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.

Hysteresis of insect acetylcholinesterase.

Pre-steady-state catalytic properties of insect acetylcholinesterase (AChE, EC 3.1.1.7) were studied with the neutral substrate N-methylindoxylacetate. Kinetics of soluble Apis mellifera and Drosophila melanogaster AChE forms showed lags (v(i)=0) before reaching the steady-state. Results were interpreted in terms of slow equilibrium between two conformational states E and E' of insect AChE. Hysteresis of insect AChE has been pointed out for the first time. The hysteretic behaviour was found to depend on the NMIA concentration and the nature of the enzyme. The maximum induction times (tau(max)) to reach the steady-state were 800 and 1000s with soluble AChE from A. mellifera and D.melanogaster, respectively. The orders of magnitude of the tau(max) were high and similar to human AChE and BuChE.

An unexpected plasma cholinesterase activity rebound after challenge with a high dose of the nerve agent VX.

Organophosphorus chemical warfare agents (nerve agents) are to be feared in military operations as well as in terrorist attacks. Among them, VX (O-ethyl-S-[2-(diisopropylamino)ethyl] methylphosphonothioate) is a low volatility liquid that represents a percutaneous as well as an inhalation hazard if aerosolized. It is a potent irreversible cholinesterase (ChE) inhibitor that causes severe signs and symptoms, including respiratory dysfunction that stems from different mechanisms. VX-induced pulmonary oedema was previously reported in dogs but mechanisms involved are not well understood, and its clinical significance remains to be assessed. An experimental model was thus developed to study VX-induced cardiovascular changes and pulmonary oedema in isoflurane-anaesthetized swine. In the course of this study, we observed a fast and unexpected rebound of plasma ChE activity following inhibition provoked by the intravenous injection of 6 and 12 microg kg(-1) of VX. In whole blood ChE activity, the rebound could stay unnoticed. Further investigations showed that the rebound of plasma esterase activity was neither related to spontaneous reactivation of ChE nor to VX-induced increase in paraoxonase/carboxylesterase activities. A bias in Ellman assay, haemoconcentration or severe liver cytolysis were also ruled out. All in all, these results suggest that the rebound was likely due to the release of butyrylcholinesterase into the blood stream from ChE producing organs. Nature of the organ(s) and mechanisms involved in enzyme release will need further investigations as it may represent a mechanism of defence, i.e. VX scavenging, that could advantageously be exploited.

Effects of mutations of active site residues and amino acids interacting with the Omega loop on substrate activation of butyrylcholinesterase.

The peripheral anionic site (PAS) of human butyrylcholinesterase is involved in the mechanism of substrate activation by positively charged substrates and ligands. Two substrate binding loci, D70 in the PAS and W82 in the active site, are connected by the Omega loop. To determine whether the Omega loop plays a role in the signal transduction between the PAS and the active site, residues involved in stabilization of the loop, N83, K339 and W430, were mutated. Mutations N83A and N83Q caused loss of substrate activation, suggesting that N83 which interacts with the D70 backbone may be an element of the transducing system. The K339M and W430A mutant enzymes retained substrate activation. Residues W82, E197, and A328 in the active site gorge have been reported to be involved in substrate activation. At butyrylthiocholine concentrations greater then 2 mM, W82A showed apparent substrate activation. Mutations E197Q and E197G strongly reduced substrate activation, while mutation E197D caused a moderate effect, suggesting that the carboxylate of residue E197 is involved in substrate activation. Mutations A328F and A328Y showed no substrate activation, whereas A328G retained substrate activation. Substrate activation can result from an allosteric effect due to binding of the second substrate molecule on the PAS. Mutation W430A was of special interest because this residue hydrogen bonds to W82 and Y332. W430A had strongly reduced affinity for tetramethylammonium. The bimolecular rate constant for reaction with diisopropyl fluorophosphate was reduced 10000-fold, indicating severe alteration in the binding area in W430A. The kcat values for butyrylthiocholine, o-nitrophenyl butyrate, and succinyldithiocholine were lower. This suggested that the mutation had caused misfolding of the active site gorge without altering the Omega loop conformation/dynamics. W430 as well as W231 and W82 appear to form the wall of the active site gorge. Mutation of any of these tryptophans disrupts the architecture of the active site.

Interaction between the peripheral site residues of human butyrylcholinesterase, D70 and Y332, in binding and hydrolysis of substrates.

Human butyrylcholinesterase displays substrate activation with positively charged butyrylthiocholine (BTC) as the substrate. Peripheral anionic site (PAS) residues D70 and Y332 appear to be involved in the initial binding of charged substrates and in activation control. To determine the contribution of PAS residues to binding and hydrolysis of quaternary substrates and activation control, the single mutants D70G/Y and Y332F/A/D and the double mutants Y332A/D70G and Y332D/D70Y were studied. Steady-state hydrolysis of the charged substrates, BTC and succinyldithiocholine, and the neutral ester o-nitrophenyl butyrate was measured. In addition, inhibition of wild-type and mutant enzymes by tetramethylammonium was investigated, at low concentrations of BTC. Single and double mutants of D70 and Y332 showed little or no substrate activation, suggesting that both residues were important for activation control. The effects of double mutations on D70 and Y332 were complex. Double-mutant cycle analysis provided evidence for interaction between these residues. The category of interaction (either synergistic, additive, partially additive or antagonistic) was found to depend on the nature of the substrate and on measured binding or kinetic parameters. This complexity reflects both the cross-talk between residues involved in the sequential formation of productive Michaelian complexes and the effect of peripheral site residues on catalysis. It is concluded that double mutations on the PAS induce a conformational change in the active site gorge of butyrylcholinesterase that can alter both substrate binding and enzyme acylation.

Polyol-induced activation by excess substrate of the D70G butyrylcholinesterase mutant.

Wild-type human butyrylcholinesterase (BuChE) has a non-Michaelian behaviour showing substrate activation with butyrylthiocholine (BTC) as the substrate. The D70G mutant has a catalytic constant identical to that of the wild-type enzyme, but a 10-fold lower affinity for BTC compared to wild-type enzyme, and it does not exhibit activation by excess BTC under conventional conditions. In the present work it was found that addition of polyols or sugars changed the kinetic behaviour of the D70G mutant with BTC. In the presence of 40% sucrose, the D70G mutant enzyme displayed marked activation by excess substrate. Because D70 is hydrogen bonded to Y332, mutants of Y332 were studied. Mutant Y332F had a behaviour similar to that of wild-type BuChE, whereas mutants Y332A, Y332A/D70G and D70G had negligible substrate activation. The behavior of wild-type, Y332F, Y332A and Y332A/D70G did not change in the presence of high concentrations of sugar. Substrate activation has been explained by binding of a second substrate molecule in the peripheral site at D70. The D70G mutant should be incapable of substrate activation, if D70 were the only residue involved in substrate activation. The ability of the D70G mutant to display substrate activation by medium engineering suggests that other residues are involved in initial substrate binding and activation by excess substrate. Osmolyte-induced change in conformation and/or hydration status of Y332 and other solvent-exposed residues may account for the non-Michaelian behaviour of the D70G mutant.

Absence of a protective effect of the oxime 2-PAM toward paraoxon-poisoned honey bees: acetylcholinesterase reactivation not at fault.

We investigated the failure of 2-PAM to protect honey bees against poisoning with paraoxon. The protective effect of the oxime 2-PAM against inhibition of acetylcholinesterase (AChE) by paraoxon was estimated in vitro and in vivo and was correlated with the mortality of paraoxon-treated bees. In vitro, 2-PAM protected 90% of AChE activity in the presence of paraoxon and reactivated more than 90% of inhibited AChE. Minor soluble and major membrane-bound forms of bee AChE presented about similar extents of reactivation, but the first order rate constant of reactivation (kobs) of the soluble form is threefold higher than that of the membrane-bound form. However, this difference did not significantly influence the reactivation kinetics of total AChE; the constant kobs of the membrane-bound form reflected that of total AChE. The linear kinetic profile of total AChE reactivation supported the conclusion that there was a single population of reactivatable species. The bimolecular rate constant of reactivation (kr), the dephosphorylation rate constant (k2), and the dissociation constant (Kd) were 646 M-1.min-1, 0.84 min-1 and 1. 30 mM, respectively. In vivo, administration of 2-PAM, after paraoxon exposure, induced a complete protection of AChE activity, but did not elicit any significant effect on mortality in paraoxon-treated bees. The inefficiency of 2-PAM to antagonize paraoxon-induced mortality was not changed by the administration of 2-PAM in pretreatment-therapy and in therapy treatments. These results indicated that the mortality of paraoxon-poisoned honey bees was not due to a lack of AChE reactivation.

Butyrylcholinesterase-catalysed hydrolysis of aspirin, a negatively charged ester, and aspirin-related neutral esters.

Although aspirin (acetylsalicylic acid) is negatively charged, it is hydrolysed by butyrylcholinesterase (BuChE). Catalytic parameters were determined in 100 mM Tris buffer, pH 7.4, in the presence and absence of metal cations. The presence of Ca2+ or Mg2+ (<100 mM) in buffer did not change the Km, but accelerated the rate of hydrolysis of aspirin by wild-type or D70G mutant BuChE by 5-fold. Turnover numbers were of the order of 5000-12000 min-1 for the wild-type enzyme and the D70G and D70K enzymes in 100 mM Tris, pH 7.4, containing 50 mM CaCl2 at 25 degreesC; Km values were 6 mM for wild-type, 16 mM for D70G and 38 mM for D70K. People with 'atypical' BuChE have the D70G mutation. The apparent inhibition seen at high aspirin concentration was not due to inhibition by excess substrate but to spontaneous hydrolysis of aspirin, causing inhibition by salicylate. The wild-type and D70G enzymes were competitively inhibited by salicylic acid; the D70K enzyme showed a complex parabolic inhibition, suggesting multiple binding. The effect of salicylate was substrate-dependent, the D70K mutant being activated by salicylate with butyrylthiocholine as substrate. Km value for wild-type enzyme was lower than for D70 mutants, suggesting that residue 70 located at the rim of the active site gorge was not the major site for the initial encounter aspirin-BuChE complex. On the other hand, the virtual absence of affinity of the W82A mutant for aspirin indicated that W82 was the major residue involved in formation of the Michaelis complex. Molecular modelling of aspirin binding to BuChE indicated perpendicular interactions between the aromatic rings of W82 and aspirin. Kinetic study of BuChE-catalysed hydrolysis of different acetyl esters showed that the rate limiting step was acetylation. The bimolecular rate constants for hydrolysis of aspirin by wild-type, D70G and D70K enzymes were found to be close to 1x106 M-1 min-1. These results support the contention that the electrostatic steering due to the negative electrostatic field of the enzyme plays a role in substrate binding, but plays no role in the catalytic steps, i.e. in the enzyme acetylation.

Resistance of butyrylcholinesterase to inactivation by ultrasound: effects of ultrasound on catalytic activity and subunit association.

The effects of 20 kHz ultrasound on catalytic activity and structure of the tetramer of wild-type human butyrylcholinesterase (BChE) from plasma and recombinant D70G mutant enzyme were studied at constant temperature. Effects on catalytic properties of both enzymes were investigated by kinetic analysis under ultrasound irradiation using a neutral substrate (o-nitrophenylbutyrate), a positively charged substrate (butyrylthiocholine), and a negatively charged substrate (aspirin). Effects on structure of highly purified wild-type BChE were followed by gel electrophoresis and activity measurements at Vmax after ultrasound treatment. Unlike hydrostatic pressure, mild ultrasound had moderate effects on catalytic parameters of BChE-catalyzed hydrolysis of substrates. For both wild-type and D70G, Km increased slightly with butyrylthiocholine and o-nitrophenylbutyrate under ultrasound irradiation, suggesting that these effects of ultrasound were not due to the periodic variation of pressure but rather to shear forces that took off substrate from the peripheral site and altered diffusion to the active site. By contrast, affinity of the D70G mutant for aspirin slightly increased with ultrasound power, suggesting that ultrasound-induced microstreaming unmasked peripheral residues involved in recognition and initial binding of the negatively charged substrate. Results support the contention that Km is a composite affinity constant, including dissociation constant of the first encounter enzyme-substrate complex on the peripheral site. Small changes in catalytic activity may have resulted from ultrasound-induced subtle conformational changes altering the active site reactivity. Short ultrasound irradiation induced a faint transient enzyme activation, but prolonged irradiation caused partial dissociation of the tetrameric enzyme and irreversible inactivation. Partial dissociation was related to enzyme microheterogeneity, i.e., nicked (C-terminal segment depleted) tetramers were less stable than native tetramers. The resistance of the native tetramer to ultrasound-induced dissociation was ascribed to the existence of an aromatic amino acid array on the apolar side of the C-terminal helical segment of subunits, the four subunits being held together in a four-helix bundle containing the aromatic zipper motifs. Aromatic/aromatic interactions between the four helical segments are thought to be enhanced by ultrasound-generated pressure.

Aging of di-isopropyl-phosphorylated human butyrylcholinesterase.

Organophosphate-inhibited cholinesterases can be reactivated by nucleophilic compounds. Sometimes phosphylated (phosphorylated or phosphonylated) cholinesterases become progressively refractory to reactivation; this can result from different reactions. The most frequent process, termed 'aging', involves the dealkylation of an alkoxy group on the phosphyl moiety through a carbocation mechanism. In attempting to determine the amino acid residues involved in the aging of butyrylcholinesterase (BuChE), the human BuChE gene was mutated at several positions corresponding to residues located at the rim of the active site gorge and in the vicinity of the active site. Mutant enzymes were expressed in Chinese hamster ovary cells. Wild-type BuChE and mutants were inhibited by di-isopropylfluorophosphate at pH 8.0 and 25 degrees C. Di-isopropyl-phosphorylated enzymes were incubated with the nucleophilic oxime 2-pyridine aldoxime methiodide and their reactivatability was determined. Reactivatability was expressed by the first-order rate constant of aging and/or the half-life of aging (t12). The t12 was found to be of the order of 60 min for wild-type BuChE. Mutations on Glu-197 increased t12 60-fold. Mutation W82A increased t12 13-fold. Mutation D70G increased t12 8-fold. Mutations in the vicinity of the active site serine residue had either moderate or no effect on aging; t12 was doubled for F329C and F329A, increased only 4-fold for the double mutant A328G+F329S, and no change was observed for the A328G mutant, indicating that the isopropoxy chain to be dealkylated does not directly interact with Ala-328 and Phe-329. These results were interpreted by molecular modelling of di-isopropylphosphorylated wild-type and mutant enzymes. Molecular dynamics simulations indicated that the isopropyl chain that is lost interacted with Trp-82, suggesting that Trp-82 has a role in stabilizing the carbonium ion that is released in the dealkylation step. This study emphasized the important role of the Glu-197 carboxylate in stabilizing the developing carbocation, and the allosteric control of the dealkylation reaction by Asp-70. Indeed, although Asp-70 does not interact with the phosphoryl moiety, mutation D70G affects the rate of aging. This indirect control was interpreted in terms of change in the conformational state of Trp-82 owing to internal motions of the Omega loop (Cys-65-Cys-92) in the mutant enzyme.

Importance of aspartate-70 in organophosphate inhibition, oxime re-activation and aging of human butyrylcholinesterase.

Asp-70 is the defining amino acid in the peripheral anionic site of human butyrylcholinesterase (BuChE), whereas acetylcholinesterase has several additional amino acids, the most important one being Trp-277 (Trp-279 in Torpedo AChE). We studied mutants D70G, D70K and A277W to evaluate the role of Asp-70 and Trp-277 in reactions with organophosphates. We found that Asp-70 was important for binding positively charged echothiophate, but not neutral paraoxon and iso-OMPA. Asp-70 was also important for binding of positively charged pralidoxime (2-PAM) and for activation of re-activation by excess 2-PAM. Excess 2-PAM had an effect similar to substrate activation, suggesting the binding of 2 mol of 2-PAM to wild-type but not to the D70G mutant. A surprising result was that Asp-70 was important for irreversible aging, the D70G mutant having a 3- and 8-fold lower rate of aging for paraoxon-inhibited and di-isopropyl fluorophosphate-inhibited BuChE. Mutants of Asp-70 had the same rate constants for phosphorylation and re-activation by 2-PAM as wild-type. The A277W mutant behaved like wild-type in all assays. Our results predict that people with the atypical (D70G) variant of BuChE will be more sensitive to the toxic effects of echothiophate, but will be equally sensitive to paraoxon and di-isopropyl fluorophosphate. People with the D70G mutation will be resistant to re-activation of their inhibited BuChE by 2-PAM, but this will be offset by the lower rate of irreversible aging of inhibited BuChE, allowing some regeneration by spontaneous hydrolysis.

Role of aspartate 70 and tryptophan 82 in binding of succinyldithiocholine to human butyrylcholinesterase.

The atypical variant of human butyrylcholinesterase has Gly in place of Asp 70. Patients with this D70G mutation respond abnormally to the muscle relaxant succinyldicholine, experiencing hours of apnea rather than the intended 3 min. Asp 70 is at the rim of the active site gorge 12 A from the active site Ser 198. An unanswered question in the literature is why the atypical variant has a 10-fold increase in Km for compounds with a single positive charge but a 100-fold increase in Km for compounds with two positive charges. We mutated residues Asp 70, Trp 82, Trp 231, Glu 197, and Tyr 332 and expressed mutant enzymes in mammalian cells. Steady-state kinetic parameters for hydrolysis of butyrylthiocholine, benzoylcholine, succinyldithiocholine, and o-nitrophenyl butyrate were determined. The wild type and the D70G mutant had identical k(cat) values for all substrates. Molecular modeling and molecular dynamics suggested that succinyldicholine could bind in two consecutive orientations in the active site gorge; formation of one complex caused a conformational change in the omega loop involving Asp 70 and Trp 82. We propose the formation of three enzyme-substrate intermediates preceding the acyl-enzyme intermediate; kinetic data support this contention. Substrates with a single positive charge interact with Asp 70 just once, whereas substrates with two positive charges, for example succinyldithiocholine, interact with Asp 70 in two complexes, thus explaining the 10- and 100-fold increases in Km in the D70G mutant.

A single amino acid substitution, Gly117His, confers phosphotriesterase (organophosphorus acid anhydride hydrolase) activity on human butyrylcholinesterase.

The G117H mutant of human butyrylcholinesterase (EC 3.1.1.8) was expressed in Chinese hamster ovary cells. Substitution of Gly 117 with His to make the G117H mutant endowed butyrylcholinesterase with the ability to catalyze the hydrolysis of organophosphate esters. G117H was still able to hydrolyze butyrylthiocholine, benzoylcholine, and o-nitrophenyl butyrate, but in addition it had acquired the ability to hydrolyze the antiglaucoma drug echothiophate and the pesticide paraoxon. Wild-type butyrylcholinesterase was irreversibly inhibited by echothiophate and paraoxon, but G117H regained 100% activity within 2-3 min following reaction with these compounds. On a polyacrylamide gel, the same bands that stained for activity with butyrylthiocholine also stained for activity with echothiophate. G117H is the only enzyme known that hydrolyzes echothiophate. Diethoxyphosphorylated G117H aged with a half-time of 5.5 h, a rate 600 times slower than the rate of hydrolysis. Echothiophate and paraoxon were hydrolyzed with the same kcat of 0.75 min-1. This calculates to a rate acceleration of 100,000-fold for hydrolysis of echothiophate and paraoxon by the G117H mutant compared to the nonenzymatic rate.

Asp7O in the peripheral anionic site of human butyrylcholinesterase.

The goal of this work was to determine what amino acids at the mouth of the active-site gorge are important for the function of human butyrylcholinesterase. Mutants D70G, Q119Y, G283D, A277W, A277H and A277W/G283D were expressed in human embryonal kidney cells and the secreted enzymes were assayed by steady-state kinetics. The result was that only one amino acid, D70 was found to be important for function. When D70 was mutated to G, the same mutation as in the naturally occurring atypical butyrylcholinesterase, the affinity for positively charged substrates and positively charged inhibitors decreased 5-30-fold. The D70G mutant had another striking abnormality in that it was virtually devoid of the phenomenon of substrate activation by excess butyrylthiocholine. Thus, though kcat was the same for wild-type and D70G mutant, being 24000 min(-1) at low butyrylthiocholine concentrations (0.01-0.1 mM), it failed to increase for the D70G mutant at 40 mM butyrylthiocholine, whereas it increased threefold for wild type. The D70G mutant was more sensitive to changes in salt concentration, its catalytic rate decreasing more than that of the wild type. The D70G mutant appeared to have a greater surface negative charge than wild type suggesting that the D70G mutant had a conformation different from that of the wild type. That D70 affects the function of butyrylcholinesterase, together with its location at the mouth of the active-site gorge, supports the hypothesis that D70 is a component of the peripheral anionic site of butyrylcholinesterase. Mutants containing aromatic amino acids at the mouth of the gorge had increased binding affinity for propidium and fasciculin, but unaltered function, suggesting that aromatic amino acids are not important to the function of the peripheral anionic site of butyrylcholinesterase.

Mutation His322Asn in human acetylcholinesterase does not alter electrophoretic and catalytic properties of the erythrocyte enzyme.

The YT blood group antigen is located on human red blood cell (RBC) acetylcholinesterase. Wild-type acetylcholinesterase, YT1, has histidine at codon 322, whereas the genetic variant of acetylcholinesterase, YT2, has asparagine. This mutation is located within exon 2 of the ACHE gene, an exon that is present in all alternatively spliced forms of acetylcholinesterase. Therefore, acetylcholinesterase in brain and muscle has the same mutation as RBC acetylcholinesterase. We compared the electrophoretic and kinetic properties of RBC acetylcholinesterases having His 322 or Asn 322. We found no differences in the isoelectric point, mobility on non-denaturing gel electrophoresis, affinity for acetylthiocholine, activity per milligram of RBC ghost protein, substrate inhibition constants, binding to the peripheral site ligand (propidium), and binding to active site ligands (tetrahydroaminoacridine and edrophonium). Thus, although the point mutation elicits antibody production in nonmatching blood transfusion recipients, it has no effect on the enzymatic properties of acetylcholinesterase.