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

---

Query: EC:3.1.1.8 (cholinesterase)
12,691 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The acidic amino acid residue required for the catalytic activity of rat pancreatic cholesterol esterase has been identified in this study by sequence comparison with other serine esterases and by site-directed mutagenesis experiments. The sequence comparison studies identified 3 acidic residues in homologous domains between cholesterol esterase, acetylcholinesterase, cholinesterase, and Geotrichum candida lipase that may potentially be the catalytic acidic residue in these proteins. The role of Glu78, Asp79, and Asp320 in the catalytic activity of rat cholesterol esterase was then addressed by mutagenesis and expression of the cDNA. Results showed that replacement of Glu78 or Asp79 with alanine has no effect on the ability of the cholesterol esterase to hydrolyze the artificial water-soluble substrate p-nitrophenyl butyrate. In contrast, the Asp320-->Ala320 substitution abolished the enzyme activity of the cholesterol esterase. The specific requirement of Asp320 for optimal enzyme activity was demonstrated by substitution of the aspartic acid with glutamic acid, thus retaining the charge unit at this position. The Asp320-->Glu320 substitution resulted in an enzyme that displayed normal interaction with bile salt. However, catalytic activity of this mutagenized protein was reduced by approximately 50%. These results strongly suggested that aspartic acid 320 is an important component of the catalytic triad of pancreatic cholesterol esterase. The specific requirement of aspartic acid, instead of glutamic acid, for optimal activity is different from that of other members of the serine esterase gene family.
...
PMID:Aspartic acid 320 is required for optimal activity of rat pancreatic cholesterol esterase. 841 37

People with genetic variants of cholinesterase (ChE) have been reported to have prolonged apnea with the use of myorelaxant succinylcholine. For the silent type variant ChE, two cases of mutation have been reported. In one case, the exon 2 of ChE gene was disrupted by a 342 bp insertion of Alu element. In the other case, a frame shift mutation was identified at Gly-117 (GGT-->GGAG) to create a stop codon at nucleotide 384. Dibucaine resistant ChE was examined and found to have a point mutation at nucleotide 209 (A-->G) that converted Asp-70 to Gly, and consequently reduced the affinity of ChE for choline esters. In addition, another two types of a point mutation reducing ChE activity were reported on K variant (Ala-539-->Thr) and a case of (Gly-365-->Arg) in a patient with liver cirrhosis.
...
PMID:[Gene analysis of human cholinesterase variants]. 846 62

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.
...
PMID:Monoclonal antibodies against a C-terminal peptide of human brain acetylcholinesterase distinguish between erythrocyte and brain acetylcholinesterases. 856 26

The atypical variant of human butyrylcholinesterase has Gly in place of Asp 70. Patients with this D70G mutation respond abnormally to the muscle relaxant succinyldicholine, experiencing hours of apnea rather than the intended 3 min. Asp 70 is at the rim of the active site gorge 12 A from the active site Ser 198. An unanswered question in the literature is why the atypical variant has a 10-fold increase in Km for compounds with a single positive charge but a 100-fold increase in Km for compounds with two positive charges. We mutated residues Asp 70, Trp 82, Trp 231, Glu 197, and Tyr 332 and expressed mutant enzymes in mammalian cells. Steady-state kinetic parameters for hydrolysis of butyrylthiocholine, benzoylcholine, succinyldithiocholine, and o-nitrophenyl butyrate were determined. The wild type and the D70G mutant had identical k(cat) values for all substrates. Molecular modeling and molecular dynamics suggested that succinyldicholine could bind in two consecutive orientations in the active site gorge; formation of one complex caused a conformational change in the omega loop involving Asp 70 and Trp 82. We propose the formation of three enzyme-substrate intermediates preceding the acyl-enzyme intermediate; kinetic data support this contention. Substrates with a single positive charge interact with Asp 70 just once, whereas substrates with two positive charges, for example succinyldithiocholine, interact with Asp 70 in two complexes, thus explaining the 10- and 100-fold increases in Km in the D70G mutant.
...
PMID:Role of aspartate 70 and tryptophan 82 in binding of succinyldithiocholine to human butyrylcholinesterase. 904 29

An unstable variant of human butyrylcholinesterase (BChE) is described in four apparently unrelated individuals sensitive to succinylcholine. Sequencing of genomic DNA revealed a single nucleotide substitution which results in the replacement of amino acid residue Gly115 by Asp. This variant can be recognized by its increased instability under extremes of temperature such as heating and also freezing and thawing, both in homozygous and heterozygous states. When in heterozygous combination with the Atypical variant, it produces dibucaine and fluoride numbers which are intermediary between those of Atypical homozygotes and heterozygotes. After repeated freezing and thawing, however, these values approach those of homozygous Atypical plasma. Measurement of activity and immunoreactive BChE protein in plasma of individuals representing different combinations of this allele indicated that the presence of the Usual or Atypical enzymes seems to partially protect this variant from denaturation in vivo. Phenotyping fresh serum or plasma samples, before they are frozen, is critical for the identification of this, and possibly some other, unstable variants.
...
PMID:Characterization of an unstable variant (BChE115D) of human butyrylcholinesterase. 911 Mar 59

Asp-70 is the defining amino acid in the peripheral anionic site of human butyrylcholinesterase (BuChE), whereas acetylcholinesterase has several additional amino acids, the most important one being Trp-277 (Trp-279 in Torpedo AChE). We studied mutants D70G, D70K and A277W to evaluate the role of Asp-70 and Trp-277 in reactions with organophosphates. We found that Asp-70 was important for binding positively charged echothiophate, but not neutral paraoxon and iso-OMPA. Asp-70 was also important for binding of positively charged pralidoxime (2-PAM) and for activation of re-activation by excess 2-PAM. Excess 2-PAM had an effect similar to substrate activation, suggesting the binding of 2 mol of 2-PAM to wild-type but not to the D70G mutant. A surprising result was that Asp-70 was important for irreversible aging, the D70G mutant having a 3- and 8-fold lower rate of aging for paraoxon-inhibited and di-isopropyl fluorophosphate-inhibited BuChE. Mutants of Asp-70 had the same rate constants for phosphorylation and re-activation by 2-PAM as wild-type. The A277W mutant behaved like wild-type in all assays. Our results predict that people with the atypical (D70G) variant of BuChE will be more sensitive to the toxic effects of echothiophate, but will be equally sensitive to paraoxon and di-isopropyl fluorophosphate. People with the D70G mutation will be resistant to re-activation of their inhibited BuChE by 2-PAM, but this will be offset by the lower rate of irreversible aging of inhibited BuChE, allowing some regeneration by spontaneous hydrolysis.
...
PMID:Importance of aspartate-70 in organophosphate inhibition, oxime re-activation and aging of human butyrylcholinesterase. 922 29

Organophosphate-inhibited cholinesterases can be reactivated by nucleophilic compounds. Sometimes phosphylated (phosphorylated or phosphonylated) cholinesterases become progressively refractory to reactivation; this can result from different reactions. The most frequent process, termed 'aging', involves the dealkylation of an alkoxy group on the phosphyl moiety through a carbocation mechanism. In attempting to determine the amino acid residues involved in the aging of butyrylcholinesterase (BuChE), the human BuChE gene was mutated at several positions corresponding to residues located at the rim of the active site gorge and in the vicinity of the active site. Mutant enzymes were expressed in Chinese hamster ovary cells. Wild-type BuChE and mutants were inhibited by di-isopropylfluorophosphate at pH 8.0 and 25 degrees C. Di-isopropyl-phosphorylated enzymes were incubated with the nucleophilic oxime 2-pyridine aldoxime methiodide and their reactivatability was determined. Reactivatability was expressed by the first-order rate constant of aging and/or the half-life of aging (t12). The t12 was found to be of the order of 60 min for wild-type BuChE. Mutations on Glu-197 increased t12 60-fold. Mutation W82A increased t12 13-fold. Mutation D70G increased t12 8-fold. Mutations in the vicinity of the active site serine residue had either moderate or no effect on aging; t12 was doubled for F329C and F329A, increased only 4-fold for the double mutant A328G+F329S, and no change was observed for the A328G mutant, indicating that the isopropoxy chain to be dealkylated does not directly interact with Ala-328 and Phe-329. These results were interpreted by molecular modelling of di-isopropylphosphorylated wild-type and mutant enzymes. Molecular dynamics simulations indicated that the isopropyl chain that is lost interacted with Trp-82, suggesting that Trp-82 has a role in stabilizing the carbonium ion that is released in the dealkylation step. This study emphasized the important role of the Glu-197 carboxylate in stabilizing the developing carbocation, and the allosteric control of the dealkylation reaction by Asp-70. Indeed, although Asp-70 does not interact with the phosphoryl moiety, mutation D70G affects the rate of aging. This indirect control was interpreted in terms of change in the conformational state of Trp-82 owing to internal motions of the Omega loop (Cys-65-Cys-92) in the mutant enzyme.
...
PMID:Aging of di-isopropyl-phosphorylated human butyrylcholinesterase. 935 35

Three different mutations at codons 330 (TTA to ATA), 365 (GGA to AGA) and 515 (CGT to TGT) of human butyrylcholinesterase (hBChE) were identified in a Japanese family. We correlated alterations in in the patient's hBChE activity with possible structural alterations in the three-dimensional structure of hBChE caused by the point mutations. This study was performed using the published computer-generated three-dimensional structure of hBChE based on the structure of acetylcholinesterase. The amino acid substitution at L330I was adjacent to hydrophobic residues that form the channel domain of the active center. This side chain faced the side opposite the active center. The amino acid substitution at G365R was located at the position most remote from the active center, and this substitution site was exposed to the surface of the BChE protein. Alpha-helical structure was present to the active center, and the guanidyl residue of native Arg 515 was hydrogen-bonded to the carboxyl group of Asp 395 in the alpha-helix. These point mutations may cause steric effects on the present patient's hBChE activity. This is the first report of three-dimensional structural analysis performed on the L330I, G365R, and R515C mutations of hBChE.
...
PMID:Three point mutations of human butyrylcholinesterase in a Japanese family and the alterations of three-dimensional structure. 1040 29

Wild-type human butyrylcholinesterase (BuChE) and Glu-197-->Asp and Asp-70-->Gly mutants (E197D and D70G respectively) were inhibited by di-isopropyl phosphorofluoridate under standard conditions of pH, temperature and pressure. The effect of hydrostatic and osmotic pressures on the aging process (dealkylation of an isopropyl chain) of phosphorylated enzymes [di-isopropylated (DIP)-BuChE] was investigated. Hydrostatic pressure markedly increased the rate of aging of wild-type enzyme. The average activation volume (DeltaV( not equal)) for the dealkylation reaction was -170 ml/mol for DIP wild-type BuChE. On the other hand, hydrostatic pressure had little effect on the aging of the DIP mutants (DeltaV( not equal)=-2.6 ml/mol for E197D and -2 ml/mol for D70G), suggesting that the transition state of the aging process was associated with an extended hydration and conformational change in wild-type BuChE, but not in the mutants. The rate of aging of wild-type and mutant enzymes decreased with osmotic pressure, allowing very large positive osmotic activation volumes (DeltaV not equal osm) to be estimated, thus probing the participation of water in the aging process. Molecular dynamics simulations performed on the active-site gorge of the wild-type DIP adduct showed that the isopropyl chain involved in aging was highly solvated, supporting the idea that water is important for stabilizing the transition state of the dealkylation reaction. Wild-type BuChE was inhibited by soman (pinacolyl methylphosphonofluoridate). Electrophoresis performed under high pressure [up to 2.5 kbar (1 bar=10(5) Pa)] showed that the soman-aged enzyme did not pass through a pressure-induced, molten-globule transition, unlike the native wild-type enzyme. Likewise, this transition was not seen for the native E197D and D70G mutants, indicating that these mutants are resistant to the penetration of water into their structure. The stability energetics of native and soman-aged wild-type BuChE were determined by differential scanning calorimetry. The pH-dependence of the midpoint transition temperature of endotherms indicated that the high difference in stabilization energy between aged and native BuChE (DeltaDeltaG=23.7 kJ/mol at pH 8.0) is mainly due to the salt bridge between protonated His-438 and PO(-), with pK(His-438)=8.3. A molecular dynamics simulation on the MIP adduct showed that there is no water molecule around the ion pair. The 'hydrostatic versus osmotic pressure' approach probed the importance of water in aging, and also revealed that Asp-70 and Glu-197 are the major residues controlling both the dynamics and the structural organization of the water/hydrogen-bond network in the active-site gorge of BuChE. In wild-type BuChE both residues function like valves, whereas in the mutant enzymes the water network is slack, and residues Gly-70 and Asp-197 function like check valves, i.e. forced penetration of water into the gorge is not easily achieved, thereby facilitating the release of water.
...
PMID:Hydration change during the aging of phosphorylated human butyrylcholinesterase: importance of residues aspartate-70 and glutamate-197 in the water network as probed by hydrostatic and osmotic pressures. 1051 Mar 1

Cholinesterases (ChE), use a Glu-His-Ser catalytic triad to enhance the nucleophilicity of the catalytic serine. It has been shown that serine proteases, which employ an Asp-His-Ser catalytic triad for optimal catalytic efficiency, decrease the hydrogen bonding distance between the Asp-His pair to form a short, strong hydrogen bond (SSHB) upon binding mechanism-based inhibitors, which form tetrahedral Ser-adducts, analogous to the tetrahedral intermediates in catalysis, or at low pH when the histidine is protonated [Cassidy, C. S., Lin, J., Frey, P. A. (1997) Biochemistry 36, 4576-4584]. Two types of mechanism-based inhibitors were bound to pure equine butyrylcholinesterase (BChE), a 364 kDa homotetramer, and the complexes were studied by (1)H NMR at 600 MHz and 25-37 degrees C. The downfield region of the (1)H NMR spectrum of free BChE at pH 7.5 showed a broad, weak, deshielded resonance with a chemical shift, delta = 16.1 ppm, ascribed to a small amount of the histidine-protonated form. Upon addition of a 3-fold excess of diethyl 4-nitrophenyl phosphate (paraoxon) and subsequent dealkylation, the broad 16.1 ppm resonance increased in intensity 4.7-fold, and yielded a D/H fractionation factor phi = 0.72+/-0.10 consistent with a SSHB between Glu and His of the catalytic triad. From an empirical correlation of delta with hydrogen-bond length in small crystalline compounds, the length of this SSBH is 2.64+/-0.04 A, in agreement with the length of 2.62+/-0.02 A independently obtained from phi. The addition of a 3-fold excess of m-(N,N, N-trimethylammonio)trifluoroacetophenone to BChE yielded no signal at 16.1 ppm, and a 640 Hz broad, highly deshielded proton resonance with a chemical shift delta = 18.1 ppm and a D/H fractionation factor phi = 0.63+/-0.10, also consistent with a SSHB. The length of this SSHB is calculated to be 2.62+/-0.04 A from delta and 2.59+/-0.03 A from phi. These NMR-derived distances agree with those found in the X-ray structures of the homologous acetylcholinesterase complexed with the same mechanism-based inhibitors, 2.60+/-0.22 and 2.66+/-0.28 A. However, the order of magnitude greater precision of the NMR-derived distances establish the presence of SSHBs. We suggest that ChEs achieve their remarkable catalytic power in ester hydrolysis, in part, due to the formation of a SSHB between Glu and His of the catalytic triad.
...
PMID:NMR evidence for a short, strong hydrogen bond at the active site of a cholinesterase. 1112 49


<< Previous 1 2 3 4 Next >>

---