A new sensitive spectrofluorimetric method for measurement of activity and kinetic study of cholinesterases.

A new spectrofluorimetric method more sensitive than the Ellman method was developed for determination of both acetylcholinesterase and butyrylcholinesterase activity and for kinetic analysis of these enzymes and their mutants. Two selected mutants of human butyrylcholinesterase (E197Q and E197G) were included in this work. As for the Ellman's method, substrates are thiocholine esters, but the chromogenic reagent, DTNB (dithio-bisnitro benzoic acid) is replaced by a fluorogenic probe, "Calbiochem Probe IV", (3-(7-Hydroxy-2-oxo-2H-chromen-3-ylcarbamoyl)acrylic acid methylester). Compared to the classical Ellman's method, the sensitivity of this new spectrofluorimetric assay is 2 orders of magnitude higher. The method allows measurement of activity in media containing <10-11 M of cholinesterase active sites at low substrate concentrations, either under first order conditions, [S] << Km, or under conditions where kinetics obeys the Michaelis-Menten model, i.e. at [S] < 1 mM for wild-type enzymes. The method adapted to titration plate reader assays is suitable for clinical and toxicological routine analyses, for high throughput screening of novel cholinesterase mutants and screening of inhibitor libraries of pharmacological interest.

Rapid Mechanistic Evaluation and Parameter Estimation of Putative Inhibitors in a Single-Step Progress-Curve Analysis: The Case of Horse Butyrylcholinesterase.

Highly efficient and rapid lead compound evaluation for estimation of inhibition parameters and type of inhibition is proposed. This is based on a single progress-curve measurement in the presence of each candidate compound, followed by the simultaneous analysis of all of these curves using the ENZO enzyme kinetics suite, which can be implemented as a web application. In the first step, all of the candidate ligands are tested as competitive inhibitors. Where the theoretical curves do not correspond to the experimental data, minimal additional measurements are added, with subsequent processing according to modified reaction mechanisms.

Fully integrated ready-to-use paper-based electrochemical biosensor to detect nerve agents.

Paper-based microfluidic devices are gaining large popularity because of their uncontested advantages of simplicity, cost-effectiveness, limited necessity of laboratory infrastructure and skilled personnel. Moreover, these devices require only small volumes of reagents and samples, provide rapid analysis, and are portable and disposable. Their combination with electrochemical detection offers additional benefits of high sensitivity, selectivity, simplicity of instrumentation, portability, and low cost of the total system. Herein, we present the first example of an integrated paper-based screen-printed electrochemical biosensor device able to quantify nerve agents. The principle of this approach is based on dual electrochemical measurements, in parallel, of butyrylcholinesterase (BChE) enzyme activity towards butyrylthiocholine with and without exposure to contaminated samples. The sensitivity of this device is largely improved using a carbon black/Prussian Blue nanocomposite as a working electrode modifier. The proposed device allows an entirely reagent-free analysis. A strip of a nitrocellulose membrane, that contains the substrate, is integrated with a paper-based test area that holds a screen-printed electrode and BChE. Paraoxon, chosen as nerve agent simulant, is linearly detected down to 3µg/L. The use of extremely affordable manufacturing techniques provides a rapid, sensitive, reproducible, and inexpensive tool for in situ assessment of nerve agent contamination. This represents a powerful approach for use by non-specialists, that can be easily broadened to other (bio)systems.

Activity and determinants of cholinesterases and paraoxonase-1 in blood of workers exposed to non-cholinesterase inhibiting pesticides.

Pesticide exposure has been associated with different adverse health effects which may be modulated to some extent by paraoxonase-1 (PON1) activity and genetic polymorphisms. This study assessed seasonal variations in PON1 activity (using paraoxon -POase-, phenylacetate -AREase-, diazoxon -DZOase- and dihydrocoumarin -DHCase- as substrates), erythrocyte acetylcholinesterase (AChE) and plasma cholinesterase (using butyrylthiocholine -BuChE- and benzoylcholine -BeChE- as substrates. The study population consisted of intensive agriculture workers regularly exposed to pesticides other than organophosphates and non-exposed controls from Almería (Southeastern Spain). The effect of common genetic polymorphisms of PON1 and BCHE on paraoxonase-1 and cholinesterase activities toward different substrates was also assessed. Linear mixed models were used to compare esterase activities in agricultural workers and control subjects over the two study periods (high and low exposure to pesticides). The significant decrease in AChE and increase in BuChE and BeChE activities observed in workers with respect to control subjects was attributed to pesticide exposure. Workers also had higher levels of AREase, DZOase and, to a lesser extent, of POase, but showed decreased DHCase activity. While PON1 Q192R and PON1 -108C/T gene polymorphisms were significantly associated with all PON1 activities, PON1 L55M showed a significant association with AREase, DZOase and DHCase. BCHE-K (Karlow variant) was significantly associated with lower BeChE activity (but not with BuChE) and BCHE-A (atypical variant) showed no significant association with any cholinesterase activity. These findings suggest that increased PON1, BuChE and BeChE activities in exposed workers might result from an adaptive response against pesticide exposure to compensate for adverse effects at the biochemical level. This response appears to be modulated by PON1 and BCHE gene polymorphisms.

New Insights into Butyrylcholinesterase Activity Assay: Serum Dilution Factor as a Crucial Parameter.

Butyrylcholinesterase (BChE) activity assay and inhibitor phenotyping can help to identify patients at risk of prolonged paralysis following the administration of neuromuscular blocking agents. The assay plays an important role in clinical chemistry as a good diagnostic marker for intoxication with pesticides and nerve agents. Furthermore, the assay is also commonly used for in vitro characterization of cholinesterases, their toxins and drugs. There is still lack of standardized procedure for measurement of BChE activity and many laboratories use different substrates at various concentrations. The purpose of this study was to validate the BChE activity assay to determine the best dilution of human serum and the most optimal concentration of substrates and inhibitors. Serum BChE activity was measured using modified Ellman's method applicable for a microplate reader. We present our experience and new insights into the protocol for high-throughput routine assays of human plasma cholinesterase activities adapted to a microplate reader. During our routine assays used for the determination of BChE activity, we have observed that serum dilution factor influences the results obtained. We show that a 400-fold dilution of serum and 5mM S-butyrylthiocholine iodide can be successfully used for the accurate measurement of BChE activity in human serum. We also discuss usage of various concentrations of dibucaine and fluoride in BChE phenotyping. This study indicates that some factors of such a multicomponent clinical material like serum can influence kinetic parameters of the BChE. The observed inhibitory effect is dependent on serum dilution factor used in the assay.

Kinetic characterization of a cocaine hydrolase engineered from mouse butyrylcholinesterase.

Mouse butyrylcholinesterase (mBChE) and an mBChE-based cocaine hydrolase (mCocH, i.e. the A¹99S/S²²7A/S²87G/A³²8W/Y³³²G mutant) have been characterized for their catalytic activities against cocaine, i.e. naturally occurring (-)-cocaine, in comparison with the corresponding human BChE (hBChE) and an hBChE-based cocaine hydrolase (hCocH, i.e. the A¹99S/F²²7A/S²87G/A³²8W/Y³³²G mutant). It has been demonstrated that mCocH and hCocH have improved the catalytic efficiency of mBChE and hBChE against (-)-cocaine by ~8- and ~2000-fold respectively, although the catalytic efficiencies of mCocH and hCocH against other substrates, including acetylcholine (ACh) and butyrylthiocholine (BTC), are close to those of the corresponding wild-type enzymes mBChE and hBChE. According to the kinetic data, the catalytic efficiency (k(cat)/K(M)) of mBChE against (-)-cocaine is comparable with that of hBChE, but the catalytic efficiency of mCocH against (-)-cocaine is remarkably lower than that of hCocH by ~250-fold. The remarkable difference in the catalytic activity between mCocH and hCocH is consistent with the difference between the enzyme-(-)-cocaine binding modes obtained from molecular modelling. Further, both mBChE and hBChE demonstrated substrate activation for all of the examined substrates [(-)-cocaine, ACh and BTC] at high concentrations, whereas both mCocH and hCocH showed substrate inhibition for all three substrates at high concentrations. The amino-acid mutations have remarkably converted substrate activation of the enzymes into substrate inhibition, implying that the rate-determining step of the reaction in mCocH and hCocH might be different from that in mBChE and hBChE.

Novel human butyrylcholinesterase variants: toward organophosphonate detoxication.

Human butyrylcholinesterase (hBChE) is currently being developed as a detoxication enzyme for stoichiometric binding and/or catalytic hydrolysis of organophosphates. Herein, we describe the use of a molecular evolution method to develop novel hBChE variants with increased resistance to stereochemically defined nerve agent model compounds of soman, sarin, and cyclosarin. Novel hBChE variants (Y332S, D340H, and Y332S/D340H) were identified with an increased resistance to nerve agent model compounds that retained robust intrinsic catalytic efficiency. Molecular dynamics simulations of these variants revealed insights into the mechanism by which these structural changes conferred nerve agent model compound resistance.

Substrate selectivity of high-activity mutants of human butyrylcholinesterase.

Cocaine is one of the most addictive drugs, and there is still no FDA (Food and Drug Administration)-approved medication specific for cocaine abuse. A promising therapeutic strategy is to accelerate cocaine metabolism, producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway, i.e. cocaine hydrolysis catalyzed by butyrylcholinesterase (BChE) in plasma. However, the native BChE has a low catalytic efficiency against the abused cocaine, i.e. (-)-cocaine. Our recently designed and discovered A199S/F227A/S287G/A328W/Y332G mutant and other mutants of human BChE have a considerably improved catalytic efficiency against (-)-cocaine. In the present study, we carried out both computational modeling and experimental kinetic analysis on the catalytic activities of these promising new BChE mutants against other known substrates, including neurotransmitter acetylcholine (ACh), acetylthiocholine (ATC), butyrylthiocholine (BTC), and (+)-cocaine, in comparison with the corresponding catalytic activity against (-)-cocaine. Both the computational modeling and kinetic analysis have consistently revealed that all the examined amino acid mutations only considerably improve the catalytic efficiency of human BChE against (-)-cocaine, without significantly improving the catalytic efficiency of the enzyme against any of the other substrates examined. In particular, all the examined BChE mutants have a slightly lower catalytic efficiency against neurotransmitter ACh compared to the wild-type BChE. This observation gives us confidence in developing an anti-cocaine enzyme therapy by using one of these BChE mutants, particularly the A199S/F227A/S287G/A328W/Y332G mutant.

Molecular and kinetic properties of two acetylcholinesterases from the western honey bee, Apis mellifera.

We investigated the molecular and kinetic properties of two acetylcholinesterases (AmAChE1 and AmAChE2) from the Western honey bee, Apis mellifera. Western blot analysis revealed that AmAChE2 has most of catalytic activity rather than AmAChE1, further suggesting that AmAChE2 is responsible for synaptic transmission in A. mellifera, in contrast to most other insects. AmAChE2 was predominately expressed in the ganglia and head containing the central nervous system (CNS), while AmAChE1 was abundantly observed not only in the CNS but also in the peripheral nervous system/non-neuronal tissues. Both AmAChEs exist as homodimers; the monomers are covalently connected via a disulfide bond under native conditions. However, AmAChE2 was associated with the cell membrane via the glycophosphatidylinositol anchor, while AmAChE1 was present as a soluble form. The two AmAChEs were functionally expressed with a baculovirus system. Kinetic analysis revealed that AmAChE2 has approximately 2,500-fold greater catalytic efficiency toward acetylthiocholine and butyrylthiocholine than AmAChE1, supporting the synaptic function of AmAChE2. In addition, AmAChE2 likely serves as the main target of the organophosphate (OP) and carbamate (CB) insecticides as judged by the lower IC(50) values against AmAChE2 than against AmAChE1. When OP and CB insecticides were pre-incubated with a mixture of AmAChE1 and AmAChE2, a significant reduction in the inhibition of AmAChE2 was observed, suggesting a protective role of AmAChE1 against xenobiotics. Taken together, based on their tissue distribution pattern, molecular and kinetic properties, AmAChE2 plays a major role in synaptic transmission, while AmAChE1 has non-neuronal functions, including chemical defense.

An evaluation of the inhibition of human butyrylcholinesterase and acetylcholinesterase by the organophosphate chlorpyrifos oxon.

Acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8) are enzymes that belong to the superfamily of alpha/beta-hydrolase fold proteins. While they share many characteristics, they also possess many important differences. For example, whereas they have about 54% amino acid sequence identity, the active site gorge of acetylcholinesterase is considerably smaller than that of butyrylcholinesterase. Moreover, both have been shown to display simple and complex kinetic mechanisms, depending on the particular substrate examined, the substrate concentration, and incubation conditions. In the current study, incubation of butyrylthiocholine in a concentration range of 0.005-3.0 mM, with 317 pM human butyrylcholinesterase in vitro, resulted in rates of production of thiocholine that were accurately described by simple Michaelis-Menten kinetics, with a K(m) of 0.10 mM. Similarly, the inhibition of butyrylcholinesterase in vitro by the organophosphate chlorpyrifos oxon was described by simple Michaelis-Menten kinetics, with a k(i) of 3048 nM(-1) h(-1), and a K(D) of 2.02 nM. In contrast to inhibition of butyrylcholinesterase, inhibition of human acetylcholinesterase by chlorpyrifos oxon in vitro followed concentration-dependent inhibition kinetics, with the k(i) increasing as the inhibitor concentration decreased. Chlorpyrifos oxon concentrations of 10 and 0.3 nM gave k(i)s of 1.2 and 19.3 nM(-1) h(-1), respectively. Although the mechanism of concentration-dependent inhibition kinetics is not known, the much smaller, more restrictive active site gorge of acetylcholinesterase almost certainly plays a role. Similarly, the much larger active site gorge of butyrylcholinesterase likely contributes to its much greater reactivity towards chlorpyrifos oxon, compared to acetylcholinesterase.

Inhibition of human plasma cholinesterase by malachite green and related triarylmethane dyes: mechanistic implications.

The inhibitory effects of the cationic triarylmethane (TAM+) dyes, pararosaniline (PR+), malachite green (MG+), and methyl green (MeG+) on human plasma cholinesterase (BChE) were studied at 25 degrees C in 100 mM Mops, pH 8.0, with butyrylthiocholine as substrate. PR+ and MG+ caused linear mixed inhibition of enzyme activity. The respective inhibitory parameters were K(i) = 1.9 +/- 0.23 microM, alpha = 13 +/- 48, beta = 0 and K(i) = 0.28 +/- 0.037 microM, alpha = 23 +/- 7.4, beta = 0. MeG+ acted as a competitive inhibitor with K(i) = 0.12 +/- 0.017 microM (alpha, infinity, beta, not applicable). The K(i) values were within the same range reported for a number of ChE inhibitors including propidium ion, donepezil, and the phenothiazines, suggesting that TAM+s are active site ligands. On the other hand, the alpha values failed to correlate with values previously reported for a number of ChE inhibitors. It appears that mixed inhibition is the combined result of more than one type of binding and S-I interference. The impact of ligands at the choline-specific and peripheral anionic sites (or, possibly, accessory structural domains) on BChE activity needs to be studied in further detail.

Pralidoxime hydrolysis of thiocholine esters.

Pralidoxime (2-PAM) hydrolyzes both acetylthiocholine and butytylthiocholine in an apparent first-order manner, with higher rates observed at pH 8.0 compared to those at pH 7.4. When the reaction is followed by the decrease in pH due to hydrolysis, pralidoxime hydrolyzes acetylthiocholine but not acetylcholine or succinylcholine. This implies that, while pralidoxime acts to reverse intoxication by organophosphate compounds due to the otherwise irreversible inhibition of acetylcholinesterase, it does not also supplement this detoxification by hydrolysis of the enzyme's substrate, acetylcholine.

High activity of human butyrylcholinesterase at low pH in the presence of excess butyrylthiocholine.

Butyrylcholinesterase is a serine esterase, closely related to acetylcholinesterase. Both enzymes employ a catalytic triad mechanism for catalysis, similar to that used by serine proteases such as alpha-chymotrypsin. Enzymes of this type are generally considered to be inactive at pH values below 5, because the histidine member of the catalytic triad becomes protonated. We have found that butyrylcholinesterase retains activity at pH

Use of whole blood for spectrophotometric determination of cholinesterase activity in dogs.

Whole blood has been compared with erythrocytes and plasma for spectrophotometric cholinesterase determination in the dog. Cholinesterase activity was characterized using two substrates: acetylthiocholine and butyrylthiocholine. Acetylcholinesterase was the only form of cholinesterase present on erythrocytes and hydrolysed only acetylthiocholine. Butyrylcholinesterase (pseudocholinesterase) was predominant in plasma, hydrolysing mainly butyrylthiocholine. Based on these results, a method based on the use of two substrates (acetylthiocholine for monitoring acetylcholinesterase and butyrylthiocholine for determining butyrylcholinesterase) in the same whole blood sample is recommended for canine cholinesterase analysis. This way of monitoring both enzymes can be easily automated, yielding good within (CVs < 5%) and between-run (CVs < 7%) precision.

Catalytic parameters for the hydrolysis of butyrylthiocholine by human serum butyrylcholinesterase variants.

Catalysed hydrolysis of butyrylthiocholine (BTCh) by the usual (UU), fluoride-resistant (FS), AK, AJ and atypical (AA) human serum butyrylcholinesterase (EC 3.1.1.8) variants was measured in phosphate buffer pH 7.4 at 25 degrees C. pS-curves for all phenotypes were S-shaped; the activities rose to a plateau with increasing substrate concentration except at 100 mM where there was a small decrease. To obtain the catalytic constants, three equations were applied: Michaelis-Menten equation (Eq. 1), Hill equation (Eq. 2) and an equation which assumes simultaneous binding of the substrate to the catalytic site and to a peripheral site on the enzyme (Eq. 3). Over a range from 0.01 to 50 mM BTCh, the activity versus substrate concentration relationship deviated from Michaelis-Menten kinetics (Eq. 1) while data fitted well with Eqs. 2 and 3. The Michaelis-Menten equation was applied separately to two BTCh concentration ranges: the corresponding Km constants for the UU, FS, AK, AJ and AA phenotypes ranged from 0.1 to 0.2 mM (at 0.01-1.0 mM BTCh) and from 0.3 to 2.0 mM (at 1.0-50 mM BTCh). Hill coefficients (nH) calculated from Eq. 2 were similar for all phenotypes (nH approximately 0.5). The dissociation constants K1 and K2 calculated from Eq. 3 for two sites on the enzyme fell between 0.02 and 0.12 mM (K1) and 0.89 and 4.9 mM (K2) for the five phenotypes. Experimental data support the assumption that the phenotypes studied have two substrate binding sites.

Crystal structures of aged phosphonylated acetylcholinesterase: nerve agent reaction products at the atomic level.

Organophosphorus acid anhydride (OP) nerve agents are potent inhibitors which rapidly phosphonylate acetylcholinesterase (AChE) and then may undergo an internal dealkylation reaction (called "aging") to produce an OP-enzyme conjugate that cannot be reactivated. To understand the basis for irreversible inhibition, we solved the structures of aged conjugates obtained by reaction of Torpedo californica AChE (TcAChE) with diisopropylphosphorofluoridate (DFP), O-isopropylmethylphosponofluoridate (sarin), or O-pinacolylmethylphosphonofluoridate (soman) by X-ray crystallography to 2.3, 2.6, or 2.2 A resolution, respectively. The highest positive difference density peak corresponded to the OP phosphorus and was located within covalent bonding distance of the active-site serine (S200) in each structure. The OP-oxygen atoms were within hydrogen-bonding distance of four potential donors from catalytic subsites of the enzyme, suggesting that electrostatic forces significantly stabilize the aged enzyme. The active sites of aged sarin- and soman-TcAChE were essentially identical and provided structural models for the negatively charged, tetrahedral intermediate that occurs during deacylation with the natural substrate, acetylcholine. Phosphorylation with DFP caused an unexpected movement in the main chain of a loop that includes residues F288 and F290 of the TcAChE acyl pocket. This is the first major conformational change reported in the active site of any AChE-ligand complex, and it offers a structural explanation for the substrate selectivity of AChE.

Mechanism of eserine action on the hydrolysis of butyrylthiocholine by butyrylcholinesterase.

The mechanism of the interaction of eserine with butyrylcholinesterase has been proposed only on the basis of analogy with acetylcholinesterase. Here the interactions was studied in detail and the results analysed by classical kinetic methods and by means of mathematical modelling. An appropriate kinetic scheme was developed, an adequate equation derived and the corresponding kinetic parameters evaluated. The findings suggest that a fast but relatively weak binding of eserine to the enzyme's active site is followed by a slow acylation step and by an even slower rate limiting deacylation step so misrepresenting eserine as an irreversible inhibitor. The proposed kinetic scheme also suggests that the reaction of eserine with a peripheral substrate site is unlikely as seen with the substrate, butyrylthiocholine.