A collaborative endeavor to design cholinesterase-based catalytic scavengers against toxic organophosphorus esters

Wild-type human butyrylcholinesterase (BuChE) has proven to be an efficient bioscavenger for protection against nerve agent toxicity. Human acetylcholinesterase (AChE) has a similar potential. A limitation to their usefulness is that both cholinesterases (ChEs) react stoichiometrically with organophosphosphorus (OP) esters. Because OPs can be regarded as pseudo-substrates for which the dephosphylation rate constant is almost zero, several strategies have been attempted to promote the dephosphylation reaction. Oxime-mediated reactivation of phosphylated ChEs generates a turnover, but it is too slow to make pseudo-catalytic scavengers of pharmacological interest. Alternatively, it was hypothesized that ChEs could be converted into OP hydrolases by using rational site-directed mutagenesis based upon the crystal structure of ChEs. The idea was to introduce a nucleophile into the oxyanion hole, at an appropriate position to promote hydrolysis of the phospho-serine bond via a base catalysis mechanism. Such mutants, if they showed the desired catalytic and pharmacokinetic properties, could be used as catalytic scavengers. The first mutant of human BuChE that was capable of hydrolyzing OPs was G117H. It had a slow rate. Crystallographic study of the G117H mutant showed that hydrolysis likely occurs by activation of a water molecule rather than direct nucleophilic attack by H117. Numerous BuChE mutants were made later, but none of them was better than the G117H mutant at hydrolyzing OPs, with the exception of soman. Soman aged too rapidly to be hydrolyzed by G117H. Hydrolysis was however accomplished with the double mutant G117H/E197Q, which did not age after phosphonylation with soman. Multiple mutations in the active center of human and Bungarus AChE led to enzymes displaying low catalytic activity towards OPs and unwanted kinetic complexities. A new generation of human AChE mutants has been designed with the assistance of molecular modelling and computational methods. According to the putative water-activation mechanism of G117H BChE, a new histidine/aspartate dyad was introduced into the active center of human AChE at the optimum location for hydrolysis of the OP adduct. Additional mutations were made for optimizing activity of the new dyad. It is anticipated that these new mutants will have OP hydrolase activity.     

The structure of G117H mutant of butyrylcholinesterase: Nerve agents scavenger

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. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Screening assays for cholinesterases resistant to inhibition by organophosphorus toxicants

Methods to measure resistance to inhibition by organophosphorus toxicants (OP) for mutants of butyrylcholinesterase (EC 3.1.1.8; BChE) and acetylcholinesterase (EC 3.1.1.7; AChE) enzymes were devised. Wild-type cholinesterases were completely inhibited by 0.1 mM echothiophate or 0.001 mM diisopropylfluorophosphate, but human BChE mutants G117H, G117D, L286H, and W231H and snake AChE mutant HFQT retained activity. Tissues containing a mixture of cholinesterases could be assayed for amount of G117H BChE. For example, the serum of transgenic mice expressing human G117H BChE contained 0.5 microg/ml human G117H BChE, 2 microg/ml wild-type mouse BChE, and 0.06 microg/ml wild-type mouse AChE. The oligomeric structure of G117H BChE in the serum of transgenic mice was determined by nondenaturing gel electrophoresis followed by staining for butyrylthiocholine hydrolysis activity in the presence of 0.1 mM echothiophate. Greater than 95% of the human G117H BChE in transgenic mouse serum was a tetramer. To visualize the distribution of G117H BChE in tissues of transgenic mice, sections of small intestine were treated with echothiophate and then stained for BChE activity. Both wild-type and G117H BChE were in the epithelial cells of the villi. These assays can be used to identify OP-resistant cholinesterases in culture medium and in animal tissues.     

X-ray crystallographic snapshots of reaction intermediates in the G117H mutant of human butyrylcholinesterase, a nerve agent target engineered into a catalytic bioscavenger

OPs (organophosphylates) exert their acute toxicity through inhibition of acetylcholinesterase, by phosphylation of the catalytic serine residue. Engineering of human butyrylcholinesterase, by substitution of a histidine residue for the glycine residue at position 117, led to the creation of OP hydrolase activity. However, the lack of structural information and poor understanding of the hydrolytic mechanism of the G117H mutant has hampered further improvements in the catalytic activity. We have solved the crystallographic structure of the G117H mutant with a variety of ligands in its active site. A sulfate anion bound to the active site suggested the positioning for an OP prior to phosphylation. A fluoride anion was found in the active site when NaF was added to the crystallization buffer. In the fluoride complex, the imidazole ring from the His117 residue was substantially shifted, adopting a relaxed conformation probably close to that of the unliganded mutant enzyme. Additional X-ray structures were obtained from the transient covalent adducts formed upon reaction of the G117H mutant with the OPs echothiophate and VX [ethyl ({2-[bis(propan-2-yl)amino]ethyl}sulfanyl](methyl)phosphinate]. The position of the His117 residue shifted in response to the introduction of these adducts, overlaying the phosphylserine residue. These structural data suggest that the dephosphylation mechanism involves either a substantial conformational change of the His117 residue or an adjacent nucleophilic substitution by water.     

Muscular dystrophy by merosin deficiency decreases acetylcholinesterase activity in thymus of Lama2dy mice

Half of congenital muscular dystrophy cases arise from laminin alpha2 (merosin) deficiency, and merosin-deficient mice (Lama2dy) exhibit a dystrophic phenotype. The abnormal development of thymus in Lama2dy mice, the occurrence of acetylcholinesterase (AChE) in the gland and the impaired distribution of AChE molecules in skeletal muscle of the mouse mutant prompted us to compare the levels of AChE mRNAs and enzyme species in thymus of control and Lama2dy mice. AChE activity in normal thymus (mean +/- SD 1.42 +/- 0.28 micromol acetylthiocholine/h/mg protein, U/mg) was decreased by approximately 50% in dystrophic thymus (0.77 +/- 0.23 U/mg) (p = 0.007), whereas butyrylcholinesterase activity was little affected. RT-PCR assays revealed variable levels of R, H and T AChE mRNAs in thymus, bone marrow and spinal cord. Control thymus contained amphiphilic AChE dimers (G2A, 64%) and monomers (G1A, 19%), as well as hydrophilic tetramers (G4H, 9%) and monomers (G1H, 8%). The dimers consisted of glycosylphosphatidylinositol-anchored H subunits. Western blot assays with anti-AChE antibodies suggested the occurrence of inactive AChE in mouse thymus. Despite the decrease in AChE activity in Lama2dy thymus, no differences between thymuses from control and dystrophic mice were observed in the distribution of AChE forms, phosphatidylinositol-specific phospholipase C sensitivity, binding to lectins and size of AChE subunits.     

Substrate activation in acetylcholinesterase induced by low pH or mutation in the pi-cation subsite

Substrate inhibition is considered a defining property of acetylcholinesterase (AChE), whereas substrate activation is characteristic of butyrylcholinesterase (BuChE). To understand the mechanism of substrate inhibition, the pH dependence of acetylthiocholine hydrolysis by AChE was studied between pH 5 and 8. Wild-type human AChE and its mutants Y337G and Y337W, as well as wild-type Bungarus fasciatus AChE and its mutants Y333G, Y333A and Y333W were studied. The pH profile results were unexpected. Instead of substrate inhibition, wild-type AChE and all mutants showed substrate activation at low pH. At high pH, there was substrate inhibition for wild-type AChE and for the mutant with tryptophan in the pi-cation subsite, but substrate activation for mutants containing small residues, glycine or alanine. This is particularly apparent in the B. fasciatus AChE. Thus a single amino acid substitution in the pi-cation site, from the aromatic tyrosine of B. fasciatus AChE to the alanine of BuChE, caused AChE to behave like BuChE. Excess substrate binds to the peripheral anionic site (PAS) of AChE. The finding that AChE is activated by excess substrate supports the idea that binding of a second substrate molecule to the PAS induces a conformational change that reorganizes the active site.     

Acetylthiocholine binds to asp74 at the peripheral site of human acetylcholinesterase as the first step in the catalytic pathway

Studies of ligand binding to acetylcholinesterase (AChE) have demonstrated two sites of interaction. An acyl-enzyme intermediate is formed at the acylation site, and catalytic activity can be inhibited by ligand binding to a peripheral site. The three-dimensional structures of AChE-ligand complexes reveal a narrow and deep active site gorge and indicate that ligands specific for the acylation site at the base of the gorge must first traverse the peripheral site near the gorge entrance. In recent studies attempting to clarify the role of the peripheral site in the catalytic pathway for AChE, we showed that ligands which bind specifically to the peripheral site can slow the rates at which other ligands enter and exit the acylation site, a feature we called steric blockade [Szegletes, T., Mallender, W. D., and Rosenberry, T. L. (1998) Biochemistry 37, 4206-4216]. We also demonstrated that cationic substrates can form a low-affinity complex at the peripheral site that accelerates catalytic hydrolysis at low substrate concentrations but results in substrate inhibition at high concentrations because of steric blockade of product release [Szegletes, T., Mallender, W. D., Thomas, P. J., and Rosenberry, T. L. (1999) Biochemistry 38, 122-133]. In this report, we demonstrate that a key residue in the human AChE peripheral site with which the substrate acetylthiocholine interacts is D74. We extend our kinetic model to evaluate the substrate affinity for the peripheral site, indicated by the equilibrium dissociation constant K(S), from the dependence of the substrate hydrolysis rate on substrate concentration. For human AChE, a K(S) of 1.9+/-0.7 mM obtained by fitting this substrate inhibition curve agreed with a K(S) of 1.3+/-1.0 mM measured directly from acetylthiocholine inhibition of the binding of the neurotoxin fasciculin to the peripheral site. For Torpedo AChE, a K(S) of 0.5+/- 0.2 mM obtained from substrate inhibition agreed with a K(S) of 0.4+/- 0.2 mM measured with fasciculin. Introduction of the D72G mutation (corresponding to D74G in human AChE) increased the K(S) to 4-10 mM in the Torpedo enzyme and to about 33 mM in the human enzyme. While the turnover number k(cat) was unchanged in the human D74G mutant, the roughly 20-fold decrease in acetylthiocholine affinity for the peripheral site in D74G resulted in a corresponding decrease in k(cat)/K(app), the second-order hydrolysis rate constant, in the mutant. In addition, we show that D74 is important in conveying to the acylation site an inhibitory conformational effect induced by the binding of fasciculin to the peripheral site. This inhibitory effect, measured by the relative decrease in the first-order phosphorylation rate constant k(OP) for the neutral organophosphate 7-[(methylethoxyphosphonyl)oxy]-4-methylcoumarin (EMPC) that resulted from fasciculin binding, decreased from 0.002 in wild-type human AChE to 0.24 in the D74G mutant.     

Molecular properties of acetylcholinesterase in mouse spleen

The presence of acetylcholinesterase (AChE) mRNA and activity in the tissues and cells involved in immune responses prompted us to investigate the level and pattern of AChE components in spleen. AChE activity was higher in mouse spleen (0.46 +/- 0.13 micromol of acetylthiocholine split per hour and per mg protein) than in muscle or heart, but lower than in brain. The spleen was essentially free of butyrylcholinesterase (BuChE) activity. About 40% of spleen AChE was extracted with a saline buffer, and a further 40% with 1% Triton X-100. Sedimentation analyses, the splitting of subunits in AChE dimers, phosphatidylinositol-specific phospholipase C (PIPLC) exposure, and phenyl-agarose chromatography showed that hydrophilic (G1H, 43%) and amphiphilic AChE monomers (G1A, 36%), as well as amphiphilic dimers (G2A, 21%), occurred in spleen. All these molecules bound to fasciculin-2-Sepharose, although the extent of binding was higher for G1H (77%) than for G1A (63%) or G2A (48%) forms. Differences in the extent to which wheat germ lectin (WGA) adsorbed with AChE of mouse spleen and of erythrocyte allowed us to discard the blood origin of spleen AChE activity. A 62 kDa protein was labeled in spleen samples using antibodies against human AChE. The protein was attributed to AChE monomers since its size was the same, regardless of whether disulfide bonds were reduced or not. Since cholinergic stimulation modulates proliferation/maturation of lymphoid cells, AChE may be important for regulating the level of acetylcholine (ACh) in the neighborhood of cholinergic receptors (AChR) in spleen and other lymphoid tissues.     

A simple, label-free AuNPs-based colorimetric ultrasensitive detection of nerve agents and highly toxic organophosphate pesticide

Here, a simple label-free colorimetric sensing method for organophosphate (OP) nerve agents and pesticide based on catalytic reaction of acetylcholine esterase (AChE) and the aggregation of lipoic acid (LA) capped AuNPs has been established, which is highly sensitive with a limit of detection (LOD) lowered to pM level. In this method, only the AChE hydrolysis product of acetylthiocholine (ATCh), i.e., cationic thiocholine (TCh) can induce the aggregation of LA capped AuNPs along with a distinct color change from red to steel-blue. When OPs as enzyme inhibitors exist, the generation of TCh can be suppressed and the color change of LA capped AuNPs is gradually diminished according to different concentrations of OPs. The feasibility of this method has been demonstrated by sensitive measurement of OP nerve agents and pesticide in a spiked fruit sample with reliable results. This distinct and rapid colorimetric response enables us to readily probe OPs without more technical demand.     

Isolation of a tripeptide showing weak acetylthiocholine hydrolysing activity from a soluble form of monkey basal ganglia acetylcholinesterase by limited trypsin digestion

Acetylcholinesterase was purified from the soluble supernatant of monkey (Macaca radiata) brain basal ganglia by a three-step affinity purification procedure. The purified enzyme showed two major protein bands corresponding to molecular weights of approximately 65 kDa and approximately 58 kDa which could be labelled by [3H]diisopropylfluorophosphate. When the purified enzyme was subjected to limited trypsin digestion followed by gel filtration on Sephadex G-75 or Sephadex G-25 column, a peptide fragment of molecular weight approximately 300 Da having a weak acetylthiocholine hydrolysing activity was isolated. The amino acid sequence analysis of this peptide showed a sequence of Gly-Pro-Ser. When the [3H]DFP labelled enzyme was subjected to limited trypsin digestion and Sephadex G-75 column chromatography, a labelled peptide corresponding to approximately 430 Da was isolated. The kinetics, inhibition characteristics and binding characteristics to lectins of this peptide were compared with the parent enzyme. A synthetic peptide of sequence Gly-Pro-Ser was also found to exhibit acetylthiocholine hydrolysing activity. The kinetics and inhibition characteristics of the synthetic peptide were similar to those of the peptide derived from the purified acetylcholinesterase, except that the synthetic peptide was more specific towards acetylthiocholine than butyrylthiocholine. The specific activity (units/mg) of the synthetic peptide was about 123700 times less than that of the purified AChE.     

Postnatal changes in the activities of acetylcholinesterase and butyrylcholinesterase in rat heart atria

Postnatal development of the activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in rat heart atria has been investigated with the use of 1.5-bis (allyldimethylammoniumphenyl) pentane-3-dibromide (BW 284 C51) as a selective inhibitor of AChE. Total cholinesterase activity (mumol acetylthiocholine hydrolysed X g-1 per hour) increased from 218 on the 1st day after birth to 426 on the 30th day and diminished to 340 in adult rats. The activity of AChE (mumol acetylthiocholine hydrolysed X g-1 per hour) underwent more dramatic changes, increasing more than 4-fold during the first month of life, from 13 on the 1st to 58 on the 30th day of life and then decreasing to 42 in adult rats. The proportion of AChE on total cholinesterase activity increased from 6% on the 1st day to 12-15% in animals aged 24 days and more. Since AChE is known to be specifically involved in the termination of the action of acetylcholine in the sinoatrial node, the observed postnatal changes in its activity are likely to play a role in the postnatal development of cardiac parasympathetic control.     

Substrate activation in acetylcholinesterase induced by low pH or mutation in the π-cation subsite

Substrate inhibition is considered a defining property of acetylcholinesterase (AChE), whereas substrate activation is characteristic of butyrylcholinesterase (BuChE). To understand the mechanism of substrate inhibition, the pH dependence of acetylthiocholine hydrolysis by AChE was studied between pH 5 and 8. Wild-type human AChE and its mutants Y337G and Y337W, as well as wild-type Bungarus fasciatus AChE and its mutants Y333G, Y333A and Y333W were studied. The pH profile results were unexpected. Instead of substrate inhibition, wild-type AChE and all mutants showed substrate activation at low pH. At high pH, there was substrate inhibition for wild-type AChE and for the mutant with tryptophan in the π-cation subsite, but substrate activation for mutants containing small residues, glycine or alanine. This is particularly apparent in the B. fasciatus AChE. Thus a single amino acid substitution in the π-cation site, from the aromatic tyrosine of B. fasciatus AChE to the alanine of BuChE, caused AChE to behave like BuChE. Excess substrate binds to the peripheral anionic site (PAS) of AChE. The finding that AChE is activated by excess substrate supports the idea that binding of a second substrate molecule to the PAS induces a conformational change that reorganizes the active site.     

Structural study of the complex stereoselectivity of human butyrylcholinesterase for the neurotoxic V-agents

Nerve agents are chiral organophosphate compounds (OPs) that exert their acute toxicity by phosphorylating the catalytic serine of acetylcholinesterase (AChE). The inhibited cholinesterases can be reactivated using oximes, but a spontaneous time-dependent process called aging alters the adduct, leading to resistance toward oxime reactivation. Human butyrylcholinesterase (BChE) functions as a bioscavenger, protecting the cholinergic system against OPs. The stereoselectivity of BChE is an important parameter for its efficiency at scavenging the most toxic OPs enantiomer for AChE. Crystals of BChE inhibited in solution or in cristallo with racemic V-agents (VX, Russian VX, and Chinese VX) systematically show the formation of the P(S) adduct. In this configuration, no catalysis of aging seems possible as confirmed by the three-dimensional structures of the three conjugates incubated over a period exceeding a week. Crystals of BChE soaked in optically pure VX(R)-(+) and VX(S)-(-) solutions lead to the formation of the P(S) and P(R) adduct, respectively. These structural data support an in-line phosphonylation mechanism. Additionally, they show that BChE reacts with VX(R)-(+) in the presence of racemic mixture of V-agents, at odds with earlier kinetic results showing a moderate higher inhibition rate for VX(S)-(-). These combined results suggest that the simultaneous presence of both enantiomers alters the enzyme stereoselectivity. In summary, the three-dimensional data show that BChE reacts preferentially with P(R) enantiomer of V-agents and does not age, in complete contrast to AChE, which is selectively inhibited by the P(S) enantiomer and ages.     

PARTIAL CHARACTERIZATION OF SOLUBLE ACETYLCHOLINESTERASE ISOENZYMES OF THE RAT BRAIN

(1) The 105,000 g supernatant fluid obtained from rat brain was separated by agar-gel electrophoresis. (2) Three isoenzymes, capable of hydrolysing acetylthiocholine, one of them also hydrolysing butyrylthiocholine, were detected. (3) The pH optima and K_m for hydrolysis of acetyl- and butyrylthiocholine by the supernatant fluid were determined. (4) After extraction of acetylcholinesterase isoenzymes from the gel, individual isoenzymes were characterized by pH optima and K_m values. (5) Two of the enzymes were characterized as acetylcholinesterase (EC 3.1.1.7) and one as butyrylcholinesterase (EC 3.1.1.8).     

Evaluation of mechanisms of azinphos-methyl resistance in the codling moth Cydia pomonella (L.)

Resistance of the codling moth Cydia pomonella (L.) to azinphos-methyl is not based on enhanced detoxifying enzymes like oxidation mediated by mixed function oxidases or by glutathione S-transferases. Synergism by S,S,S-tributylphosphoro-trithioate was evident, but the overall activity of general esterases using p-nitrophenyl acetate as the substrate was similar in resistant and susceptible insects. In comparison to acetylcholinesterase (AChE) from susceptible adult codling moth, the enzyme of insects resistant to azinphos-methyl has low affinities (higher K(m) values) to the substrates acetylthiocholine (ATCh) and propionylthiocholine. This difference indicates a possible amino acid alteration at the catalytic or anionic binding sites of the resistant enzyme. Inhibition studies revealed no apparent differences in sensitivity of AChE enzymes from resistant and susceptible moths to organophosphorus compounds (OPs), carbamate insecticides and quaternary ammonium ligands. MEPQ (7-Methylethoxyphosphinyloxy)-1-methylquinolinium) is the most powerful OP inhibitor acting at a nM range, while chlopyrifos oxon, azinphos-methyl oxon and paraoxon are less inhibitory by 22.9, 82.3 and 475 fold, respectively. The codling moth AChE is a typical enzyme that displays substrate inhibition by ATCh, negligible hydrolysis of butyrylthiocholine, very high sensitivity to the bisquaternary ammonium compound BW284c51 and it is not inhibited by the powerful butyrylcholinesterase inhibitor iso-OMPA. Of the three carbamates examined, only carbaryl was inhibitory at the mM range while pirimicarb and aldicarb were inactive. Of the quaternary ammonium ligands (except for the powerful BW284c51), edrophonium and decamethonium displayed appreciable inhibition rates, while d-tubocuraine was practically inactive.     

A single nucleotide polymorphism in the acetylcholinesterase gene of the predatory mite Kampimodromus aberrans (Acari: Phytoseiidae) is associated with chlorpyrifos resistance

The predatory mite Kampimodromus aberrans (Oudemans) (Acari: Phytoseiidae) is one of the most important biocontrol agents of herbivorous mites in European perennial crops. The use of pesticides, such as organophosphate insecticides (OPs), is a major threat to the success of biocontrol strategies based on predatory mites in these cropping systems. However, resistance to OPs in K. aberrans has recently been reported. The present study investigated the target site resistance mechanisms that are potentially involved in OP insensitivity. In the herbivorous mite Tetranychus urticae Koch (Acari: Tetranychidae), resistance to OPs is due to a modified and insensitive acetylcholinesterase (AChE; EC: 3.1.1.7) that bears amino acid substitution F331W (AChE Torpedo numbering). To determine whether the predators and prey have evolved analogous molecular mechanisms to withstand the same selective pressure, the AChE cDNA from a putative orthologous gene was cloned and sequenced from susceptible and resistant strains of K. aberrans. No synonymous mutation coding for a G119S substitution was determined to be strongly associated with the resistant phenotype instead of the alternative F331W. Because the same mutation in T. urticae AChE was not associated with comparable levels of chlorpyrifos resistance, the role of the G119S substitution in defining insensitive AChE in K. aberrans remains unclear. G119S AChE genotyping can be useful in ecological studies that trace the fate of resistant strains after field release or in marker-assisted selection of improved populations of K. aberrans to achieve multiple resistance phenotypes through gene pyramiding. The latent complexity of the target site resistance in K. aberrans vs. that of T. urticae is also discussed in the context of data from the genome project of the predatory mite Metaseiulus occidentalis (Nesbitt) (Acari: Phytoseiidae).     

Determinants of substrate specificity of a second non-neuronal secreted acetylcholinesterase from the parasitic nematode Nippostrongylus brasiliensis.

We recently reported on a non-neuronal secreted acetylcholinesterase (AChE B) from the nematode parasite Nippostrongylus brasiliensis. Here we describe the primary structure and enzymatic properties of a second secreted variant, termed AChE C after the designation of native AChE isoforms from this parasite. As for the former enzyme, AChE C is truncated at the carboxyl terminus in comparison with the Torpedo AChE, and three of the 14 aromatic residues that line the active site gorge are substituted by nonaromatic residues, corresponding to Tyr70 (Ser), Trp279 (Asn) and Phe288 (Met). A recombinant form of AChE C was highly expressed by Pichia pastoris. The enzyme was monomeric and hydrophilic, and displayed a marked preference for acetylthiocholine as substrate. A double mutation (W302F/W345F, corresponding to positions 290 and 331 in Torpedo) rendered the enzyme 10-fold less sensitive to excess substrate inhibition and two times less susceptible to the bis quaternary inhibitor BW284C51, but did not radically affect substrate specificity or sensitivity to the 'peripheral site' inhibitor propidium iodide. In contrast, a triple mutant (M300G/W302F/W345F) efficiently hydrolysed propionylthiocholine and butyrylthiocholine in addition to acetylthiocholine, while remaining insensitive to the butyrylcholinesterase-specific inhibitor iso-OMPA and displaying a similar profile of excess substrate inhibition as the double mutant. These data highlight a conserved pattern of active site architecture for nematode secreted AChEs characterized to date, and provide an explanation for the substrate specificity that might otherwise appear inconsistent with the primary structure in comparison to other invertebrate AChEs.     

Acetyl- and butyrylcholinesterase molecular forms in normal and streptozotocin-diabetic rat retinal pigment epithelium.

We studied the composition of molecular forms of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in normal and streptozotocin-induced diabetic rat retinal pigment epithelium (RPE). Tissues were sequentially extracted with saline (S(1)) and saline-detergent buffers (S(2)). About a 50% decrease in AChE molecular forms was observed in the diabetic RPE compared to the controls. Approximately 70% of the BChE activity in normal RPE was brought into solution and evenly distributed in S(1) and S(2). Analysis of the fractions from RPE revealed the presence of G(A)(1), G(A)(4) and a small proportion of G(H)(4) BChE forms in S(1); whereas G(A)(4) and G(A)(1) molecules predominate in S(2). A 40% decrease in the activity of G(A)(4) in S(2) was observed in the diabetic RPE. Our results show that diabetes caused a remarkable decrease in the activity of cholinesterases molecular forms in the RPE. This might be related to the alterations observed in diabetic retinopathy.     

Kinetic analysis of effector modulation of butyrylcholinesterase-catalysed hydrolysis of acetanilides and homologous esters

The effects of tyramine, serotonin and benzalkonium on the esterase and aryl acylamidase activities of wild-type human butyrylcholinesterase and its peripheral anionic site mutant, D70G, were investigated. The kinetic study was carried out under steady-state conditions with neutral and positively charged aryl acylamides [o-nitrophenylacetanilide, o-nitrotrifluorophenylacetanilide and m-(acetamido) N,N,N-trimethylanilinium] and homologous esters (o-nitrophenyl acetate and acetylthiocholine). Tyramine was an activator of hydrolysis for neutral substrates and an inhibitor of hydrolysis for positively charged substrates. The affinity of D70G for tyramine was lower than that of the wild-type enzyme. Tyramine activation of hydrolysis for neutral substrates by D70G was linear. Tyramine was found to be a pure competitive inhibitor of hydrolysis for positively charged substrates with both wild-type butyrylcholinesterase and D70G. Serotonin inhibited both esterase and aryl acylamidase activities for both positively charged and neutral substrates. Inhibition of wild-type butyrylcholinesterase was hyperbolic (i.e. partial) with neutral substrates and linear with positively charged substrates. Inhibition of D70G was linear with all substrates. A comparison of the effects of tyramine and serotonin on D70G versus the wild-type enzyme indicated that: (a) the peripheral anionic site is involved in the nonlinear activation and inhibition of the wild-type enzyme; and (b) in the presence of charged substrates, the ligand does not bind to the peripheral anionic site, so that ligand effects are linear, reflecting their sole interaction with the active site binding locus. Benzalkonium acted as an activator at low concentrations with neutral substrates. High concentrations of benzalkonium caused parabolic inhibition of the activity with neutral substrates for both wild-type butyrylcholinesterase and D70G, suggesting multiple binding sites. Benzalkonium caused linear, noncompetitive inhibition of the positively charged aryl acetanilide m-(acetamido) N,N,N-trimethylanilinium for D70G, and an unusual mixed-type inhibition/activation (alpha > beta > 1) for wild-type butyrylcholinesterase with this substrate. No fundamental difference was observed between the effects of ligands on the butyrylcholinesterase-catalysed hydrolysis of esters and amides. Thus, butyrylcholinesterase uses the same machinery, i.e. the catalytic triad S198/H448/E325, for the hydrolysis of both types of substrate. The differences in response to ligand binding depend on whether the substrates are neutral or positively charged, i.e. the differences depend on the function of the peripheral site in wild-type butyrylcholinesterase, or the absence of its function in the D70G mutant. The complex inhibition/activation effects of effectors, depending on the integrity of the peripheral anionic site, reflect the allosteric 'cross-talk' between the peripheral anionic site and the catalytic centre.     

DNA damage in horticultural farmers: a pilot study showing an association with organophosphate pesticide exposure

A study of horticultural farmers exposed to organophosphate pesticides (OPs) and controls investigated the relationships between OP exposure, DNA damage and oxidative stress. Blood acetylcholinesterase (AChE) and urinary dialkylphosphate (DAP) levels determined exposure and 8-hydroxy-29- deoxyguanosine (8OHdG) indicated oxidative stress status. The farmers had approximately 30% lower AChE activity and increased DAP levels compared with the controls, reflecting moderate OP exposure. They had higher DNA damage than the controls and there was a significant positive relationship between DAP and DNA damage with greater than 95% power. The farmers also had a significant positive relationship between urinary DAP and 8OHdG levels.