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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)

Inferences about the catalytic mechanism of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) are frequently made on the basis of a presumed analogy with chymotrypsin, EC 3.4.21.1. Although both enzymes are serine hydrolases, several differences in the steady-state kinetic properties of the two have been observed. In this report particular attention is focused on the second-order reaction constant, kcat/Kapp. While the reported pH dependence and deuterium oxide isotope effect associated with this parameter for chymotrypsin are generally consistent with simple models involving rate-limiting general acid-base catalysis, this study finds a more complicated situation with acetylcholinesterase. The apparent pKa of kcat/Kapp for acetylcholinesterase varies between 5.5 and 6.3 for neutral substrates and involves nonlinear inhibition by [H+]. Deuterium oxide isotope effects for kcat/Kapp range from 1.1 for acetylcholine to 1.9 for p-nitrophenyl acetate. The bimolecular reaction rate appears rate-limiting for acetylcholine at low concentrations, while a rate-limiting induced-fit step is proposed to account for apparent pKa values and low deuterium oxide isotope effects associated with low concentrations of phenyl acetate and isoamyl acetate.
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PMID:Catalysis by acetylcholinesterase: evidence that the rate-limiting step for acylation with certain substrates precedes general acid-base catalysis. 0 Jun 68

1. In a previous ESR study of a membrane acetylcholinesterase (EC 3.1.1.7) we found, contrary to observations by other authors, spectra indicating that the active serine might be located in a pocket of the enzyme surface. In order to inquire into this possibility, ESR spectra were studied under the influence of different physico-chemical factors known to cause an unfolding of proteins. 2. The active serine of the postsynaptic membrane acetylcholinesterase of Torpedo marmorata electric organ was spin labeled using 1-oxyl-2, 2, 6, 6-tetramethyl-4-piperidinyletoxyphosphonofluoridate. 3. The effect of the chosen physico-chemical factors was an increase in the rotational freedom of spin labels; this result corroborates the suggestion that the active center of our acetylcholinesterase preparation is located in a pocket.
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PMID:An ESR study of the influence of some physico-chemical factors on the conformation of a postsynaptic acetylcholinesterase. 0 30

Acetylcholinesterase from Banded krait (Bungarus multicinctus) venom has been purified by CM-Sephadex chromatography and affinity chromatography to a specific activity of 4290 U/mg. The purified enzyme is a glycoprotein. It is free of electrophoretically detectable contaminating proteins. A molecular weight of 140,000 +/- 5,000 has been determined by gradient gel electrophoresis for the native enzyme. It is split into two equal-sized subunits (Mr 70,000 +/- 2,000) by SDS treatment. The N-terminal amino acid analysis gave glycine and serine. The purified acetylcholinesterase can be resolved by disc gel electrophoresis into four and by isoelectric focusing into six isozymes. The pI value of the main isozyme has been found to be 5.98 +/- 0.05.
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PMID:[The acetylcholinesterase of Bungarus multicinctus venom. Purification and properties (author's transl)]. 15 47

The acetylcholinesterase was purified by CM-Sephadex chromatography and affinity chromatography on Sepharose bound m-[6-(6-aminocaproylamino)caproylamino]phenyltrimethylammonium bromide. The purified enzyme was obtained with a specific activity of 5470 U/mg (1160-fold purification) and a 89% yield. The molecular weight of the native enzyme was estimated to be 144,000. The enzyme is split into two subunits of approximately equal molecular weight (Mr 69,000) by SDS treatment. It is a glycoprotein and can be resolved by disc gel electrophoresis into seven and by isoelectric focusing into more than ten multiple forms. The N-terminal amino acid is serine.
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PMID:[Purification by affinity chromatography and properties of the acetylcholinesterase of formosan cobra (Naja naja atra) venom (author's transl)]. 53 51

Aminoalkyl benzenesulfonyl fluorides, like organophosphates, act as irreversible inhibitors of serine proteinases by splitting off hydrogen fluoride to form an enzyme-inhibitor complex, stable in the physiological pH region. Several of these compounds are characterized by a higher rate of inhibition when trypsin is used and the second order rate constants are compared with those of organophosphates. On the other hand, upon inhibition of human serum cholinesterase by DFP and 4-nitrophenyl diethyl phosphate, some orders of magnitude higher than that of benzenesul fonyl fluorydes are observed. As shown by an oral toxicity study in mice similar differences exist with respect to LD50 values.
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PMID:[Inhibition of the activity of human serum cholinesterase by aminoalkyl benzenesulfonyl fluorides]. 102 6

A bis-quaternary fluorescence probe, propidium diiodide, has been found to exhibit a tenfold enhancement of fluorescence when bound to acetylcholinesterase from Torpedo california. The complex is characterized by a high affinity, KD = 3.0 times 10-7 M, and 1:1 stoichiometry with the 82,000 molecular weight subunit of acetylcholinesterase. A wide variety of other quaternary ammonium ligands such as decamethonium, gallamine, d-tubocurarine, tetraethylammonium, and tetramethylammonium will completely dissociate propidium from the enzyme as will monovalent and divalent inorganic cations. The competitive dissociation does not show cooperative behavior or a distinct, requirement for occupation of multiple sites of different affinity to produce displacement. While a directly competitive relationship can be illustrated macroscopically, the various quaternary ligands show a different susceptibility toward inorganic cation displacement. The affinity of propidium relative to gallamine increases with ionic strength. This finding indicates that there is not complete equivalence in the negative subsites to which quaternary groups bind. Although edrophoniumwill also displace propidium from the enzyme, the dissociation constant obtained from this competitive relationship is 3.5 orders of magnitude greater than the constants obtained for inhibition of catalysis. By competitive displacement titrations it is shown that the primary binding site of edrophonium is distinct from that of propidium and a ternary complex with the two ligands can form on each subunit. In contrast to edrophonium, the binding of propidium is unaffected by methanesulfonylation of the active center serine and is uncompetitive with the carbamylating substrate, N-methyl-7-dimethylcarbamoxyquinolinium. Thus, it appears that propidium associates with a peripheral anionic center on the enzyme. Although propidium and edrophonium associate at separate sites on acetylcholinesterase, bis-quaternary ligands where the quaternary nitrogens are separated by 14 A displace both ligands from the enzyme with equal effectiveness.
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PMID:Interaction of fluorescence probes with acetylcholinesterase. The site and specificity of propidium binding. 112 7

The glycophospholipid-linked, amphiphilic form of acetylcholinesterase (AChE) from Torpedo californica and the hydrophilic form from mouse were overexpressed in Sf9 insect cells using the baculovirus expression system. Recombinant baculovirus, constructed by inserting AChE cDNA's into the genome of Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedron promoter, yielded recombinant enzyme varying between 0.5 and 3.8 mg/L. The recombinant enzyme was glycosylated although it migrated slightly more rapidly in SDS gel electrophoresis than enzyme purified from the electric organ of Torpedo. Kinetic properties of the recombinant DNA- and tissue-derived enzymes are identical. The detailed catalytic properties and susceptibility to inhibitors were examined for two enzyme mutations of the glutamate residue N-terminal to the active site serine. The Glu199 to Gln mutation shifted both the Km and Kss to higher substrate concentrations and resulted in a kcat of 28% of the wild type. Mutation of Glu199 to Asp also yielded a reduction in kcat but with no change in Km. Substrate inhibition normally apparent in wild-type AChE was eliminated with the Asp mutation, suggesting that substrate catalysis and substrate inhibition are not directly linked. Both mutations decreased the affinity of reversible inhibitors and reduced the rates of phosphorylation and carbamoylation; these changes were more striking with the Gln199 mutation. Decarbamoylation rates were unaffected by these mutations. Glu199 is the charged residue found deep within the active center gorge close to the site of acetylcholine binding, and our findings indicate it influences, but is not essential for, efficient catalysis.
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PMID:Expression of recombinant acetylcholinesterase in a baculovirus system: kinetic properties of glutamate 199 mutants. 135 36

The bases of using blood enzyme activity measurements [e.g. AChE, non-specific cholinesterase (BChE), carboxylesterase] as markers of organophosphate ester (OP) exposure are inhibition of activity by the binding of OPs to serine active sites in the enzymes, and the accessibility of the enzymes in RBCs and serum. The methods used to determine esterases in the blood of humans, experimental animals, and wildlife are outlined with emphasis on the acetylcholinesterase (AChE) of the red blood cell. Adaptations of an acetylthiocholine ester assay of Ellman et al. (1961) are common, but other colorimetric procedures, radiometric assays, and pH methods are also in use. Optimized, standardized methods are needed to assess exposures and provide a solid basis for risk assessment analyses. Useful adjuncts to ChE measurements are oxime reactivation tests and assay of neuropathy target esterase, an enzyme associated with organophosphate-induced delayed neuropathy. Determination of urinary metabolites compliments, but does not substitute for, the information obtained from blood ChE studies. Future assays are likely to involve antibodies to OP-protein complexes. Improvements in techniques permit the detection of small decreases in ChE activities. Whether or not such small decreases in ChE activities can, by themselves, constitute an adverse effect for input into risk assessment analyses is a controversial matter.
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PMID:Blood esterase determinations as markers of exposure. 141 Jun 89

Introduction of the triple bond in the leaving group of the organophosphorus inhibitor molecule gives a sharp raise of the inhibitor activity but does not change principal characteristics of the cholinesterase inhibition mechanism. The reactivation experiments suggest that inactivation of cholinesterases by these compounds occurs due to phosphorylating of the serine hydroxyl by the corresponding phosphoric acid. A close similarity was shown between acetylenic and saturated organophosphorus inhibitors in altering ka upon change of pH and tetraalkylammonium ions action. It is demonstrated that S-alkynyl esters of thioacetic acid are slowly hydrolyzed by acetylcholinesterase and cholinesterase without irreversible inhibition of the enzymes.
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PMID:[The mechanism of anticholinesterase action of acetylene organophosphorus inhibitors]. 144 34

The distribution of amino acids between plasma, liver and brain was studied in adult male rats, fed a diet containing 8.7, 17 (control animals), 32 and 51% of protein during 15 days. The caloric intake was nearly equal in all groups. The highest food intake was observed in the animals on the low protein diet. Changes in plasma amino acids were variable. In contrast to the behavior of most amino acids in plasma, the branched chain amino acids were highest in the animals fed the 51% protein diet. Despite the low protein intake in the animals fed a 8.7% protein diet, the concentration of serine, glutamic acid, glutamine, glycine, alanine, methionine, isoleucine, leucine, phenylalanine and ornithine were significantly higher compared to control animals, whereas in those receiving a high protein diet, valine, leucine, tyrosine, tryptophan and histidine increased in relation to the increased protein and amino acid intake. The plasma amino acid patterns are not greatly influenced by the amino acid distribution in the food and the amount ingested. Alanine aminotransferase, aspartate aminotransferase, glutamate dehydrogenase and cholinesterase showed a two- to fivefold increased activity in the liver of animals consuming a high protein diet. In the brain, the concentration of valine, leucine, isoleucine, phenylalanine and tyrosine in animals receiving the low protein diet was higher than in controls and increased further with increasing protein content of the diet. Glutamine was increased in all dietary groups. The predicted influx of amino acids showed increasing influx rates in dependence of the plasma amino acid concentration. The entry of tyrosine and tryptophan and their brain concentration was inversely proportional to the protein content of the diet. In the present study which considers long-term adaptation to an increasing protein and amino acid intake in comparison to a balanced control protein diet, the levels of the indispensable amino acids were maintained within narrow limits in the brain and liver. The results indicate that inspite of a variable protein intake, the body tends to keep organ amino acids in relatively narrow limits favoring in this way amino acid homeostasis.
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PMID:Effect of different protein diets on the distribution of amino acids in plasma, liver and brain in the rat. 159 Jun 69


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