Esterase with broad substrate specificity. Contributes to the inactivation of the neurotransmitter acetylcholine. Can degrade neurotoxic organophosphate esters.
Organophosphate ester (OP) compounds are known for their ubiquitous use as insecticides. At the same time, these chemicals are highly toxic and can be used as nerve agents. G117H mutant of human Butyrylcholinesterase (BChE) was found to be capable of hydrolyzing certain OPs and protect against their toxicity. However, for therapeutic use, the rate of hydrolysis is too low. Its catalytic power can be improved by rational design, but the structure of the G117H mutant is first required. In this work, we determined, computationally, the three dimensional structure of the G117H BChE mutant. The structure was then validated by simulating acetylation of acetylthiocholine (ATC). Several plausible conformers of G117H BChE were examined but only the (62,-75) conformer fully reproduced catalytic effect. The (62,-75) conformer is, therefore, suggested as the structure adopted by the G117H BChE mutant. This conformer is shown to explain the loss of esterase activity observed for the G122H Acetylcholinesterase mutant together with its recovery when additional mutations are placed turning the enzyme also into an OP hydrolase. Furthermore, similarity of the structure to the structure of RNase A, which is known to hydrolyze the O--P bond in RNA, grants it further credibility and suggests a mechanism for the OP hydrolysis.
Human serum butyrylcholinesterase (Hu BChE) is a promising therapeutic against the toxicity of chemical warfare nerve agents. We have showed previously that recombinant (r) Hu BChE can be expressed at very high levels, 400 to 600 U/ml in mouse blood, by delivering the Hu BChE gene using adenovirus (Ad). Here, we report the biochemical properties of the Ad-expressed full-length and truncated rHu BChE in mouse blood. The molecular sizes of the full-length rHu BChE subunit and its oligomers were similar to those of native Hu BChE, although only a small portion of the full-length rHu BChE subunit underwent assembly into dimers and tetramers. As expected, Ad containing the truncated Hu BChE gene transduced the expression of monomeric rHu BChE only. Compared with 415 U of rHu BChE per milliliter in blood, tissues including liver, lung, heart, brain, kidney, muscle, intestine, diaphragm, salivary gland, and fat expressed <10 U/g of rHu BChE activity. Ad-expressed rHu BChE in mouse blood neutralized soman and O-ethyl S-2-N,N-diisopropylaminoethyl methylphosphonothiolate at rates similar to those of native Hu BChE and rHu BChE expressed in vitro. Because the expression of rHu BChE rapidly decreased 6 days after virus administration, sera were assayed for the presence of anti-Hu BChE antibodies. Anti-Hu BChE antibodies were detected on day 7 and in increased amounts thereafter, which coincided with the loss of Hu BChE expression in sera. In conclusion, the delivery of Hu BChE gene using Ad can be a promising strategy that can provide protection against multiple lethal doses of chemical warfare nerve agents in vivo.
1. Acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BuChE, EC 3.1.1.8) are enzymes that catalyze the hydrolysis of esters of choline. 2. Both AChE and BuChE have been shown to copurify with peptidases. 3. BuChE has also been shown to copurify with other proteins such as transferrin, with which it forms a stable complex. In addition, BuChE is found in association with beta-amyloid protein in Alzheimer brain tissues. 4. Since BuChE copurifies with peptidases, we hypothesized that BuChE interacts with these enzymes and that this association had an influence on their catalytic activities. One of the peptidases that copurifies with cholinesterases has specificity similar to trypsin, hence, this enzyme was used as a model to test this hypothesis. 5. Purified BuChE causes a concentration-dependent enhancement of the catalytic activity of trypsin while trypsin does not influence the catalytic activity of BuChE. 6. We suggest that, in addition to its esterase activity, BuChE may assume a regulatory role by interacting with other proteins.
Catalysis of a biochemical reaction at physiological temperatures. In biologically catalyzed reactions, the reactants are known as substrates, and the catalysts are naturally occurring macromolecular substances known as enzymes. Enzymes possess specific binding sites for substrates, and are usually composed wholly or largely of protein, but RNA that has catalytic activity (ribozyme) is often also regarded as enzymatic.
1. Acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BuChE, EC 3.1.1.8) are enzymes that catalyze the hydrolysis of esters of choline. 2. Both AChE and BuChE have been shown to copurify with peptidases. 3. BuChE has also been shown to copurify with other proteins such as transferrin, with which it forms a stable complex. In addition, BuChE is found in association with beta-amyloid protein in Alzheimer brain tissues. 4. Since BuChE copurifies with peptidases, we hypothesized that BuChE interacts with these enzymes and that this association had an influence on their catalytic activities. One of the peptidases that copurifies with cholinesterases has specificity similar to trypsin, hence, this enzyme was used as a model to test this hypothesis. 5. Purified BuChE causes a concentration-dependent enhancement of the catalytic activity of trypsin while trypsin does not influence the catalytic activity of BuChE. 6. We suggest that, in addition to its esterase activity, BuChE may assume a regulatory role by interacting with other proteins.
Organophosphate ester (OP) compounds are known for their ubiquitous use as insecticides. At the same time, these chemicals are highly toxic and can be used as nerve agents. G117H mutant of human Butyrylcholinesterase (BChE) was found to be capable of hydrolyzing certain OPs and protect against their toxicity. However, for therapeutic use, the rate of hydrolysis is too low. Its catalytic power can be improved by rational design, but the structure of the G117H mutant is first required. In this work, we determined, computationally, the three dimensional structure of the G117H BChE mutant. The structure was then validated by simulating acetylation of acetylthiocholine (ATC). Several plausible conformers of G117H BChE were examined but only the (62,-75) conformer fully reproduced catalytic effect. The (62,-75) conformer is, therefore, suggested as the structure adopted by the G117H BChE mutant. This conformer is shown to explain the loss of esterase activity observed for the G122H Acetylcholinesterase mutant together with its recovery when additional mutations are placed turning the enzyme also into an OP hydrolase. Furthermore, similarity of the structure to the structure of RNase A, which is known to hydrolyze the O--P bond in RNA, grants it further credibility and suggests a mechanism for the OP hydrolysis.
1. Acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BuChE, EC 3.1.1.8) are enzymes that catalyze the hydrolysis of esters of choline. 2. Both AChE and BuChE have been shown to copurify with peptidases. 3. BuChE has also been shown to copurify with other proteins such as transferrin, with which it forms a stable complex. In addition, BuChE is found in association with beta-amyloid protein in Alzheimer brain tissues. 4. Since BuChE copurifies with peptidases, we hypothesized that BuChE interacts with these enzymes and that this association had an influence on their catalytic activities. One of the peptidases that copurifies with cholinesterases has specificity similar to trypsin, hence, this enzyme was used as a model to test this hypothesis. 5. Purified BuChE causes a concentration-dependent enhancement of the catalytic activity of trypsin while trypsin does not influence the catalytic activity of BuChE. 6. We suggest that, in addition to its esterase activity, BuChE may assume a regulatory role by interacting with other proteins.
The chemical reactions and pathways involving choline (2-hydroxyethyltrimethylammonium), an amino alcohol that occurs widely in living organisms as a constituent of certain types of phospholipids and in the neurotransmitter acetylcholine.
The chemical reactions and pathways involving cocaine, an alkaloid obtained from the dried leaves of the shrub Erythroxylon coca. It is a cerebral stimulant and narcotic.
Life Sci. 58, PL257-61-PL257-61 (1996)[PubMed:8622553]
In humans, the plasma enzyme, butyrylcholinesterase (E.C. 3.1.1.8), metabolizes cocaine to the water-soluble, pharmacologically inactive compounds, ecgonine methylester and benzoic acid. Homogeneous enzyme was purified from human plasma and used to determine the enzyme kinetic parameters of Km and Vmax with cocaine as the substrate. The KM (11.9 microM) indicates that cocaine is tightly bound to the four active sites of the native tetramer. The Vmax (1.17 microM/min) is 50-fold greater than cocaine catalytic antibodies. Administration of purified human butyrylcholinesterase to a cocaine-intoxicated patient would be expected to shift the metabolism to the inactive metabolites and reduce the toxicity.
hBChE [human BChE (butyrylcholinesterase)] naturally scavenges OPs (organophosphates). This bioscavenger is currently in Clinical Phase I for pretreatment of OP intoxication. Phosphylated ChEs (cholinesterases) can undergo a spontaneous time-dependent process called 'aging' during which the conjugate is dealkylated, leading to creation of an enzyme that cannot be reactivated. hBChE inhibited by phosphoramidates such as tabun displays a peculiar resistance to oxime-mediated reactivation. We investigated the basis of oxime resistance of phosphoramidyl-BChE conjugates by determining the kinetics of inhibition, reactivation (obidoxime {1,1'-(oxybis-methylene) bis[4-(hydroxyimino) methyl] pyridinium dichloride}, TMB-4 [1,3-trimethylene-bis(4-hydroxyiminomethylpyridinium) dibromide], HLö 7 {1-[[[4-(aminocarbonyl) pyridinio]methoxy]methyl]-2,4-bis-[(hydroxyimino)methyl] pyridinium dimethanesulfonate)}, HI-6 {1-[[[4-(aminocarbonyl) pyridinio] methoxy] methyl]-2-[(hydroxyimino)methyl]pyridinium dichloride monohydrate} and aging, and the crystal structures of hBChE inhibited by different N-monoalkyl and N,N-dialkyl tabun analogues. The refined structures of aged hBChE conjugates show that aging proceeds through O-dealkylation of the P(R) enantiomer of N,N-diethyl and N-propyl analogues, with subsequent formation of a salt bridge preventing reactivation, similarly to a previous observation made on tabun-ChE conjugates. Interestingly, the N-methyl analogue projects its amino group towards the choline-binding pocket, so that aging proceeds through deamination. This orientation results from a preference of hBChE's acyl-binding pocket for larger than 2-atoms linear substituents. The correlation between the inhibitory potency and the N-monoalkyl chain length is related to increasingly optimized interactions with the acyl-binding pocket as shown by the X-ray structures. These kinetics and X-ray data lead to a structure-activity relationship that highlights steric and electronic effects of the amino substituent of phosphoramidate. This study provides the structural basis to design new oximes capable of reactivating phosphoramidyl-hBChE conjugates after intoxication, notably when hBChE is used as pretreatment, or to design BChE-based catalytic bioscavengers.
Organophosphate ester (OP) compounds are known for their ubiquitous use as insecticides. At the same time, these chemicals are highly toxic and can be used as nerve agents. G117H mutant of human Butyrylcholinesterase (BChE) was found to be capable of hydrolyzing certain OPs and protect against their toxicity. However, for therapeutic use, the rate of hydrolysis is too low. Its catalytic power can be improved by rational design, but the structure of the G117H mutant is first required. In this work, we determined, computationally, the three dimensional structure of the G117H BChE mutant. The structure was then validated by simulating acetylation of acetylthiocholine (ATC). Several plausible conformers of G117H BChE were examined but only the (62,-75) conformer fully reproduced catalytic effect. The (62,-75) conformer is, therefore, suggested as the structure adopted by the G117H BChE mutant. This conformer is shown to explain the loss of esterase activity observed for the G122H Acetylcholinesterase mutant together with its recovery when additional mutations are placed turning the enzyme also into an OP hydrolase. Furthermore, similarity of the structure to the structure of RNase A, which is known to hydrolyze the O--P bond in RNA, grants it further credibility and suggests a mechanism for the OP hydrolysis.
hBChE [human BChE (butyrylcholinesterase)] naturally scavenges OPs (organophosphates). This bioscavenger is currently in Clinical Phase I for pretreatment of OP intoxication. Phosphylated ChEs (cholinesterases) can undergo a spontaneous time-dependent process called 'aging' during which the conjugate is dealkylated, leading to creation of an enzyme that cannot be reactivated. hBChE inhibited by phosphoramidates such as tabun displays a peculiar resistance to oxime-mediated reactivation. We investigated the basis of oxime resistance of phosphoramidyl-BChE conjugates by determining the kinetics of inhibition, reactivation (obidoxime {1,1'-(oxybis-methylene) bis[4-(hydroxyimino) methyl] pyridinium dichloride}, TMB-4 [1,3-trimethylene-bis(4-hydroxyiminomethylpyridinium) dibromide], HLö 7 {1-[[[4-(aminocarbonyl) pyridinio]methoxy]methyl]-2,4-bis-[(hydroxyimino)methyl] pyridinium dimethanesulfonate)}, HI-6 {1-[[[4-(aminocarbonyl) pyridinio] methoxy] methyl]-2-[(hydroxyimino)methyl]pyridinium dichloride monohydrate} and aging, and the crystal structures of hBChE inhibited by different N-monoalkyl and N,N-dialkyl tabun analogues. The refined structures of aged hBChE conjugates show that aging proceeds through O-dealkylation of the P(R) enantiomer of N,N-diethyl and N-propyl analogues, with subsequent formation of a salt bridge preventing reactivation, similarly to a previous observation made on tabun-ChE conjugates. Interestingly, the N-methyl analogue projects its amino group towards the choline-binding pocket, so that aging proceeds through deamination. This orientation results from a preference of hBChE's acyl-binding pocket for larger than 2-atoms linear substituents. The correlation between the inhibitory potency and the N-monoalkyl chain length is related to increasingly optimized interactions with the acyl-binding pocket as shown by the X-ray structures. These kinetics and X-ray data lead to a structure-activity relationship that highlights steric and electronic effects of the amino substituent of phosphoramidate. This study provides the structural basis to design new oximes capable of reactivating phosphoramidyl-hBChE conjugates after intoxication, notably when hBChE is used as pretreatment, or to design BChE-based catalytic bioscavengers.
Human butyrylcholinesterase (hBChE) hydrolyzes or scavenges a wide range of toxic esters, including heroin, cocaine, carbamate pesticides, organophosphorus pesticides, and nerve agents. Organophosphates (OPs) exert their acute toxicity through inhibition of acetylcholinesterase (AChE) by phosphorylation of the catalytic serine. Phosphylated cholinesterase (ChE) can undergo a spontaneous, time-dependent process called "aging", during which the OP-ChE conjugate is dealkylated. This leads to irreversible inhibition of the enzyme. The inhibition of ChEs by tabun and the subsequent aging reaction are of particular interest, because tabun-ChE conjugates display an extraordinary resistance toward most current oxime reactivators. We investigated the structural basis of oxime resistance for phosphoramidated ChE conjugates by determining the crystal structures of the non-aged and aged forms of hBChE inhibited by tabun, and by updating the refinement of non-aged and aged tabun-inhibited mouse AChE (mAChE). Structures for non-aged and aged tabun-hBChE were refined to 2.3 and 2.1 A, respectively. The refined structures of aged ChE conjugates clearly show that the aging reaction proceeds through O-dealkylation of the P(R) enantiomer of tabun. After dealkylation, the negatively charged oxygen forms a strong salt bridge with protonated His438N epsilon2 that prevents reactivation. Mass spectrometric analysis of the aged tabun-inhibited hBChE showed that both the dimethylamine and ethoxy side chains were missing from the phosphorus. Loss of the ethoxy is consistent with the crystallography results. Loss of the dimethylamine is consistent with acid-catalyzed deamidation during the preparation of the aged adduct for mass spectrometry. The reported 3D data will help in the design of new oximes capable of reactivating tabun-ChE conjugates.
The poorly known mechanism of inhibition of cholinesterases by inorganic mercury (HgCl2) has been studied with a view to using these enzymes as biomarkers or as biological components of biosensors to survey polluted areas. The inhibition of a variety of cholinesterases by HgCl2 was investigated by kinetic studies, X-ray crystallography, and dynamic light scattering. Our results show that when a free sensitive sulfhydryl group is present in the enzyme, as in Torpedo californica acetylcholinesterase, inhibition is irreversible and follows pseudo-first-order kinetics that are completed within 1 h in the micromolar range. When the free sulfhydryl group is not sensitive to mercury (Drosophila melanogaster acetylcholinesterase and human butyrylcholinesterase) or is otherwise absent (Electrophorus electricus acetylcholinesterase), then inhibition occurs in the millimolar range. Inhibition follows a slow binding model, with successive binding of two mercury ions to the enzyme surface. Binding of mercury ions has several consequences: reversible inhibition, enzyme denaturation, and protein aggregation, protecting the enzyme from denaturation. Mercury-induced inactivation of cholinesterases is thus a rather complex process. Our results indicate that among the various cholinesterases that we have studied, only Torpedo californica acetylcholinesterase is suitable for mercury detection using biosensors, and that a careful study of cholinesterase inhibition in a species is a prerequisite before using it as a biomarker to survey mercury in the environment.
Enzyme which catalyzes hydrolysis reaction, i.e. the addition of the hydrogen and hydroxyl ions of water to a molecule with its consequent splitting into two or more simpler molecules.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
My favorites 
My labels 
Login
