protected by TAP, corresponding to Asn-533. The slow inactivation of eel AChE by reaction of [14C]BrPin at His-440 contrasts with that of AChE from T. nobiliana, where it reacts rapidly with a free cysteine, Cys-231, not present in eel AChE. For both AChEs, inactivation by BrPin prevents subsequent reaction with [3H]DFP, and prior inactivation by DFP does not prevent reactions with [14C]BrPin.
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PMID:Active-site peptides of acetylcholinesterase of Electrophorus electricus: labelling of His-440 by 1-bromo-[2-14C]pinacolone and Ser-200 by tritiated diisopropyl fluorophosphate. 794 65

The active site of acetylcholinesterase is near the bottom of a long and narrow gorge. The dimensions of the gorge and the strong electrostatic field generated by the enzyme appear inconsistent with the enzyme's high turnover rate. Consequently, a "back door" mechanism involving movement of the reaction products through a transient opening near the active center was recently suggested. We investigated this hypothesis in human acetylcholinesterase by testing mutants at key residues (Glu-84, Trp-86, Asp-131, and Val-132) located near or along the putative back door channel. The turnover rates of all mutants tested, and in particular of V132K, where the channel is expected to be sealed by salt bridge Lys-132-Glu-452, are similar to that of the wild type enzyme. This indicates that the proposed back door is not a route for product clearance from the active site gorge of acetylcholinesterase and is probably of no functional relevance to its catalytic activity.
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PMID:The "back door" hypothesis for product clearance in acetylcholinesterase challenged by site-directed mutagenesis. 796 9

1. It was recently proposed that acetylcholinesterase (AChE), in addition to its esteratic activity, has proteolytic activity such that it may cleave the beta-amyloid precursor (beta-APP) within the beta-amyloid sequence. The purpose of this paper was to examine further whether AChE or butyrylcholinesterase (BuChE) had associated proteinase activity that was involved in the metabolism of beta-APP. 2. The ability of various preparations of AChE and BuChE to hydrolyze two synthetic fragments of beta-APP695 as model substrates containing the normal and aberrant cleavage sites was studied. 3. Digestion of these synthetic substrates with commercial preparations of Electrophorus electricus AChE indicated the presence of a trypsin-like proteolytic activity cleaving each peptide at the carboxy-terminal side of an internal lysine residue. 4. Purification of the trypsin-like proteinase activity by aminobenzamidine affinity chromatography yielded a preparation that was devoid of AChE activity but retained all of the proteinase activity. 5. Amino-terminal sequence analysis of this preparation showed that the first 13 amino acid residues were identical to beta-pancreatic trypsin. 6. These data indicate that the proteinase activity found in these commercial preparations of AChE is due to contamination with trypsin.
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PMID:Proteolysis at the secretase and amyloidogenic cleavage sites of the beta-amyloid precursor protein by acetylcholinesterase and butyrylcholinesterase using model peptide substrates. 824 91

Dendroaspis angusticeps (green mamba) has two toxins, fasciculins, that are non-competitive inhibitors of acetylcholinesterase. Amino groups of fasciculin 2 were acetylated with acetic anhydride. The monoacetyl derivatives of the epsilon-amino groups (Lys 25, 32, 51 and 58) retained between 28 and 43% of the initial activity and that of the alpha-amino group 72%. Acetylation of Lys 25 that has the most reactive amino group decreased the activity by 65% apparently without producing structural perturbations, since the circular dichroism spectrum was not affected. The three-dimensional structure shows a cationic cluster formed by Lys 32, 51, Arg 24 and 28. A comparison of 175 sequences of homologous toxins shows that Lys 32 is unique for fasciculin. Acetylation of lysine residues in the cluster had a large effect and reduced the activity by 72% (Lys 32) and 57% (Lys 51). This suggests an important role for the cationic cluster. Lys 25 together with Lys 32 and 51 were, therefore, assumed to be in the active site.
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PMID:Study of structure-activity relationship of fasciculin by acetylation of amino groups. 828 Jul 46

The precise localization of an endothelin (ET) receptor subtype, the ETB receptor, in porcine lung was elucidated by in vitro microautoradiography using a novel ETB-selective radioligand, [125I]BQ-3020 ([125I-Tyr]-N-acetyl-Leu-Met-Asp-Lys-Glu-Ala-Val-Tyr-Phe-Ala-His-Leu-Asp -Ile-Ile-Trp). Of the labeled native ET isopeptides, [125I]ET-3 is selective for ETB receptors. However, [125I]ET-3 was not suitable for autoradiography due to its high degree of non-specific binding. On the other hand, [125I]BQ-3020 showed extremely low non-specific binding on autoradiography. The distribution of [125I]BQ-3020 binding in porcine lung was clearly different from that of [125I]ET-1, which showed more widespread binding than [125I]BQ-3020 due to a high affinity to both ETA and ETB receptors. [125I]BQ-3020 was found to bind to parenchyma, parasympathetic ganglia, pulmonary and submucosal plexuses, but bound only slightly to circular smooth muscle layers and the epithelium of airway tracts. Although [125I]ET-1 bound to the smooth muscle layer of all blood vessels, the binding of [125I]BQ-3020 differed among blood vessels. [125I]BQ-3020 binding in blood vessels paralleled acetylcholinesterase activity, suggesting that ETB receptors in blood vessels are located on parasympathetic nerves. Thus, the radioligand [125I]BQ-3020 is very useful for studying the precise localization of ETB receptors.
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PMID:A novel ligand, [125I]BQ-3020, reveals the localization of endothelin ETB receptors. 851 85

Monoclonal antibodies (mAbs) were raised against a peptide of the 10 C-terminal amino acids of human brain acetylcholinesterase (AChE): H-Tyr-Ser-Lys-Gln-Asp-Arg-Cys-Ser-Asp-Leu-OH. Two positive clones (mAbs 190-1 and 190-2) were selected and tested for their ability to distinguish between mammalian brain and erythrocyte AChEs. In a solid-phase enzyme antigen immunoassay as well as by Western- and dot-blot analysis, both antibodies showed clear binding to AChE from human and bovine brain but not to AChE from erythrocytes. MAbs 190-1 and 190-2 reacted with neither AChE from electric eel nor butyrylcholinesterase from human serum. Both antibodies were used in a quantitative assay for AChE in amniotic fluids, where AChE activity could be found only in samples from open neural tube-defect pregnancies, but not in fluids from normal pregnancies or in artificially blood-contaminated samples.
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PMID:Monoclonal antibodies against a C-terminal peptide of human brain acetylcholinesterase distinguish between erythrocyte and brain acetylcholinesterases. 856 26

Phosphotriesterase from Pseudomonas diminuta catalyzes the hydrolysis of paraoxon and related acetylcholinesterase inhibitors with rate enhancements that approach 10(12). The enzyme requires a binuclear metal center for activity and as isolated contains 2 equiv of zinc per subunit. Here we describe the three-dimensional structure of the Zn2+/Zn2+-substituted enzyme complexed with the substrate analog diethyl 4-methylbenzylphosphonate. Crystals employed in the investigation belonged to the space group C2 with unit cell dimensions of a = 129.6 A, b = 91.4 A, c = 69.4 A, beta = 91.9 degrees, and two subunits in the asymmetric unit. The model was refined by least-squares analysis to a nominal resolution of 2.1 A and a crystallographic R-factor of 15.4% for all measured X-ray data. As in the previously reported structure of the cadmium-containing enzyme, the bridging ligands are a carbamylated lysine residue (Lys 169) and a hydroxide. The zinc ions are separated by 3.3 A. The more buried zinc ion is surrounded by His 55, His 57, Lys 169, Asp 301, and the bridging hydroxide in a trigonal bipyramidal arrangement as described for the cadmium-substituted enzyme. Unlike the octahedral coordination observed for the more solvent-exposed cadmium ion, however, the second zinc is tetrahedrally ligated to Lys 169, His 201, His 230, and the bridging hydroxide. The diethyl 4-methylbenzylphosphonate occupies a site near the binuclear metal center with the phosphoryl oxygen of the substrate analog situated at 3.5 A from the more solvent-exposed zinc ion. The aromatic portion of the inhibitor binds in a fairly hydrophobic pocket. A striking feature of the active site pocket is the lack of direct electrostatic interactions between the inhibitor and the protein. This most likely explains the broad substrate specificity exhibited by phosphotriesterase. The position of the inhibitor within the active site suggests that the nucleophile for the hydrolysis reaction is the metal-bound hydroxide.
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PMID:Three-dimensional structure of the zinc-containing phosphotriesterase with the bound substrate analog diethyl 4-methylbenzylphosphonate. 863 43

As deduced from cDNA clones, the catalytic domain of Bungarus fasciatus venom acetylcholinesterase (AChE) is highly homologous to those of other AChEs. It is, however, associated with a short hydrophilic carboxyl-terminal region, containing no cysteine, that bears no resemblance to the alternative COOH-terminal peptides of the GPI-anchored molecules (H) or of other homomeric or heteromeric tailed molecules (T). Expression of complete and truncated AChE in COS cells showed that active hydrophilic monomers are produced and secreted in all cases, and that cleavage of a very basic 8-residue carboxyl-terminal fragment occurs upon secretion. The COS cells produced Bungarus AChE about 30 times more efficiently than an equivalent secreted monomeric rat AChE. The recombinant Bungarus AChE, like the natural venom enzyme, showed a distinctive ladder pattern in nondenaturing electrophoresis, probably reflecting a variation in the number of sialic acids. By mutagenesis, we showed that two differences (methionine instead of tyrosine at position 70; lysine instead of aspartate or glutamate at position 285) explain the low sensitivity of Bungarus AChE to peripheral site inhibitors, compared to the Torpedo or mammalian AChEs. These results illustrate the importance of both the aromatic and the charged residues, and the fact that peripheral site ligands (propidium, gallamine, D-tubocurarine, and fasciculin 2) interact with diverse subsets of residues.
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PMID:Cloning and expression of acetylcholinesterase from Bungarus fasciatus venom. A new type of cooh-terminal domain; involvement of a positively charged residue in the peripheral site. 866 67


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