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)

A protein isolated from sciatic nerves of adult chickens promotes the morphological maturation and maintenance of embryonic avian skeletal muscle cells in the absence of innervation and is required for normal myogenesis in vitro. This trophic protein, sciatin, has been purified by ion exchange column chromatography on DEAE-cellulose followed by gel filtration on Sephadex G-100. Sciatin migrated as a single polypeptide chain of molecular weight 84,000 on sodium dodecyl sulfategel electrophoresis. The native molecular weight of sciatin as determined by sedimentation equilibrium centrifugation was 86,400. Amino acid analysis revealed that sciatin is relatively deficient in tryptophan, histidine, glycine, and arginine, but enriched in cysteine, methionine, alanine, and lysine. Carbohydrate determination showed that sciatin in composed of 11% sugar by weight with no detectable N-acetylneuraminic acid residues. Sedimentation velocity centrifugation studies revealed an S20,w0 of 5.11 with a frictional coefficient of 1.31. Sciatin had no detectable protease or acetylcholinesterase activity. The results of the present study provide new biochemical information on a macromolecule with biological activities similar to those expressed by the "maintenance" group of growth factors which includes such proteins as nerve growth factor.
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PMID:Sciatin: purification and characterization of a myotrophic protein from chicken sciatic nerves. 699 6

Endogenous calcitonin was altered in rats by thyroparathyroidectomy, followed by supplementation with thyroxine and calcium. As a result, a reduction in the content of 5-hydroxytryptamine in the brain together with an increase in the concentration of tryptophan in the plasma were observed. The changes were accompanied by a diminution in the activity of both choline acetyltransferase and acetylcholinesterase in the cerebral hemispheres. The results are consistent with those produced by exogenous hormone and suggest that calcitonin probably plays a role in the regulation of 5-hydroxytryptamine levels in the brain.
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PMID:Response of rat brain to calcitonin alteration. 709 70

We exposed human red blood cell (RBC) membranes to low levels of ozone and measured the oxidative damage that occurs to the proteins and the unsaturated lipids that are present. Oxidative damage to proteins causes significant decreases in the content of thiol groups, the fluorescence of protein-tryptophan residues, and the activity of membrane-bound acetylcholinesterase. Oxidative damage to lipids causes changes in some of the unsaturated fatty acids (UFA) in the lipid fraction of these RBC membranes. Significant amounts of hexanal, heptanal, and nonanal are formed from the ozonation of UFA. Although no decrease in the amount of oleate is detected, it does undergo ozonation to yield nonanal; thus, as would be expected, product appearance is a more sensitive measure of ozonation than is substrate disappearance. These results imply that both proteins and unsaturated lipids undergo simultaneous and competitive ozonation in human RBC membranes when ozone is the limiting reactant. The ratios of reaction of ozone with different targets can be predicted in reasonably good agreement with the observed values using calculations (W. A. Pryor and R. M. Uppu (1993) J. Biol. Chem. 268, 3120-3126; R. M. Uppu and W. A. Pryor (1994) Chem. Res. Toxicol. 7, 47-55) that take into account the reactivities and relative amounts of protein and lipid functionalities present in the RBC membranes. Similar calculations are used to predict the reaction of ozone with unsaturated lipids and proteins at the air/lung interface, and both UFA and proteins are predicted to react with ozone in the lung, as in RBC membranes.
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PMID:What does ozone react with at the air/lung interface? Model studies using human red blood cell membranes. 777 93

A large-scale preparation of a recombinant human acetylcholinesterase (rhAChE) mutant harbouring a CyS580-->Ser substitution, expressed in Escherichia coli, was refolded following solubilization of the inclusion bodies. Refolded active rhAChE was purified by DEAE-Sepharose and affinity chromatography to apparent homogeneity with a specific activity (4572 units/mg) similar to that of erythrocyte AChE. The stability of the purified enzyme at 22-37 degrees C was dependent on the presence of 0.5 mg/ml BSA, and the optimum pH for stability was 9.0. rhAChE has a UV-absorbance spectrum typical of a tryptophan-rich protein, with a distinct shoulder at 290 nm and a high absorption coefficient at 280 nm (epsilon 1% = 23.1). The tryptophan residues in active rhAChE are located in an apolar environment, characteristic of a globular molecule. The difference in amino acid composition between red-blood-cell-derived and recombinant hAChE is probably reflected in their different pI values, namely 5.5-5.8 and 4.6-5.2 respectively. The CD spectrum of rhAChE is typical for an alpha/beta protein, indicating 39% alpha-helix and 22% beta-sheet. This secondary structure is similar to that determined for the Torpedo (electric fish) AChE, by both CD and X-ray crystallography. On the other hand, a purified misfolded and inactive molecule displays a decrease in alpha-helical content to 24%, accompanied by an increase in beta-sheet up to 42%, indicative of extensive changes in the conformation of the protein. On the whole, the recombinant enzyme has been refolded into a native-like conformation possessing full activity, and is thus similar to the naturally occurring red-blood-cell-derived hAChE.
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PMID:Recombinant human acetylcholinesterase expressed in Escherichia coli: refolding, purification and characterization. 779 33

Substitution of Trp-86, in the active center of human acetylcholinesterase (HuAChE), by aliphatic but not by aromatic residues resulted in a several thousandfold decrease in reactivity toward charged substrate and inhibitors but only a severalfold decrease for noncharged substrate and inhibitors. The W86A and W86E HuAChE enzymes exhibit at least a 100-fold increase in the Michaelis-Menten constant or 100-10,000-fold increase in inhibition constants toward various charged inhibitors, as compared to W86F HuAChE or the wild type enzyme. On the other hand, replacement of Glu-202, the only acidic residue proximal to the catalytic site, by glutamine resulted in a nonselective decrease in reactivity toward charged and noncharged substrates or inhibitors. Thus, the quaternary nitrogen groups of substrates and other active center ligands, are stabilized by cation-aromatic interaction with Trp-86 rather than by ionic interactions, while noncharged ligands appear to bind to distinct site(s) in HuAChE. Analysis of the Y133F and Y133A HuAChE mutated enzymes suggests that the highly conserved Tyr-133 plays a dual role in the active center: (a) its hydroxyl appears to maintain the functional orientation of Glu-202 by hydrogen bonding and (b) its aromatic moiety maintains the functional orientation of the anionic subsite Trp-86. In the absence of aromatic interactions between Tyr-133 and Trp-86, the tryptophan acquires a conformation that obstructs the active site leading, in the Y133A enzyme, to several hundredfold decrease in rates of catalysis, phosphorylation, or in affinity to reversible active site inhibitors. It is proposed that allosteric modulation of acetylcholinesterase activity, induced by binding to the peripheral anionic sites, proceeds through such conformational change of Trp-86 from a functional anionic subsite state to one that restricts access of substrates to the active center.
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PMID:Contribution of aromatic moieties of tyrosine 133 and of the anionic subsite tryptophan 86 to catalytic efficiency and allosteric modulation of acetylcholinesterase. 783 36

The enzyme acetylcholinesterase generates a strong electrostatic field that can attract the cationic substrate acetylcholine to the active site. However, the long and narrow active site gorge seems inconsistent with the enzyme's high catalytic rate. A molecular dynamics simulation of acetylcholinesterase in water reveals the transient opening of a short channel, large enough to pass a water molecule, through a thin wall of the active site near tryptophan-84. This simulation suggests that substrate, products, or solvent could move through this "back door," in addition to the entrance revealed by the crystallographic structure. Electrostatic calculations show a strong field at the back door, oriented to attract the substrate and the reaction product choline and to repel the other reaction product, acetate. Analysis of the open back door conformation suggests a mutation that could seal the back door and thus test the hypothesis that thermal motion of this enzyme may open multiple routes of access to its active site.
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PMID:Open "back door" in a molecular dynamics simulation of acetylcholinesterase. 812 10

Through site-specific mutagenesis, we examined the determinants on acetylcholinesterase which govern the specificity and reactivity of three classes of substrates: enantiomeric alkyl phosphonates, trifluoromethyl acetophenones, and carboxyl esters. By employing cationic and uncharged pairs of enantiomeric alkyl methylphosphonyl thioates of known absolute stereochemistry, we find that an aspartate residue near the gorge entrance (D74) is responsible for the enhanced reactivity of the cationic organophosphonates. Removal of the charge with the mutation D74N causes a near equal reduction in the reaction rate constants for the Rp and Sp enantiomers and exerts a greater influence on the cationic organophosphonates than on the charged trimethylammonio trifluoromethyl acetophenone and acetylthiocholine. This pattern of reactivity suggests that the orientation of the leaving group for both enantiomers is directed toward the gorge exit and in apposition to Asp 74. Replacement of tryptophan 86 with alanine in the choline subsite also diminishes the reaction rates for cationic organophosphonates, although to a lesser extent than with the D74N mutation, while not affecting the reactions with the uncharged compounds. Hence, reaction with cationic OPs depends to a lesser degree on Trp 86 than on Asp 74. Docking of Sp and Rp cycloheptyl methylphosphonyl thiocholines and thioethylates in AChE as models of the reversible complex and transition state using molecular dynamics affords structural insight into the spatial arrangement of the substituents surrounding phosphorus prior to and during reaction. The leaving group of the Rp and Sp enantiomers, regardless of charge, is directed to the gorge exit and toward Asp 74, an orientation unique to tetrahedral ligands.
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PMID:Aspartate 74 as a primary determinant in acetylcholinesterase governing specificity to cationic organophosphonates. 871 93

The major type of acetylcholinesterase in vertebrates (AChET) is characterized by the presence of a short C-terminal domain of 40 residues, the 'tryptophan amphiphilic tetramerization' (WAT) domain. The presence of this domain is not necessary for catalytic activity but is responsible for hydrophobic interactions and for the capacity of AChET subunits to form quaternary associations with anchoring proteins, thereby conditioning their functional localization. In the collagen tail of asymmetric forms, we characterized a small conserved region that is sufficient for binding an AChET tetramer, the proline-rich attachment domain (PRAD). We show that the WAT domain alone is sufficient for association with the PRAD, and that it can attach foreign proteins (alkaline phosphatase, GFP) to a PRAD-containing construct with a glycophosphatidylinositol anchor (GPI), and thus anchor them to the cell surface. Furthermore, we show that isolated WAT domains, or proteins containing a WAT domain, can replace individual AChET subunits in PRAD-linked tetramers. This suggests that the four WAT domains interact with the PRAD in a similar manner. These quaternary interactions can form without intercatenary disulfide bonds. The common catalytic domains of AChE are not necessary for tetrameric assembly, although they may contribute to the stability of the tetramer.
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PMID:A four-to-one association between peptide motifs: four C-terminal domains from cholinesterase assemble with one proline-rich attachment domain (PRAD) in the secretory pathway. 979 27

3-Alkylpyridinium polymers (poly-APS), composed of 29 or 99 N-butyl-3-butyl pyridinium units, were isolated from the marine sponge Reniera sarai. They act as potent cholinesterase inhibitors. The inhibition kinetics pattern reveals several successive phases ending in irreversible inhibition of the enzyme. To provide more information on mechanism of inhibition, interaction of poly-APS and N-butyl-3-butyl pyridinium iodide (NBPI) with soluble dimeric and monomeric insect acetylcholinesterase (AChE) was studied by using enzyme intrinsic fluorescence and light scattering, conformational probes ANS and trypsin, and SDS-PAGE. Poly-APS quenched tryptophan fluorescence emission of AChE more extensively than NBPI. Both inhibitors exhibited a pseudo-Lehrer type of quenching. Interaction of poly-APS with dimeric AChE did not induce significant changes of the enzyme conformation as assayed by using the hydrophobic probe ANS and trypsin digestion. In contrast to NBPI, titration of both monomeric and dimeric AChE with poly-APS resulted in the appearance of large complexes detected by measuring light scattering. An excess of poly-APS produced AChE precipitation as proved on SDS-PAGE. None of the effects were observed with trypsin as a control. It was concluded that AChE aggregation and precipitation rather than the enzyme conformational changes accounted for the observed irreversible component of poly-APS inhibition.
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PMID:Interaction of 3-alkylpyridinium polymers from the sea sponge Reniera sarai with insect acetylcholinesterase. 1039 43

The role of active-site residues in the dealkylation reaction in the P(S)C(S) diastereomer of 2-(3,3-dimethylbutyl)methylphosphonofluoridate (soman)-inhibited Torpedo californica acetylcholinesterase (AChE) was investigated by full-scale molecular dynamics simulations using CHARMM: >400 ps equilibration was followed by 150-200 ps production runs with the fully solvated tetracoordinate phosphonate adduct of the wild-type, Trp84Ala and Gly199Gln mutants of AChE. Parallel simulations were carried out with the tetrahedral intermediate formed between serine-200 Ogamma of AChE and acetylcholine. We found that the NepsilonH in histidine H(+)-440 is positioned to protonate the oxygen in choline and thus promote its departure. In contrast, NepsilonH in histidine H(+)-440 is not aligned for a favourable proton transfer to the pinacolyl O to promote dealkylation, but electrostatic stabilization by histidine H(+)-440 of the developing anion on the phosphonate monoester occurs. Destabilizing interactions between residues and the alkyl fragment of the inhibitor enforce methyl migration from Cbeta to Calpha concerted with C-O bond breaking in soman-inhibited AChE. Tryptophan-84, phenyalanine-331 and glutamic acid-199 are within 3.7-3.9 A (1 A=10(-10) m) from a methyl group in Cbeta, 4.5-5.1 A from Cbeta and 4.8-5.8 A from Calpha, and can better stabilize the developing carbenium ion on Cbeta than on Calpha. The Trp84Ala mutation eliminates interactions between the incipient carbenium ion and the indole ring, but also reduces its interactions with phenylalanine-331 and aspartic acid-72. Tyrosine-130 promotes dealkylation by interacting with the indole ring of tryptophan-84. Glutamic acid-443 can influence the orientation of active-site residues through tyrosine-421, tyrosine-442 and histidine-440 in soman-inhibited AChE, and thus facilitate dealkylation.
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PMID:Molecular dynamics study of active-site interactions with tetracoordinate transients in acetylcholinesterase and its mutants. 1117 Oct 62


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