Theoretical conformational analysis in the determination of productive conformations of substrates for acetylcholinesterase and butyrylcholinesterase

All the equilibrium conformations of 34 analogues of acetylcholine (ACh) with the general formula R-C(O)O-Alk-N+(CH3)3 are calculated by the method of molecular mechanics. In the series R-C(O)O-(CH2)2-N+(CH3)3, a reliable correlation is found between the molecular volume of the substrate and the rate of its hydrolysis by acetylcholinesterase (AChE); the absence of such a correlation is demonstrated for butyrylcholinesterase (BChE). Theoretical conformational analysis confirms that the completely extended tt conformation of ACh is productive for the hydrolysis by AChE, which agrees with the results of X-ray analysis of AChE. AChE is shown to hydrolyze only those substrates that form equilibrium conformers compatible in the mutual arrangement of trimethylammonium group, carbonyl carbon, and carbonyl oxygen with the tt conformation of ACh; in this case, the rate of substrate hydrolysis depends on the total population of these conformers. A reliable correlation was found between the population of the semifolded (tg-) conformation of the choline moiety of substrate molecules and the rate of their BChE hydrolysis. In a series of CH3-C(O)O-Alk-N+(CH3)3, the rate of BChE hydrolysis is demonstrated to depend on the total population of conformations compatible in the mutual arrangement of functionally important atoms with the tg- conformation of ACh. The tg- conformation of ACh is concluded to be productive for BChE hydrolysis. Similar orientations of the substrate molecules relative to the catalytic triads of both AChE and BChE are proven to coincide upon the substrate productive sorption in their active sites. It is hypothesized that the sorption stage is rate-limiting in cholinesterase hydrolysis and the enzyme hydrolyzes the ACh molecule in its energetically favorable conformation.     

Theoretical Conformational Analysis in the Determination of Productive Conformations of Substrates for Acetylcholinesterase and Butyrylcholinesterase

All the equilibrium conformations of 34 analogues of acetylcholine (ACh) with the general formula R-C(O)O-Alk-N⁺(CH₃)₃ are calculated by the method of molecular mechanics. In the series R-C(O)O-(CH₂)₂-N⁺(CH₃)₃, a reliable correlation is found between the molecular volume of the substrate and the rate of its hydrolysis by acetylcholinesterase (AChE); the absence of such a correlation is demonstrated for butyryl-cholinesterase (BChE). Theoretical conformational analysis confirms that the completely extended tt conformation of ACh is productive for the hydrolysis by AChE, which agrees with the results of X-ray analysis of AChE. AChE is shown to hydrolyze only those substrates that form equilibrium conformers compatible in the mutual arrangement of trimethylammonium group, carbonyl carbon, and carbonyl oxygen with the tt conformation of ACh; in this case, the rate of substrate hydrolysis depends on the total population of these conformers. A reliable correlation was found between the population of the semifolded (tg^?) conformation of the choline moiety of substrate molecules and rate of their BChE hydrolysis. In a series of CH₃-C(O)O-Alk-N⁺(CH₃)₃, the rate of BChE hydrolysis is demonstrated to depend on the total population of conformations compatible in the mutual arrangement of functionally important atoms with the tg^? conformation of ACh. The tg^? conformation of ACh is concluded to be productive for BChE hydrolysis. Similar orientations of the substrate molecules relative to the catalytic triads of both AChE and BChE are proven to coincide upon the substrate productive sorption in their active sites. It is hypothesized that the sorption stage is rate-limiting in cholinesterase hydrolysis and the enzyme hydrolyzes the ACh molecule in its energetically favorable conformation.     

Differential perturbation of erythrocyte membrane-associated transport and enzyme activities by structurally related lipophilic compounds

The alteration of two erythrocyte plasma membrane functions, acetylcholine hydrolysis and glucose exchange, by a series of structurally related small lipophilic compounds which exhibit similar antihemolytic behavior was studied. 2-Methyldimethylaminoazobenzene is a more potent inhibitor of acetylcholinesterase than the 3′-methyl analogue, while the unsubstituted compound fails to inhibit. Esterase inhibition by the 2-methyl compound is noncompetitive and dependent on the anion composition of the assay buffer. The temperature dependence of acetylcholinesterase activity in the presence of the 2-methyl compound suggests that interaction with inhibitor is influenced by the state of lipids tightly bound to the enzyme. Glucose exchange is inhibited to the same extent by both methyl derivatives but not by the unsubstituted dye, and the temperature dependence in the presence of inhibitor is not grossly altered. The lack of correlation between inhibition of membrane function and stabilization of erythrocytes against osmotic hemolysis is discussed.     

Silicon compounds as substrates and inhibitors of acetylcholinesterase

Several trimethylsilyl derivatives were found to be ligands of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7): trimethylsilylethyl acetate (III) and trimethylsilylmethyl acetate (V) are substrates of the enzyme, whereas trimethylsilylethanol (VIII) is a competitive inhibitor. The silicon compounds have kinetic parameters similar to those of their carbon analogues, except for trimethylsilylmethyl acetate, which is a substrate of acetylcholinesterase, whereas its carbon analogue is not susceptible to enzymic hydrolysis.     

Modeling evolution of hydrogen bonding and stabilization of transition states in the process of cocaine hydrolysis catalyzed by human butyrylcholinesterase

Molecular dynamics (MD) simulations and quantum mechanical/molecular mechanical (QM/MM) calculations were performed on the prereactive enzyme-substrate complex, transition states, intermediates, and product involved in the process of human butyrylcholinesterase (BChE)-catalyzed hydrolysis of (-)-cocaine. The computational results consistently reveal a unique role of the oxyanion hole (consisting of G116, G117, and A199) in BChE-catalyzed hydrolysis of cocaine, compared to acetylcholinesterase (AChE)-catalyzed hydrolysis of acetylcholine. During BChE-catalyzed hydrolysis of cocaine, only G117 has a hydrogen bond with the carbonyl oxygen (O31) of the cocaine benzoyl ester in the prereactive BChE-cocaine complex, and the NH groups of G117 and A199 are hydrogen-bonded with O31 of cocaine in all of the transition states and intermediates. Surprisingly, the NH hydrogen of G116 forms an unexpected hydrogen bond with the carboxyl group of E197 side chain and, therefore, is not available to form a hydrogen bond with O31 of cocaine in the acylation. The NH hydrogen of G116 is only partially available to form a weak hydrogen bond with O31 of cocaine in some structures involved in the deacylation. The change of the estimated hydrogen-bonding energy between the oxyanion hole and O31 of cocaine during the reaction process demonstrates how the protein environment can affect the energy barrier for each step of the BChE-catalyzed hydrolysis of cocaine. These insights concerning the effects of the oxyanion hole on the energy barriers provide valuable clues on how to rationally design BChE mutants with a higher catalytic activity for the hydrolysis of (-)-cocaine.     

Length of the acyl carbonyl bond in acyl-serine proteases correlates with reactivity

Resonance Raman (RR) spectroscopy has been used to obtain the vibrational spectrum of the acyl carbonyl group in a series of acylchymotrypsins and acylsubtilisins at the pH of optimum hydrolysis. The acyl-enzymes, which utilize arylacryloyl acyl groups, include three oxyanion hole mutants of subtilisin BPN', Asn155Leu, Asn155Gln, and Asn155Arg, and encompass a 500-fold range of deacylation rate constants. For each acyl-enzyme a RR carbonyl band has been identified which arises from a population of carbonyl groups undergoing nucleophilic attack in the active site. As the deacylation rate (k3) increases through the series of acyl-enzymes, the carbonyl stretching band (vC = O) is observed to shift to lower frequency, indicating an increase in single bond character of the reactive acyl carbonyl group. Experiments involving the oxyanion hole mutants of subtilisin BPN' indicate that a shift of vC = O to lower frequency results from stronger hydrogen bonding of the acyl carbonyl group in the oxyanion hole. A plot of log k3 against vC = O is linear over the range investigated, demonstrating that the changes in vC = O correlate with the free energy of activation for the deacylation reaction. By use of an empirical correlation between carbonyl frequency (vC = O) and carbonyl bond length (rC = O) it is estimated that rC = O increases by 0.015 A as the deacylation rate increases 500-fold through the series of acyl-enzymes. This change in rC = O is about 7% of that expected for going from a formal C = O double bond in the acyl-enzyme to a formal C-O single bond in the tetrahedral intermediate for deacylation. The data also allow us to estimate the energy needed to extend the acyl carbonyl group along its axis to be 950 kJ mol-1 A-1.     

Kinetics of 13 new cholinesterase inhibitors

Kinetics of hydrolysis of acetylcholine and acetylthiocholine by two types of acetylcholinesterase and butyrylcholinesterase inhibited by 13 new inhibitors (5 carbamates and 8 carbazates--hydrazinium derivatives) was measured in vitro in a batch reactor at 25 degrees C, pH 8, ionic strength 0.11 M and enzyme activity 3.5 U by four nondependent analytical methods. Sevin, rivastigmin (Exelon) and galantamin (Reminyl) served as comparative inhibiting standards. Kinetics of hydrolyses inhibited by all studied carbamates, sevin, carbazates (with exceptions) and rivastigmin (with exceptions) can be simulated by the competitive inhibition model with irreversible reaction between enzyme and inhibitor. Galantamin does not fulfil this model. In positive simulations, the value of inhibition (carbamoylation) rate constant k3 was calculated, describing the reaction velocity between the given enzyme and inhibitor. Physiologically important hydrolyses of acetylcholine catalyzed by acetylcholinesterase from electric eel or bovine erythrocytes and butyrylcholinesterase from horse plasma can be most quickly inhibited by carbamoylation of the mentioned enzymes by the 3-N,N-diethylaminophenyl-N'-(1-alkyl) carbamates 4 and 5. Probably this is due to a long enough hydrocarbon aliphatic substituent (hexyl and octyl) on the amidic nitrogen atom. The tested carbazates failed as inhibitors of cholinesterases. The regeneration ability of the inhibited enzymes was not measured.     

Luminescent nanoparticles for rapid monitoring of endogenous acetylcholine release in mice atria

The present work introduces for the first time a nanoparticulate approach for ex vivo monitoring of acetylcholinesterase‐catalyzed hydrolysis of endogenous acetylcholine released from nerve varicosities in mice atria. Amino‐modified 20‐nm size silica nanoparticles (SNs) doped by luminescent Tb(III) complexes were applied as the nanosensors. Their sensing capacity results from the decreased intensity of Tb(III)‐centred luminescence due to the quenching effect of acetic acid derived from acetylcholinesterase‐catalyzed hydrolysis of acetylcholine. Sensitivity of the SNs in monitoring acetylcholine hydrolysis was confirmed by in vitro experiments. Isolated atria were exposed to the nanosensors for 10 min to stain cell membranes. Acetylcholine hydrolysis was monitored optically in the atria samples by measuring quenching of Tb(III)‐centred luminescence by acetic acid derived from endogenous acetylcholine due to its acetylcholinesterase‐catalyzed hydrolysis. The reliability of the sensing was demonstrated by the quenching effect of exogenous acetylcholine added to the bath solution. Additionally, no luminescence quenching occurred when the atria were pre‐treated with the acetylcholinesterase inhibitor paraoxon.     

Synthesis and hydrolytic evaluation of acid-labile imine-linked cytotoxic isatin model systems

In this study a series of isatin-based, pH-sensitive aryl imine derivatives with differing aromatic substituents and substitution patterns were synthesised and their acid-catalysed hydrolysis evaluated. These derivatives were functionalised at the C3 carbonyl group of a potent N-substituted isatin cytotoxin and were stable at physiological pH but readily cleaved at pH 4.5. Observed rates of hydrolysis for the embedded imine-acid moiety were in the order para-phenylpropionic acid>phenylacetic acid (para>meta)>benzoic acid (meta>para). The ability to fine-tune hydrolysis rates in this way has potential implications for optimising imine linked, tumour targeting cytotoxin-protein conjugates.     

CNDO calculations on the conformational structure of acetylcholine

Molecular orbital theory in the CNDO framework has been used to calculate the torsional angles which lead to minimum energy conformations in acetylcholine. The calculated angles agree well with the experimental observations on acetylcholine and its derivatives. The results have been compared with the earlier predictions based on extended Hückel theory and van der Waal pairwise interactions.     

Pulsefluorimetry of tyrosyl peptides

The fluorescence quantum yield and the fluorescence decay of aqueous solutions of derivatives containina a single tyrosine residue have been measured at different pH. In these derivatives tyrosine was substituted on its amino end (series I) or/and, on its carboxyl end (series II), by acyl, amino or amino acyl groups. The fluorescence decays of series I derivatives are monoexponential regardless to the ionization state of their amino group. Upon deprotonation of the α-amino group, the quantum yields and the lifetimes increase in the case of dipeptides, and slightly decrease, for the tripeptides. The quantum yield and the lifetime increase with the side chain length of the aliphatic residue adjacent to the tyrosine residue, (the fluorescence of Val Tyr anion being identical to that of free Tyrosine). Quite different is the behavior of series II derivatives: their decays at pH 5.5 must be described by two exponential terms, one of them decaying with a short time constant (about 0.5 ns) and little side chain effect is observed. The fluorescence intensity increases upon deprolonalion of the α-amino proup (though to a lesser extent than for series I derivatives); a nearly monoexponential decay is observed at basic pH for dipeptides. but not for tyrosine amide, amide or dipeptides, or tripeptides. The following interpretation of our results is proposed: fluorescence quenching occurs in molecular conformations in which a peptide carbonyl can come in contact with the phenolic chromophore. This condition depends mainly on the value of the angle x₁ which determines the conformation of the tyrosyl residue around its C_α-C_β bond. It appears that the rotamer in which quenching occurs are not the same for series I and series II derivatives, which can explain the different behavior of these two kinds of compounds. The interpretation of the fluorescence properties is developed taking into account on one side the relative population of the rotamers in the ground state, which is given by studies of crystals and of solutions, and on the other side the possibility of an exchange between these rotamers during the excited state time. In this scheme the protonated α-amino groups would act to reinforce the quenching efficiency of the carbonyl. At last it is found that the radiative lifetime of the phenolic chromophore is the same for all the compounds studies.     

Isologous Oxygen,Sulfur, and Selenium Compounds as Probes of Acetylcholine Receptors

Evidence is presented that while the conformations of acetylcholine and acetylthiolcholine are different, acetylthiolcholine and acetylselenolcholine are structurally and conformationally very similar. Experiments with sulfur and selenium isologs of acetylcholine, choline, and local anesthetics suggest that the active sites of receptors of the electroplax and of electric eel acetylcholinesterase are different, but are compatible with the postulate that acetylcholine receptors of axonal and synaptic excitable membranes are similar.     

A theoretical conformational analysis of several substrates of cholinesterase having a cyclic ammonium group structure]

Conformational possibilities of pirrolidine analogues of acetylcholine beta-(N-methyl pirrolidinium)-ethyl ester of acetic acid and beta-(N-ethyl pirrolidinium)-ethyl ester of acetic acid and beta-(N-ethyl pirrolidinium)-ethyl ester of acetic acid were investigated by the method of atomic potentials. The conformational energy was considered as a sum of non-bonded and electrostatical interactions, torsional energy and distortions of bond angles. It has been shown that the replacement of the nitrogen methyl group to ethyl group results in decrease of the average barrier height between two gauche conformations of the O--C--C--N fragment. Comparison of conformational properties of some cholinesterase substrates permit to draw a suggestion that the barrier height influences the rate of the enzymatic hydrolysis.     

Acetylcholinesterase-catalyzed hydrolysis of an amide.

In this paper we report that acetylcholinesterase catalyzes hydrolysis of amides, an observation which had not been made previously. The amide used is an analog of acetylcholine, 2-acetoaminoethyltrimethylammonium iodide. The experiments were performed with an enzyme preparation obtained from electroplax of Electrophorus electricus. Inhibition of the enzyme by a specific organic phosphate inhibitor abolished both the esterase and the amidase activity of the enzyme. The effect of hydrogen ions between pH 5 and pH 10 on the steady-state kinetic parameters, Km and kcat, has been investigated. These parameters show essentially the same dependence on pH as is observed in catalytic hydrolysis of acetylcholine. k-cat is controlled by an ionizing group of the enzyme with an apparent pK of approximately 6.3, and reaches a pH-independent maximum value of 3.6 sec- minus 1 above pH 8. The value for Km of 1 mM at pH 7 and 25 degrees is about five times greater than that for catalytic hydrolysis of the ester at the same pH and temperature. Preliminary electrophysiological experiments indicate that the amide analog binds to the receptor less well, by several orders of magnitude, than acetylcholine does.     

The monoclonal antibody AE-2 modulates fetal bovine serum acetylcholinesterase substrate hydrolysis

The monoclonal antibody (mAb) AE-2 decreases the rate of hydrolysis of acetylthiocholine (ATC) by fetal bovine serum acetylcholinesterase (acetylcholine acetylhydrolase EC 3.1.1.7) (FBS-AChE) (Doctor, B.P. et al. (1989) Proc. 32nd Oholo Conf., Eilat, Israel, in press), but increases the rate of hydrolysis (Vmax) of the nonpolar substrate, indophenyl acetate (IPA) approx. 15-fold. The affinity (Km) of FBS-AChE for IPA changes minimally in comparison with the increase in the rate of hydrolysis. The complex is dissociated, and the modulation of substrate hydrolysis is reversed by the active-center ligand, 1-methyl-2-hydroxyiminomethylpyridinium chloride (2-PAM).     

Kinetic analysis of calcium activation of brain acetylcholinesterase forms.

Calcium activation of acetylcholine hydrolysis by bovine brain acetylcholinesterase (Acetylcholine hydrolase, EC 3.1.1.7) forms has been analyzed in terms of changes in kinetic constants and thermodynamic activation parameters. De-acetylation was determined to be the major rate-influencing step in acetylcholine hydrolysis by both 60 000- and 240 000-dalton forms of the brain enzyme and 10 mM Ca2+ increased the rate constant for this step (k+3) by approximately 30% for both forms. For the smaller acetylcholinesterase form the effects of Ca2+ on de-acetylation was equivalent to its effect on the overall rate constant (k) and occurred without an effect on pK. In the case of the 240 000-dalton species, the overall rate constant was increased by Ca2+ by 33% at pH 8.0 and 81% at pH 7.25 and involved a pK shift of -0.2 pH units. For both enzyme forms the rate constants for acetylation (k+2) were increased by Ca2+. Thermodynamic analysis suggested that Ca2+ activation of the acetylation step was entropically driven. Differences between the two enzymes forms in terms of Ca2+ appear to result from association of low molecular weight species.     

The mechanism of ageing of phosphonylated acetylcholinesterase

1. The extent of potential reactivation of organophosphate-inhibited acetylcholinesterase decreases with time, a phenomenon called ageing. Ageing is due to dealkylation of the alkoxyl group of the residue bound to the enzyme. The rate of ageing is proportional to the electron-donating capacity of the alkyl group. 2. The ageing of phosphophonylated cholinesterase cal also be demonstrated using a phrenic nerve-diaphragm preparation. The same relationship between the rate of ageing and the structure of the alkyl group was observed. 3. Ageing occurs much faster in electrically stimulated preparations than in resting preparations. This may be due to production of a more acidic environment for the enzyme at the active centre by the products of hydrolysis of the acetylcholine released by stimulation.     

A Computer-Aided Radiopharmaceutical Drug Design Study Using Ab Initio and Molecular Mechanics Methods

This is an investigation of technetium ligands and their complexes with [TcO]³⁺ using ab initio population analysis and molecular mechanics conformational searching methods. Calculated atomic electronic populations on the technetium atom in complexes with a number of ligands gauge the degree of covalent bonding between technetium and these ligands. Here a reduction in the positive charge on the [TcO]³⁺ moiety by complexation with a given ligand is correlated with covalent bonding. Our ab initio results suggest that ligands with more sulphur atoms have better covalent bonding to technetium than do other ligands. A conformational analysis of the uncomplexed ligands indicates that conformational reorganization before complexation correlates inversely with stable complex formation. This conformational analysis shows that ligands with ethylene carbonyl bridges have low energy conformations closer to the final complexation geometries than do ligands with ethylene, propylene or propylene carbonyl bridges. The presence of these low energy conformations facilitates a faster complexation of the ethylene carbonyl [TcO]³⁺ moiety. This result produces a kinetic explaination why ethylene carbonyl bridged ligands form stable complexes while many other ligands do not [1]. The conclusion is that kinetic and thermodynamic considerations play a role in stable complex formation between these ligands and technetium.     

Boundaries of the butyrylcholinesterase anion site from data of a conformational analysis of substrates

By means of molecular mechanics, theoretical conformational analysis has been made of 19 substrates of butyrylcholinesterase - acetylcholine derivatives with different structure of the ammonium group. It was concluded that the anionic point is located in the cavity of the enzymic molecule. Dimensions and shape of this cavity were established which provide satisfactory correlation between its filling by substrate conformers and the rate of their enzymic hydrolysis. Some suggestions were made with respect to the mechanism of the effect of non-productive sorbtion of the substrates on the rate of their enzymic hydrolysis.     

Ligands of cholinesterases of ephedrine and pseudoephedrine structure

The paper is a review of literature data on interaction of erythrocytic acetylcholinesterase and of mammalian blood serum butyrylcholinesterase with a group of isomeric complex ester derivatives (acetates, propionates, butyrates, valerates, and isobutyrates) of bases and iodomethylates of ephedrine and its enantiomer pseudoephedrine. For 20 alkaloid monoesters, parameters of enzymatic hydrolysis are determined and their certain specificity toward acetylcholinesterase is revealed, whereas 5 diesters of iodomethylates of pseudoephedrine were submitted to hydrolysis only by butyrylcholinesterase. It turned out that 20 alkaloid diesters and 10 trimethylammonium derivatives were uncompetitive reversible inhibitors of acetylcholinesterase and competitive inhibitors of butyrylcholinesterase. The performed for the first time isomer and enantiomer analysis “structure—efficiency” has shown that in most caes it is possible to state the greater complementarity of catalytical surface of enzymes for ligands of pseudoephedrine structure, such differentiation being more often manifested.