Direct correlation between molecular dynamics and enzymatic stability: a comparative neutron scattering study of native human butyrylcholinesterase and its "aged" soman conjugate.

An incoherent elastic neutron scattering study of the molecular dynamics of native human butyrylcholinesterase and its "aged" soman-inhibited conjugate revealed a significant change in molecular flexibility on an angstrom-nanosecond scale as a function of temperature. The results were related to the stability of each state as established previously by differential scanning calorimetry. A striking relationship was found between the denaturation behavior and the molecular flexibility of the native and inhibited enzymes as a function of temperature. This was reflected in a quantitative correlation between the atomic mean-square displacements on an angstrom-nanosecond scale determined by neutron spectroscopy and the calorimetric specific heat. By the application of a simple two-state model that describes the transition from a folded to a denatured state, the denaturation temperatures of the native and the inhibited enzyme were correctly extracted from the atomic mean-square displacements. Furthermore, the transition entropy and enthalpy extracted from the model fit of the neutron data were, within the experimental accuracy, compatible with the values determined by differential scanning calorimetry.

Celastrus paniculatus seed oil and organic extracts attenuate hydrogen peroxide- and glutamate-induced injury in embryonic rat forebrain neuronal cells.

Seed oil of Celastrus paniculatus Willd. (CP) has been reported to improve memory and the methanolic extract (ME) of CP was shown to exhibit free-radical-scavenging properties and anti-oxidant effects in human non-immortalized fibroblasts. In the present study, we have investigated the free-radical-scavenging capacity of CP seed oil (CPO) and two extracts, an ethanolic extract (EE) and a ME. CPO and EE showed dose-dependent, free-radical-scavenging capacity, but to a lesser degree than observed for ME. Oxidative stress involves the generation of free radicals and free radical scavenging is one of the mechanisms of neuroprotection. We therefore investigated the effects of CPO, ME, and EE for protection against hydrogen peroxide (H(2)O(2))- and glutamate-induced neurotoxicity in embryonic rat forebrain neuronal cells (FBNC). Pre-treatment of neuronal cells with CPO dose-dependently attenuated H(2)O(2)-induced neuronal death. Pre-treatment with ME and EE partially attenuated H(2)O(2)-induced toxicity, but these extracts were less effective than CPO for neuronal survival. In H(2)O(2)-treated cells, cellular superoxide dismutase (SOD) activity was unaffected, but catalase activity was decreased and levels of malondialdehyde (MDA) were increased. Pre-treatment with CPO, ME, or EE increased catalase activity and decreased MDA levels significantly. Also, CPO pre-treatment attenuated glutamate-induced neuronal death dose-dependently. The activity of cellular acetylcholinesterase (AChE) was not affected by CPO, ME, or EE, suggesting that the neuroprotection offered by CPO was independent of changes in AChE activity. Taken together, the data suggest that CPO, ME, and EE protected neuronal cells against H(2)O(2)-induced toxicity in part by virtue of their antioxidant properties, and their ability to induce antioxidant enzymes. However, CPO, which exhibited the least antioxidant properties, was the most effective in preventing neuronal cells against H(2)O(2)- and glutamate-induced toxicities. Thus, in addition to free-radical scavenging attributes, the mechanism of CP seed component (CP-C) neuroprotection must be elucidated.

Polyethylene glycosylation prolongs the circulatory stability of recombinant human butyrylcholinesterase.

Previous studies in rodents and non-human primates have demonstrated that pretreatment of animals with cholinesterases could provide significant protection against organophosphate (OP) nerve agent toxicity. Gene delivery/therapy is emerging as an approach to achieve high-level expression of proteins in vivo that are very similar to their native counterparts. Recently, adenoviral (Ad) vectors have proven to be excellent vehicles for delivering genes to cells in vitro and in vivo. In this study, we explored the use of the newly designed AdenoVATOR system for the expression of recombinant human butyrylcholinesterase (rHu BChE) in human embryonic kidney 293A (HEK-293A) cells. In these cells, rHu BChE was expressed as mostly tetrameric form by the simultaneous expression of proline-rich attachment domain. By optimizing the culture conditions, 1.5-2.0 U/ml of rHu BChE could be expressed in HEK-293A cells. Recombinant Hu BChE was purified to homogeneity by ammonium sulfate fractionation followed by affinity column chromatography using procainamide Sepharose and cobalt Sepharose gels. The enzymatic and physico-chemical properties of purified rHu BChE were similar to those of native serum-derived Hu BChE. To determine the suitability of this preparation for use as an antidote against highly toxic nerve agents, its pharmacokinetics were evaluated in mice. Recombinant Hu BChE exhibited a mean residence time of 18.3 h which was 2.5-fold shorter than that observed for native Hu BChE in mice. However, rHu BChE chemically modified with polyethyleneglycol (PEG) displayed a mean residence time of 36.2 h suggesting that PEG-modification can prolong the circulatory stability of rHu BChE. The efficacy of Ad-Hu BChE to induce the production of therapeutic levels of bioscavenger in vivo is under evaluation.

Effects of soman inhibition and of structural differences on cholinesterase molecular dynamics: a neutron scattering study.

Incoherent elastic neutron scattering experiments on members of the cholinesterase family were carried out to investigate how molecular dynamics is affected by covalent inhibitor binding and by differences in primary and quaternary structure. Tetrameric native and soman-inhibited human butyrylcholinesterase (HuBChE) as well as native dimeric Drosophila melanogaster acetylcholinesterase (DmAChE) hydrated protein powders were examined. Atomic mean-square displacements (MSDs) were found to be identical for native HuBChE and for DmAChE in the whole temperature range examined, leading to the conclusion that differences in activity and substrate specificity are not reflected by a global modification of subnanosecond molecular dynamics. MSDs of native and soman-inhibited HuBChE were identical below the thermal denaturation temperature of the native enzyme, indicating a common mean free-energy surface. Denaturation of the native enzyme is reflected by a relative increase of MSDs consistent with entropic stabilization of the unfolded state. The results suggest that the stabilization of HuBChE phosphorylated by soman is due to an increase in free energy of the unfolded state due to a decrease in entropy.

In vitro and in vivo characterization of recombinant human butyrylcholinesterase (Protexia) as a potential nerve agent bioscavenger.

Previous studies in rodents and nonhuman primates have demonstrated that pretreatment with cholinesterases can provide significant protection against behavioral and lethal effects of nerve agent intoxication. Human butyrylcholinesterase (HuBuChE) purified from plasma has been shown to protect against up to 5 x LD50s of nerve agents in guinea pigs and non-human primates, and is currently being explored as a bioscavenger pretreatment for human use. A recombinant form of HuBuChE has been expressed in the milk of transgenic goats as a product called Protexia. Protexia was supplied by Nexia Biotechnologies (Que., Canada) as a purified solution with a specific activity of 600 U/mg. Initial in vitro studies using radiolabeled 3H-soman or 3H-DFP (diisopropyl fluorophosphate) demonstrated that these inhibitors specifically bind to Protexia. When Protexia was mixed with soman, sarin, tabun or VX using varying molar ratios of enzyme to nerve agent (8:1, 4:1, 1:1 and 1:4, respectively), the data indicated that 50% inhibition of enzyme activity occurs around the 1:1 molar ratio for each of the nerve agents. Protexia was further characterized for its interaction with pyridostigmine bromide and six unique carbamate inhibitors of cholinesterase. IC50 and Ki values for Protexia were determined to be very similar to those of HuBuChE purified from human plasma. These data suggest that Protexia has biochemical properties very similar to those HuBuChE when compared in vitro. Together these data the continued development of the goat milk-derived recombinant HuBuChE Protexia as a potential bioscavenger of organophosphorus nerve agents.

Transcriptional induction of cholinesterase expression and protection against chemical warfare nerve agents.

We investigated whether transcriptional inducers could enhance the expression of acetylcholinesterase (AChE) in cell lines to achieve protection against organophosphate (OP) poisoning. Trichostatin A (TSA), an inhibitor of histone deacetylase that de-condenses chromatin and increases the binding of transcription factors and mRNA synthesis, induced three- to four-fold extracellular and 8-10-fold intracellular AChE expression at the optimal dose of 165-333 nM in Neuro 2A cells. Pre-treatment with TSA protected against OP exposure. Thus, transcriptional inducers, such as TSA, up-regulate AChE, which then can scavenge the OP and protect the cells from OP-induced toxicity, and are potential novel ways to treat chemical warfare nerve agent (CWNA) exposure.

Safety and pharmacokinetics of human serum butyrylcholinesterase in guinea pigs.

Human serum butyrylcholinesterase (Hu BChE) has been demonstrated to be a highly effective detoxifying enzyme for counteracting the acute toxicity of organophosphorus (OP) nerve agents. In order to initiate an investigational new drug (IND) application for human use, the safety and pharmacokinetic properties of the enzyme were assessed in guinea pigs. Sixty milligrams per kilogram of Hu BChE was administered to guinea pigs by either i.p. or i.m. injection. Blood was drawn at various time points for up to 2 weeks following enzyme injection for the measurement of blood BChE activity. Hu BChE displayed a mean residence time of 110 h, regardless of the route of administration and the enzyme activity remained almost 10-fold above baseline level even after 2 weeks post enzyme injection. Fourteen days post Hu BChE administration, all animals were subjected to 20 panel serum chemistry, hematology, and complete gross/histopathology examination. Results showed no toxic effects as measured by general observation, serum chemistry, hematology, and gross and histological tissue changes. In conclusion, Hu BChE displays a long-lasting stability in the circulation of guinea pigs, and is devoid of any toxic side effects. These results provide convincing data for the safe and effective use of Hu BChE as a bioscavenger to protect humans against all OP nerve agents.

The influence of solvent composition on global dynamics of human butyrylcholinesterase powders: a neutron-scattering study.

A major result of incoherent elastic neutron-scattering experiments on protein powders is the strong dependence of the intramolecular dynamics on the sample environment. We performed a series of incoherent elastic neutron-scattering experiments on lyophilized human butyrylcholinesterase (HuBChE) powders under different conditions (solvent composition and hydration degree) in the temperature range from 20 to 285 K to elucidate the effect of the environment on the enzyme atomic mean-square displacements. Comparing D(2)O- with H(2)O-hydrated samples, we were able to investigate protein as well as hydration water molecular dynamics. HuBChE lyophilized from three distinct buffers showed completely different atomic mean-square displacements at temperatures above approximately 200 K: a salt-free sample and a sample containing Tris-HCl showed identical small-amplitude motions. A third sample, containing sodium phosphate, displayed highly reduced mean-square displacements at ambient temperature with respect to the other two samples. Below 200 K, all samples displayed similar mean-square displacements. We draw the conclusion that the reduction of intramolecular protein mean-square displacements on an Angstrom-nanosecond scale by the solvent depends not only on the presence of salt ions but also on their type.

The NMDA receptor ion channel: a site for binding of Huperzine A.

Huperzine A (HUP-A), first isolated from the Chinese club moss Huperzia serrata, is a potent, reversible and selective inhibitor of acetylcholinesterase (AChE) over butyrylcholinesterase (BChE) (Life Sci. 54: 991-997). Because HUP-A has been shown to penetrate the blood-brain barrier, is more stable than the carbamates used as pretreatments for organophosphate poisoning (OP) and the HUP-A:AChE complex has a longer half-life than other prophylactic sequestering agents, HUP-A has been proposed as a pretreatment drug for nerve agent toxicity by protecting AChE from irreversible OP-induced phosphonylation. More recently (NeuroReport 8: 963-968), pretreatment of embryonic neuronal cultures with HUP-A reduced glutamate-induced cell death and also decreased glutamate-induced calcium mobilization. These results suggest that HUP-A might interfere with and be beneficial for excitatory amino acid overstimulation, such as seen in ischemia, where persistent elevation of internal calcium levels by activation of the N-methyl-D-aspartate (NMDA) glutamate subtype receptor is found. We have now investigated the interaction of HUP-A with glutamate receptors. Freshly frozen cortex or synaptic plasma membranes were used, providing 60-90% specific radioligand binding. Huperzine A (< or =100 microM) had no effect on the binding of [3H]glutamate (low- and high-affinity glutamate sites), [3H]MDL 105,519 (NMDA glycine regulatory site), [3H]ifenprodil (NMDA polyamine site) or [3H]CGS 19755 (NMDA antagonist). In contrast with these results, HUP-A non-competitively (Hill slope < 1) inhibited [3H]MK-801 and [3H]TCP binding (co-located NMDA ion channel PCP site) with pseudo K(i) approximately 6 microM. Furthermore, when neuronal cultures were pretreated with HUP-A for 45 min prior to NMDA exposure, HUP-A dose-dependently inhibited the NMDA-induced toxicity. Although HUP-A has been implicated to interact with cholinergic receptors, it was without effect at 100 microM on muscarinic (measured by inhibition of [3H]QNB or [3H]NMS binding) or nicotinic [3H]epibatidine binding) receptors; also, HUP-A did not perturb adenosine receptor binding [3H]PIA or [3H]NECA). Therefore, HUP-A most likely attenuates excitatory amino acid toxicity by blocking the NMDA ion channel and subsequent Ca2+ mobilization at or near the PCP and MK-801 ligand sites. Thus, on the one hand, HUP-A could be used as a pretreatment against OPs and it might also be a valuable therapeutic intervention in a variety of acute and chronic disorders by protecting against overstimulation of the excitatory amino acid pathway. By blocking NMDA ion channels without psychotomimetic side-effects, HUP-A may protect against diverse neurodegenerative states observed during ischemia or Alzheimer's disease.

Synthesis, chiral chromatographic separation, and biological activities of the enantiomers of 10,10-dimethylhuperzine A.

(+/-)-10,10-Dimethylhuperzine A (2, DMHA) has been synthesized, and its enantiomers have been separated using chiral HPLC. (-)-DMHA inhibits AChE with a Ki value approaching that of (-)-huperzine A, whereas (+)-DMHA shows no AChE inhibitory activity. On the other hand, both enantiomers are equally potent against glutamate-induced neurotoxicity when tested in neurons.

Phosphoryl oxime inhibition of acetylcholinesterase during oxime reactivation is prevented by edrophonium.

Reactivation of organophosphate (OP)-inhibited acetylcholinesterase (AChE) is a key objective in the treatment of OP poisoning. This study with native, wild-type, and mutant recombinant DNA-expressed AChEs, each inhibited by representative OP compounds, establishes a relationship between edrophonium acceleration of oxime-induced reactivation of OP-AChE conjugates and phosphoryl oxime inhibition of the reactivated enzyme that occurs during reactivation by pyridinium oximes LüH6 and TMB4. No such recurring inhibition could be observed with HI-6 as the reactivator due to the extreme lability of the phosphoryl oximes formed by this oxime. Phosphoryl oximes formed during reactivation of the ethoxy methylphosphonyl-AChE conjugate by LüH6 and TMB4 were isolated for the first time and their structures confirmed by (31)P NMR. However, phosphoryl oximes formed during the reactivation of the diethylphosphoryl-AChE conjugate were not sufficiently stable to be detected by (31)P NMR. The purified ethoxy methylphosphonyl oximes formed during the reactivation of ethoxy methylphosphonyl-AChE conjugate with LüH6 and TMB4 are 10- to 22-fold more potent than MEPQ as inhibitors of AChE and stable for several hours at pH 7.2 in HEPES buffer. Reactivation of both ethoxy methylphosphonyl- and diethylphosphoryl-AChE by these two oximes was accelerated in the presence of rabbit serum paraoxonase, suggesting that organophosphorus hydrolase can hydrolyze phosphoryl oxime formed during the reactivation. Our results emphasize that certain oximes, such as LüH6 and TMB4, if used in the treatment of OP pesticide poisoning may cause prolonged inhibition of AChE due to formation of phosphoryl oximes.

Differences in active-site gorge dimensions of cholinesterases revealed by binding of inhibitors to human butyrylcholinesterase.

We examined the role of A328(F330) in the binding of various inhibitors to cholinesterases (ChEs) using human butyrylcholinesterase (BChE) mutants to determine if the conclusions drawn from studies with acetylcholinesterase (AChE) mutants could be extended to BChE. For huperzine A and edrophonium, the results obtained with AChE mutants could be directly correlated with those obtained with native ChEs and site-specific mutants of human BChE. Inhibition studies of ethopropazine with BChE mutants, where A328 was modified to either F or Y, suggested that A328 was not solely responsible for the selectivity of ethopropazine. Volume calculations for the active-site gorge showed that the poor inhibitory activity of ethopropazine towards AChE was due to the smaller dimension of the active-site gorge. The volume of the BChE active-site gorge is approximately 200 A3 larger than that of the AChE gorge, which allows the accommodation of ethopropazine in two different orientations as demonstrated by rigid-body refinement and molecular dynamics calculations. These results suggest that, although the overall scaffolding of the two enzymes may be highly similar, the dimensions and the micro-environment of the gorge play a significant role in determining the selectivity of substrate and inhibitors for ChEs.

Role of edrophonium in prevention of the re-inhibition of acetylcholinesterase by phosphorylated oxime.

We examined the role of edrophonium in the acceleration phenomenon using mouse wild-type and mutant D74N AChE inhibited with 7-(O,O-diethyl-phosphinyloxy)-1-methylquinolinium methylsulfate (DEPQ). With DEPQ-inhibited wild-type mouse acetylcholinesterase (AChE), the reactivation kinetic profile demonstrated one-phase exponential association only when 2-[hydroxyimino methyl]-1-methylpyridinium chloride (2-PAM) and 1-(2-hydroxy-iminomethyl-1-pyridinium)-1-(4-carboxy-aminopyridi nium)-dimethyl ether hydrochloride (HI-6) were used as reactivators. When 1,1[oxybis-methylene)bis[4-(hydroxyimino)methyl] pyridinium dichloride (LüH6) and 1,1-trimethylene bis(4-hydroxyimino methyl) pyridinium dichloride (TMB4) were used, the reactivation kinetic profile was biphasic in nature. Edrophonium had no effect on reactivation by 2-PAM and HI-6, but significantly accelerated LüH6- and TMB4-induced reactivation of DEPQ-inhibited wild-type mouse AChE. Comparison of the initial and overall reactivation rate constants with five oximes indicated that acceleration by edrophonium may be due to the prevention of re-inhibition of the reactivated enzyme by the phosphorylated oxime (POX) produced during the reactivation. With LüH6 and TMB4, about 2.5-fold increase in the reactivation rate constants was observed in the presence of edrophonium, but little or no effect was observed with the other three oximes. The initial reactivation rate constants were 5.4- and 4.2-fold of the overall rate constants with LüH6 and TMB4 as reactivators respectively, however, very little change was found between the initial and overall rate constants with the other three oximes. In experiments with D74N AChE, for which the inhibition potency of charged organophosphate (OP) was two to three orders less than wild-type enzyme, edrophonium had no effect on the reactivation by LüH6 and TMB4 and the time courses of reactivation were monophasic. The data from mutant enzyme substantiate the involvement of edrophonium in protecting POX re-inhibition of reactivated enzyme formed during the reactivation of OP-inhibited AChE.

Improvements in scavenger protection against organophosphorus agents by modification of cholinesterases.

The ability of stoichiometric scavengers, such as ChEs, to protect against a variety of OP agents has been demonstrated in several in vivo models. To improve the detoxification of OP agents by ChEs, several approaches have been recently used to increase the stoichiometry, stability, and in vivo effectiveness of ChEs as OP scavengers. For example, the in vitro stoichiometric neutralization of sarin by AChE was increased from 1:1 to 3200:1 by the addition of the oxime HI-6, while the in vivo stoichiometry was increased to 57:1 in mice by HI-6. The aging rate of soman-inhibited mouse AChE was reduced 12-fold in a mutant AChE (E202Q) which resulted in a two-fold increase in oxime-assisted detoxification of soman. To improve the duration of scavenger protection provided by ChEs, the mean residence times of five tissue-derived and two recombinant ChEs injected i.v. in mice were compared with their oligosaccharide profiles. The mean residence times of these ChEs were found to increase with molecular weight and with the levels of oligosaccharide sialylation. The stability of AChE in non-physiological environments was improved by immobilizing it in a polyurethane foam matrix that allowed AChE to retain enzymatic activity at high temperature (75 degrees C) where soluble enzyme denatured. These developments in scavenger technology have improved the in vivo protection provided by OP scavengers and extended their applicability to provide external decontamination of chemical agents and pesticides.

Organophosphate skin decontamination using immobilized enzymes.

We previously demonstrated that a combination of cholinesterase (ChE) pre-treatment with an oxime is an effective measure against soman and sarin. We describe here a novel approach for the preparation of covalently linked ChEs which are immobilized to a polyurethane matrix. Such preparation of ChE-sponges enhances the stability and usefulness of the enzymes in non-physiological environments. The ChE-sponges, which can be molded to any form, can effectively be used to remove and decontaminate organophosphates (OPs) from surfaces, biological (skin or wounds) or otherwise (clothing or sensitive medical equipment), or the environment. The ChE-sponges retained their catalytic activity under conditions of temperature, time, and drying where the native soluble enzyme would rapidly denature, and can be reused in conjunction with oximes many times. The ChE-sponge in the presence of oxime repeatedly detoxified OPs such as DFP or MEPQ. These developments in ChE technology have extended the applicability of OP scavengers from in vivo protection, to a variety of external detoxification and decontamination schemes. In addition to treatment of OP-contaminated soldiers, the ChE-sponge could protect medical personnel from secondary contamination while attending chemical casualties, and civilians exposed to pesticides or highly toxic nerve agents such as sarin.

The pH dependence of dealkylation in soman-inhibited cholinesterases and their mutants: further evidence for a push-pull mechanism.

Bimolecular rate constants for the inactivation of recombinant (r) human (Hu) butyrylcholinesterase (BChE) with P(S)C(S)- and P(S)C(R)-2-(3,3-dimethylbutyl) methylphosphonofluoridate (soman) are (92 +/- 7) x 10(6) M-1 min-1 and (13.7 +/- 0.8) x 10(6) M-1 min-1 at pH 7.4, mu = 0.1 M and 25 degreesC. Mutations of E197(199) to D or Q and W82(84) to A result in reductions in the rate constants for inactivation with P(S)C(S)-soman 4.3-, 11.8-, and 263-fold and with P(S)C(R)-soman by 6.5-, 47.3-, and 685-fold, respectively. The pH dependence of dealkylation (aging) in r mouse (Mo) acetylcholinesterase (AChE) and rHu BChE and their mutants inactivated with P(S)C(S)- and P(S)C(R)-soman was compared. Best-fit parameters for the asymmetric bell curves for the adducts of wild-type Mo AChE are pK1 = pK2 = 4.0-4.9 and pK3 = 5.2-6.6. These pKs are consistent with the involvement of two carboxylic acids, possibly E202(199) and either E334(327) or E450(443), and H447(440)H+ in the dealkylation of AChE. E202Q MoAChE inactivated with the soman diastereomers yielded pK3 = 5.5-5.8. Nearly symmetric pH curves for soman-inhibited wild-type and E197D Hu BChE gave pK2 = 3.7-4.6 and pK3 = 7.3-8.0, but much lower, pK3 approximately 5, for the corresponding adduct of the E197Q mutant. Dealkylation in soman-inhibited BChE is consistent with the participation of one carboxylic acid side chain and H438(440)H+. Maximal rate constants for dealkylation (kmax) are 1-6 min-1 for AChE and 2 min-1 for BChE at 25 degreesC. The W82 to A mutation in BChE results in the largest reduction, 2500-6000-fold, in the rate constant for dealkylation. The reduction in the rate constants for dealkylation in the E197 mutants is highly pH dependent. The solvent isotope effects at the pH maxima are 1.3-1.4, indicating unlikely preprotonation or proton in "flight" at the enzymic transition states. The new results support the push-pull mechanism of dealkylation in soman-inhibited cholinesterases proposed previously.

Inhibition of acetylcholinesterase and butyrylcholinesterase by chlorpyrifos-oxon.

Phosphorothionate insecticides such as parathion (O,O-diethyl O-p-nitrophenyl phosphorothioate) and chlorpyrifos (CPS; O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate; Dursban) are metabolically converted by oxidative desulfuration into paraoxon and chlorpyrifos-oxon (CPO). The insecticidal action of chlorpyrifos stems from inhibition of acetylcholinesterase (AChE) by CPO, resulting in severe cholinergic toxicity. Sensory peripheral neuropathy was observed in people exposed environmentally to chlorpyrifos sprayed in confined areas. We have examined the kinetics of inhibition of AChE and butyrylcholinesterase (BChE) by paraoxon and CPO. The bimolecular rate constants (ki) for inhibition by paraoxon of recombinant human (rH) AChE, recombinant mouse (rM) AChE, and fetal bovine serum (FBS) AChE were 7.0, 4.0, and 3.2 x 10(5) M(-1) min(-1). The ki values for the inhibition by CPO of rH AChE, fetal bovine serum AChE, human RBC AChE, Torpedo AChE, and recombinant mouse (rM) AChE were 9.3, 2.2, 3.8, 8.0, and 5.1 x 10(6) M(-1) min(-1), respectively. Inhibition of human serum BChE, rH BChE, and rM BChE by CPO yielded ki values of 1.65, 1.67, and 0.78 x 10(9) M(-1) min(-1), respectively. The ki values obtained for BChE from various species were 160- to 750-fold larger than those of AChE from parallel sources. Inhibition of the single-site mutant A328Y of rH BChE by CPO displayed a 21-fold lower rate than that of wild-type rH BChE (ki, 7.9 x 10(7) vs 1.67 x 10(9) M(-1) min(-1)). The double mutant of acyl pocket residues of rH AChE, F295L/F297V, was inhibited by CPO with a 150-fold larger ki than wild type (1.5 x 10(9) vs 1.0 x 10(7) M(-1) min(-1)). The increased rate obtained with the double mutant displaying characteristics of the BChE active center provides a rationale for higher efficacy of CPO scavenging by BChE, compared with AChE.

Acceleration of oxime-induced reactivation of organophosphate-inhibited fetal bovine serum acetylcholinesterase by monoquaternary and bisquaternary ligands.

Reactivation of organophosphate (OP)-inhibited acetylcholinesterase (AChE) by oximes is the primary reason for their effectiveness in the treatment of OP poisoning. Reactivation is reported to accelerate by quaternary ligands such as decamethonium, which is devoid of nucleophilicity. The mechanism of this enhancement is not known. To better understand the acceleration phenomenon, we examined ligand modulations of oxime-induced reactivation of methylphosphonylated AChE using 7-(methylethoxyphosphinyloxy)-1-methylquinolinium iodide and fetal bovine serum AChE. Edrophonium, decamethonium, and propidium, three quaternary AChE ligands of different types, were tested as potential accelerators. Experiments were carried out with both soluble enzyme preparation and AChE conjugated to polyurethane. Kinetic measurements with oximes 2-[hydroxyiminomethyl]-1-methylpyridinium chloride, 1,1'-trimethylene bis-(4-hydroxyimino methyl)-pyridinium dibromide, and 1, 1'-[oxybis-methylene)bis[4-(hydroxyimino)methyl]pyridiniu um dichloride showed that in the presence of 50 microM edrophonium, the reactivation rate constants increased 3.3-12.0-fold; 200 microM decamethonium produced a 1.6-3.0-fold enhancement of reactivation rate constants by the same oximes. Reactivation of the inhibited enzyme by 1-(2-hydroxyiminomethyl-1-pyridinium)-1-(4-carboxy-aminopyridinium )-d imethyl ether hydrochloride, 1-(2-hydroxyiminomethyl-1-pyridinium)-1-(3-carboxy-aminopyridinium )-d imethyl ether hydrochloride, and 1-[[[4-(aminocarbonyl)pyridino]methoxy]methyl]-2, 4, -bis(hydroxyimino)methyl pyridinium dichloride was not affected by either ligand. Propidium slowed the reactivation of 7-(methylethoxyphosphinyloxy)-1- methylquinolinium iodide-inhibited AChE by all oximes. Results suggest that the accelerator site may reside inside the catalytic gorge rather than at its entrance and acceleration may be due to the prevention of reinhibition of the regenerated enzyme by the putative product, the phosphonylated oxime. In addition to the nucleophilic property of the oximate anion, some of the reactivators may carry an accelerating determinant, as characterized with respect to edrophonium and decamethonium. Results offer possible explanations for the superiority of 1-(2-hydroxyiminomethyl-1-pyridinium)-1-(4-carboxy-aminopyridinium )-d imethyl ether hydrochloride over other oximes in the reactivation of specific AChE-OP conjugates.

Stable complexes involving acetylcholinesterase and amyloid-beta peptide change the biochemical properties of the enzyme and increase the neurotoxicity of Alzheimer's fibrils.

Brain acetylcholinesterase (AChE) forms stable complexes with amyloid-beta peptide (Abeta) during its assembly into filaments, in agreement with its colocalization with the Abeta deposits of Alzheimer's brain. The association of the enzyme with nascent Abeta aggregates occurs as early as after 30 min of incubation. Analysis of the catalytic activity of the AChE incorporated into these complexes shows an anomalous behavior reminiscent of the AChE associated with senile plaques, which includes a resistance to low pH, high substrate concentrations, and lower sensitivity to AChE inhibitors. Furthermore, the toxicity of the AChE-amyloid complexes is higher than that of the Abeta aggregates alone. Thus, in addition to its possible role as a heterogeneous nucleator during amyloid formation, AChE, by forming such stable complexes, may increase the neurotoxicity of Abeta fibrils and thus may determine the selective neuronal loss observed in Alzheimer's brain.

Allosteric control of acetylcholinesterase activity by monoclonal antibodies.

Previous studies showed that monoclonal antibodies raised against phosphorylated fetal bovine serum acetylcholinesterase appeared to modulate the catalytic activity of the enzyme by binding to a conformational epitope located at or near the region of the peripheral anionic site. The mechanism of inhibition of acetylcholinesterase by these monoclonal antibodies was further investigated by determining their effect on (i) substrate inhibition due to the binding of excess substrate to the peripheral anionic site and (ii) binding of peripheral anionic site ligands, such as propidium and fasciculin. Results of these experiments demonstrate that the accessibility of substrate to the peripheral anionic site in these complexes was restricted but not completely blocked, as none of the monoclonal antibodies eliminated the phenomenon of excess substrate inhibition. The results also show that propidium clearly slowed the inhibition of fetal bovine serum acetylcholinesterase by all six inhibitory monoclonal antibodies but to different levels. Complexation of fetal bovine serum acetylcholinesterase with monoclonal antibodies 25B1, 4E5, 6H9, and 5E8 interfered with the binding of fasciculin to the complexed enzyme, suggesting that part of their epitope overlapped with the fasciculin binding site. These monoclonal antibodies bind, in part, at the peripheral anionic site, since polyclonal anti-idiotypic antibodies generated against two monoclonal antibodies, 25B1 and 6H9, bound stoichiometric amounts of propidium. Like fasciculin, binding of these monoclonal antibodies in the vicinity of the peripheral anionic site at the rim of the active site gorge allosterically affects the orientation of W86 located at the base of the gorge, resulting in inhibition of enzyme activity.