Interaction of human butyrylcholinesterase variants with bambuterol and terbutaline

Bambuterol, a dimethylcarbamate, carbamoylates butyrylcholinesterase (BChE; EC 3.1.1.8). The carbamoylated enzyme is not very stable and the final product of the two-step hydrolysis is a bronchodilator drug, terbutaline (1-(3,5-dihydroxyphenyl)-2-t-butylamino-ethanol sulphate). Both bambuterol and terbutaline inhibit BChE, but their affinities differ in human serum BChE variants (U, A, F, K and S) due to their positive charge. Bambuterol inhibition rate constants for the homozygous usual (UU), Kalow (KK), fluoride-resistant (FF) or atypical (AA) variant ranged from 4.4 to 0.085min (-1)microM(-1). Terbutaline showed competitive reversible inhibition for all BChE variants. The dissociation constants for UU, FF and AA homozygotes were 0.18, 0.31 and 3.3 mM, respectively. The inhibition rate or dissociation constants for heterozygotes were distributed between the respective constants for the corresponding homozygotes. A 50-fold difference in inhibition between the UU and AA enzyme might affect terbutaline release in humans. The affinity of all studied BChE variants for terbutaline was low, which suggests that terbutaline originating from bambuterol hydrolysis should not affect the hydrolysis of bambuterol by BChE.     

Stereoselective inhibition of human, mouse, and horse cholinesterases by bambuterol enantiomers

Bambuterol is a chiral carbamate and a selective inhibitor of butyrylcholinesterase (BChE, EC 3.1.1.8). In order to relate bambuterol selectivity and stereoselectivity of BChE and acetylcholinesterase (AChE, EC 3.1.1.7) of different species, we studied the inhibition of human, mouse, and horse BChE, as well as AChE of human and mouse by (R)- and (S)-bambuterol. AChE and BChE of all studied species were progressively inhibited by both bambuterol enantiomers, with a preference for the (R)-bambuterol whose inhibition rate constants were about five times higher than that of (S)-bambuterol. We observed no significant difference between human and mouse in bambuterol enantiomer BChE inhibition. However, (R)-bambuterol inhibited horse BChE about 14 times slower than human and mouse BChE, and the inhibition rate for (S)-bambuterol was about 18 times slower. Although the primary structure of horse BChE differs from the other two species in 15 amino acids, we presumed that differences in inhibition rates could be attributed to threonine at position 69 located close to the peripheral site of BChE. Since BChE inhibition by bambuterol enantiomers was at least 8000 times faster than that of AChE, both bambuterol enantiomers proved to be selective BChE inhibitors, as was previously shown for racemate.     

Amino acid residues involved in the interaction of acetylcholinesterase and butyrylcholinesterase with the carbamates Ro 02-0683 and bambuterol, and with terbutaline.

In order to identify amino acids involved in the interaction of acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BChE; EC 3.1.1.8) with carbamates, the time course of inhibition of the recombinant mouse enzymes BChE wild-type (w.t.), AChE w.t. and of 11 site-directed AChE mutants by Ro 02-0683 and bambuterol was studied. In addition, the reversible inhibition of cholinesterases by terbutaline, the leaving group of bambuterol, was studied. The bimolecular rate constant of AChE w.t. inhibition was 6.8 times smaller by Ro 02-0683 and 16000 times smaller by bambuterol than that of BChE w.t. The two carbamates were equipotent BChE inhibitors. Replacement of tyrosine-337 in AChE with alanine (resembling the choline binding site of BChE) resulted in 630 times faster inhibition by bambuterol. The same replacement decreased the inhibition by Ro 02-0683 ten times. The difference in size of the choline binding site in the two w.t. enzymes appeared critical for the selectivity of bambuterol and terbutaline binding. Removal of the charge with the mutation D74N caused a reduction in the reaction rate constants for Ro 02-0683 and bambuterol. Substitution of tyrosine-124 with glutamine in the AChE peripheral site significantly increased the inhibition rate for both carbamates. Substitution of phenylalanine-297 with alanine in the AChE acyl pocket decreased the inhibition rate by Ro 02-0683. Computational docking of carbamates provided plausible orientations of the inhibitors inside the active site gorge of mouse AChE and human BChE, thus substantiating involvement of amino acid residues in the enzyme active sites critical for the carbamate binding as derived from kinetic studies.     

Amino acid residues involved in stereoselective inhibition of cholinesterases with bambuterol

Bambuterol is a chiral carbamate known as selective inhibitor of butyrylcholinesterase (BChE). In order to relate bambuterol selectivity and stereoselectivity of cholinesterases to the active site residues, we studied the inhibition of recombinant mouse BChE, acetylcholinesterase (AChE) and six AChE mutants, employed to mimic BChE active site residues, by bambuterol enantiomers. Both enantiomers selectively inhibited BChE about 8000 times faster than AChE. The largest inhibition rate increase in comparison to AChE w.t. was observed with the F295L/Y337A mutant, showing that leucine 295 and alanine 337 are crucial residues in BChE for high bambuterol selectivity. All studied enzymes preferred inhibition by the R- over the S-bambuterol. The enlargement of the AChE choline binding site and of the acyl pocket by single or double mutations (Y337A, F295L/Y337A and F297I/Y337A) increased, in comparison to w.t. enzymes, inhibition rate constants of R- bambuterol more than that of S- bambuterol resulting in four times higher stereoselectivity. Peripheral site mutations (Y124Q and Y72N/Y124Q/Y337A) increased inhibition rate by S- more than R-bambuterol and consequently diminished the stereoselectivity.     

Correlation between butyrylcholinesterase variants and sensitivity to soman toxicity

Butyrylcholinesterase (BChE) is synthesized in the liver and found in high concentrations in blood plasma, liver, heart, pancreas, vascular endothelium, skin, brain white matter, smooth muscle cells and adipocytes. BChE is a non specific enzyme that hydrolyzes different choline esters (succinylcholine, mivacurium) and many other drugs such as aspirin, cocaine and procaine. The enzyme is also considered as a bioscavenger due to its ability to neutralize the toxic effects of organophosphorus compounds (nervous system fs agents) such as soman. BChE displays several polymorphisms that influence its serum activity; therefore they could determine the individual sensitivity to chemical nerve agents. In this study, we investigated the correlation between BChE variants and the degree of enzyme inhibition and reactivation after soman application on blood samples of 726 individuals. The blood samples of individuals expressing abnormal variants, were more sensitive to soman compared to variants of homozygotes and heterozygotes for U-allele. We found significant differences in the degree of enzyme reactivation between different variants (with and without U-presence).     

Mechanism of stereoselective interaction between butyrylcholinesterase and ethopropazine enantiomers

Stereoselectivity of reversible inhibition of butyrylcholinesterase (BChE; EC 3.1.1.8) by optically pure ethopropazine [10-(2-diethylaminopropyl)phenothiazine hydrochloride] enantiomers and racemate was studied with acetylthiocholine (0.002–250 mM) as substrate. Molecular modelling resulted in the reaction between BChE and ethopropazine starting with the binding of ethopropazine to the enzyme peripheral anionic site. In the next step ethopropazine ‘slides down’ the enzyme gorge, resulting in interaction of the three rings of ethopropazine through π–π interactions with W82 in BChE. Inhibition mechanism was interpreted according to three kinetic models: A, B and C. The models differ in the type and number of enzyme–substrate, enzyme–inhibitor and enzyme–substrate–inhibitor complexes, i.e., presence of the Michaelis complex and/or acetylated BChE. Although, all three models reproduced well the BChE activity in absence of ethopropazine, model A was poor in describing inhibition with ethopropazine, while models B and C were better, especially for substrate concentrations above 0.2 mM. However model C was singled out because it approaches fulfilment of the one step-one event criteria, and confirms the inhibition mechanism derived from molecular modelling. Model C resulted in dissociation constants for the complex between BChE and ethopropazine: 61, 140 and 88 nM for R-enantiomer, S-enantiomer and racemate, respectively. The respective dissociation constants for the complexes between acetylated BChE and ethopropazine were 268, 730 and 365 nM. Butyrylcholinesterase had higher affinity for R-ethopropazine.     

Active site mutant acetylcholinesterase interactions with 2-PAM, HI-6, and DDVP

We used mouse recombinant wild-type acetylcholinesterase (AChE; EC 3.1.1.7), butyrylcholinesterase (BChE; EC 3.1.1.8), and AChE mutants with mutations (Y337A, F295L, F297I, Y72N, Y124Q, and W286A) that resemble residues found at structurally equivalent positions in BChE, to find the basis for divergence between AChE and BChE in following reactions: reversible inhibition by two oximes, progressive inhibition by the organophosphorus compound DDVP, and oxime-assisted reactivation of the phosphorylated enzymes. The inhibition enzyme-oxime dissociation constants of AChE w.t. were 150 and 46 microM, of BChE 340 and 27 microM for 2-PAM and HI-6, respectively. Introduced mutations lowered oxime binding affinities for both oximes. DDVP progressively inhibited cholinesterases yielding symmetrical dimethylphosphorylated enzyme conjugates at rates between 104 and 105/min/M. A high extent of oxime-assisted reactivation of all conjugates was achieved, but rates by both oximes were up to 10 times slower for phosphorylated mutants than for AChE w.t.     

Human serum butyrylcholinesterase interactions with cisplatin and cyclophosphamide

Human serum Butyrylcholinesterase (BChE) is an important enzyme in detoxification with its capacity for hydrolyzing esters. The inhibitory effect of cisplatin (CDDP) and cyclophosphamide (CY) on BChE is characterized. Time dependent inhibition of BChE with both chemotherapeutics was rapid, reversible. CY was found as non-competitive inhibitor with Ki of 503.6 ± 50.4 μM. Time dependent CDDP studies displayed progressive inhibition. The constants for apparent dissociation (Ka), first order constant for the break down of the Michaelis complex (k + 2), and bimolecular rate (ka) were calculated as 6.38 × 10⁻⁵ M⁻¹ min⁻¹, 0.063 min⁻¹, and 9.83 × 10⁻⁴ M, respectively. Enzyme protection could be achieved with moderate butyrylthiocholine concentrations (0.3 mM) but higher concentrations increased CDDP inhibition. Apparent Ki value for CDDP was 191.8 ± 71.2 μM. These results suggest that used in combination therapy, CY and CDDP cause considerable BChE inhibition and may aggravate conditions observed during chemotherapy.     

Steady-state kinetic analysis of human cholinesterases over wide concentration ranges of competing substrates

Substrate competition for human acetylcholinesterase (AChE) and human butyrylcholinesterase (BChE) was studies under steady-state conditions using wide range of substrate concentrations. Competing couples of substates were acetyl-(thio)esters. Phenyl acetate (PhA) was the reporter substrate and competitor were either acetylcholine (ACh) or acetylthiocholine (ATC). The common point between investigated substrates is that the acyl moiety is acetate, i.e. same deacylation rate constant for reporter and competitor substrate.Steady-state kinetics of cholinesterase-catalyzed hydrolysis of PhA in the presence of ACh or ATC revealed 3 phases of inhibition as concentration of competitor increased: a) competitive inhibition, b) partially mixed inhibition, c) partially uncompetitive inhibition for AChE and partially uncompetitive activation for BChE. This sequence reflects binding of competitor in the active centrer at low concentration and on the peripheral anionic site (PAS) at high concentration. In particular, it showed that binding of a competing ligand on PAS may affect the catalytic behavior of AChE and BChE in an opposite way, i.e. inhibition of AChE and activation of BChE, regardless the nature of the reporter substrate.For both enzymes, progress curves for hydrolysis of PhA at very low concentration (?K_m) in the presence of increasing concentration of ATC showed that: a) the competing substrate and the reporter substrate are hydrolyzed at the same time, b) complete hydrolysis of PhA cannot be reached above 1 mM competing substrate. This likely results from accumulation of hydrolysis products (P) of competing substrate and/or accumulation of acetylated enzyme·P complex that inhibit hydrolysis of the reporter substrate.     

Novel irreversible butyrylcholinesterase inhibitors: 2-chloro-1-(substituted-phenyl)ethylphosphonic acids

2-Chloroethylphosphonic acid (ethephon) as the dianion phosphorylates butyrylcholinesterase (BChE) at its active site. In contrast, the classical organophosphorus esterase inhibitors include substituted-phenyl dialkylphosphates (e.g., paraoxon) with electron-withdrawing aryl substituents. The chloroethyl and substituted-phenyl moieties are combined in this study as 2-chloro-1-(substituted-phenyl)ethylphosphonic acids (1) to define the structure--activity relationships and mechanism of BChE inhibition by ethephon and its analogues. Phenyl substituents considered are 3- and 4-nitro, 3- and 4-dimethylamino, and 3- and 4-trimethylammonium. Phosphonic acids were synthesized via the corresponding O,O-diethyl phosphonate precursors followed by deprotection with trimethylsilyl bromide. They decompose under basic conditions about 100-fold faster than ethephon to yield the corresponding styrene derivatives. Electron-withdrawing substituents on the phenyl ring decrease the hydrolysis rate while electron-donating substituents increase the rate. The 4-trimethylammonium analogue has the highest affinity (K(i)=180 microM) and potency (IC(50)=19 microM) in first binding reversibly at the substrate site (possibly with stabilization in a dianion--monoanion environment) and then progressively and irreversibly inhibiting the enzyme activity. These observations suggest dissociation of chloride as the first and rate-limiting step both in the hydrolysis and by analogy in phosphorylation of BChE by bound at the active site.     

A specific substrate-inhibitor, a 2'-deoxy-2'-fluorouridine-containing oligoribonucleotide, against human RNase L

We examined the properties of RNA analogues containing 2'-deoxy-2'-alpha-fluorouridine (1) or 2'-O-methyluridine (2) as inhibitors against human RNase L, that cleaves a single-stranded RNA in the presence of 2',5'-linked oligoadenylate (2-5A). The RNA analogue, FF, containing two molecules of 1 in place of uridine efficiently inhibited the RNase L-catalyzed RNA cleavage reaction, whereas the analogue, MM, containing two molecules of 2 was found not to have affinity for the enzyme. The k(cat) value for FF was 1/100 of that for an unmodified RNA, UU, whereas the K(m) value of FF was only twice as great as that of UU. Thus, it was found that the analogue, FF, containing 1 is an efficient inhibitor against human RNase L.     

Synthesis of some tetrahydropyrimidine-5-carboxylates,determination of their metal chelating effects and inhibition profiles against acetylcholinesterase,butyrylcholinesterase and carbonic anhydrase

2-(Methacryloyloxy)ethyl 6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate, is a cyclic urea derivative synthesized from urea, 2-(methacryloyloxy) ethyl acetoacetate and substituted benzaldehyde, and tested in terms of the inhibition of two physiologically relevant carbonic anhydrase (CA) isozymes I and II. Acetylcholinesterase (AChE) is found in high concentrations in the red blood cells and brain. Butyrylcholinesterase (BChE) is another enzyme abundantly present in the liver and released into blood in a soluble form. Also, they were tested for the inhibition of AChE and BChE enzymes and demonstrated effective inhibition profiles with Ki values in the range of 429.24–530.80?nM against hCA I, 391.86–530.80?nM against hCA II, 68.48–97.19?nM against AChE and 104.70–214.15?nM against BChE. On the other hand, acetazolamide clinically used as CA inhibitor, showed Ki value of 281.33?nM against hCA I, and 202.70?nM against hCA II. Also, Tacrine inhibited AChE and BChE showed Ki values of 396.03 and 209.21?nM, respectively.     

Unexpected AChE inhibitory activity of (2E)α,β-unsaturated fatty acids

A small library of (E) α,β-unsaturated fatty acids was prepared, and 20 different saturated and mono-unsaturated fatty acids differing in chain length were subjected to Ellman’s assays to determine their ability to act as inhibitors for AChE or BChE. While the compounds were only very weak inhibitors of BChE, seven molecules were inhibitors of AChE holding IC₅₀?=?4.3–12.8?M with three of them as significant inhibitors of this enzyme. The results have shown trans 2-mono-unsaturated fatty acids are better inhibitors for AChE than their saturated analogs. Furthermore, the screening results indicate that the chain length is crucial for obtaining an inhibitory efficacy. The best results were obtained for (2E) eicosenoic acid (14) showing inhibition constants K_i?=?1.51?±?0.09?M and K_i′?=?7.15?±?0.55?M. All tested compounds were mixed-type inhibitors with a dominating competitive part. Molecular modelling calculations indicate a different binding mode of active/inactive compounds for the enzymes AChE and BChE.     

Design of high-activity mutants of human butyrylcholinesterase against (-)-cocaine: structural and energetic factors affecting the catalytic efficiency

The present study was aimed to explore the correlation between the protein structure and catalytic efficiency of butyrylcholinesterase (BChE) mutants against (-)-cocaine by modeling the rate-determining transition state (TS1), i.e., the transition state for the first step of chemical reaction process, of (-)-cocaine hydrolysis catalyzed by various mutants of human BChE in comparison with the wild type. Molecular modeling of the TS1 structures revealed that mutations on certain nonactive site residues can indirectly affect the catalytic efficiency of the enzyme against (-)-cocaine through enhancing or weakening the overall hydrogen bonding between the carbonyl oxygen of (-)-cocaine benzoyl ester and the oxyanion hole of the enzyme. Computational insights and predictions were supported by the catalytic activity data obtained from wet experimental tests on the mutants of human BChE, including five new mutants reported for the first time. The BChE mutants with at least ～1000-fold improved catalytic efficiency against (-)-cocaine compared to the wild-type BChE are all associated with the TS1 structures having stronger overall hydrogen bonding between the carbonyl oxygen of (-)-cocaine benzoyl ester and the oxyanion hole of the enzyme. The combined computational and experimental data demonstrate a reasonable correlation relationship between the hydrogen-bonding distances in the TS1 structure and the catalytic efficiency of the enzyme against (-)-cocaine.     

Substrate Inhibition Competes with Halide Inhibition in Polyphenol Oxidase

Polyphenol oxidase (PPO) is a ubiquitous enzyme important in the food industry. Although PPO activity followed Michaelis?CMenten kinetics at catechol concentrations of up to 1?mM, it slowly decreased at catechol concentrations above 2?mM. This result indicated that in addition to the active site (site A), the enzyme possesses a second catechol-binding site (site B) that exerts an inhibitory effect on PPO activity. Halides inhibit PPO activity in such a way that substrate inhibition is lessened when halide concentration is increased. Furthermore, elevated concentrations of catechol diminished the degree of inhibition by halides. These findings suggest that halides also bind to site B to inhibit PPO activity. A steady-state kinetic analysis demonstrated that the dissociation constant between catechol and PPO depended on the binding of halides to site B. The dissociation constants were greatest when chloride bound to the site. Bromide and iodide yielded lower dissociation constants, in that order. These data indicate that the binding of halide to site B modulated the structure of site A, thereby exerting an inhibitory effect.     

Improved octyl glucoside synthesis using immobilized β-glucosidase on PA-M with reduced glucose surplus inhibition

A β-glucosidase extracted from bitter almond (Prunus dulcis var. amara) was immobilized on polyamine microspheres (PA-M) for catalytic octyl glucoside (OG) synthesis from glucose and octanol through reversed hydrolysis. The immobilization increased the activity of enzyme at pH 6.0–7.0, and the optimal reaction temperature for immobilized enzyme was identical to the free enzyme. The thermal stability and solvent tolerance of enzyme were increased by its immobilization. In the co-solvent system using 10% t-butyl alcohol and 10% (v/v) water, the yield of OG was increased by 1.7-fold compared to the yield from the system without co-solvent. Based on dynamic and Dixon plot analyses, the initial reaction velocity (V₀) increased approximately three-fold on immobilization and the OG synthesis was inhibited by surplus glucose. The inhibition dissociation constants for free and immobilized enzyme were 219?mM and 116?mM, respectively. A fed-batch mode was applied in the OG synthesis to minimize substrate inhibition. After 336?h of reaction, the OG yield and the conversion rate of glucose reached 134?mM and 59.6%, respectively. Compared to the batch operation, the fed-bath operation increased the OG yield and the conversion rate of glucose by 340% and 381%, respectively.     

Two-step binding mechanism for HIV protease inhibitors.

Rate constants for binding of five inhibitors of human immunodeficiency virus (HIV) protease were determined by stopped-flow spectrofluorometry. The two isomers of quinoline-2-carbonyl-Asn-Phe psi-[CH(OH)CH2N]Pro-O-t-Bu (R diastereomer = 1R; S diastereomer = 1S) quenched the protein fluorescence of HIV protease and thus provided a spectrofluorometric method to determine their binding rate constants. The dissociation rate constants for acetyl-Thr-Ile-Leu psi(CH2NH)Leu-Gln-Arg-NH2 (2), (carbobenzyloxy)-Phe psi[CH(OH)CH2N]Pro-O-t-Bu (3), and pepstatin were determined by trapping free enzyme with 1R as 2, 3, and pepstatin dissociated from the respective enzyme.inhibitor complex. Association rate constants of 1R, 2, and pepstatin were calculated from the time-dependent inhibition of protease-catalyzed hydrolysis of the fluorescent substrate (2-aminobenzoyl)-Thr-Ile-Nle-Phe(NO2)-Gln-Arg-NH2 (4). The kinetic data for binding of 1S to the protease fit a two-step mechanism. Kd values for these inhibitors were calculated from the rate constants for binding and were similar to the respective steady-state Ki values.     

Cholinestrase inhibitory effects of geranylated flavonoids from Paulownia tomentosa fruits

Alzheimer's disease is rapidly becoming one of the most prevalent human diseases. Inhibition of human acetylcholinestrase (hAChE) and butyrylcholinestrase (BChE) has been linked to amelioration of Alzheimer's symptoms and research into inhibitors is of critical importance. Purification of the methanol extract of Paulownia tomentosa fruits yielded potent hAChE and BChE inhibitory flavonoids (1-9). A comparative activity screen indicated that a geranyl group at C6 is crucial for both hAChE and BChE. For example, diplacone (8) showed 250-fold higher efficacy than its parent eriodictyol (12). IC(50)s of diplacone (8) were 7.2 μM for hAChE and 1.4 μM for BChE. Similar trends were also observed for 4'-O-methyldiplacone (4) (vs its parent, hesperetin 10) and mimulone (7) (vs its parent, naringenin 11). Representative inhibitors (1-8) showed mixed inhibition kinetics as well as time-dependent, reversible inhibition toward hAChE. The binding affinities of these compounds to hAChE were investigated by monitoring quenching of inherent enzyme fluorescence. The affinity constants (K(SA)) increased in proportion to inhibitory potencies.