The role of the functional architecture of the HuAChE (human acetylcholinesterase) in reactivity toward the carbamates pyridostigmine, rivastigmine and several analogues of physostigmine, that are currently used or considered for use as drugs for Alzheimer's disease, was analysed using over 20 mutants of residues that constitute the interaction subsites in the active centre. Both steps of the HuAChE carbamylation reaction, formation of the Michaelis complex as well as the nucleophilic process, are sensitive to accommodation of the ligand by the enzyme. For certain carbamate/HuAChE combinations, the mode of inhibition shifted from a covalent to a noncovalent type, according to the balance between dissociation and covalent reaction rates. Whereas the charged moieties of pyridostigmine and rivastigmine contribute significantly to the stability of the corresponding HuAChE complexes, no such effect was observed for physostigmine and its analogues, phenserine and cymserine. Moreover, physostigmine-like ligands carrying oxygen instead of nitrogen at position −1 of the tricyclic moiety (physovenine and tetrahydrofurobenzofuran analogues) displayed comparable structure–function characteristics toward the various HuAChE enzymes. The essential role of the HuAChE hydrophobic pocket, comprising mostly residues Trp86 and Tyr337, in accommodating (−)-physostigmine and in conferring ∼300-fold stereoselectivity toward physostigmines, was elucidated through examination of the reactivity of selected HuAChE mutations toward enantiomeric pairs of different physostigmine analogues. The present study demonstrates that certain charged and uncharged ligands, like analogues of physostigmine and physovenine, seem to be accommodated by the enzyme mostly through hydrophobic interactions.

INTRODUCTION

The enzyme AChE (acetylcholinesterase) is presently the most important molecular target for therapeutic intervention in symptomatic treatment of senile dementia in AD (Alzheimer's disease) [1]. The ongoing effort to develop more therapeutically efficacious AChE inhibitors is currently driven by the remarkable progress, made during the last 15 years, in elucidating the structural and functional properties of the enzyme through X-ray crystallography [2,3] and site-directed mutagenesis [48]. Combination of these two powerful techniques allowed for the detailed mapping of the HuAChE (human AChE) active centre, delineating the functional subsites involved in reactivity toward substrates and other covalent modifiers as well as toward noncovalent ligands specific for the active centre. These include the catalytic triad (Ser203, His447 and Glu334), the ‘oxyanion hole’ consisting of residues Gly120, Gly121 and Ala204, as well as different combinations of the 14 aromatic amino acids which line approx. 40% of the HuAChE active-centre gorge surface: e.g. the acyl pocket (Phe295 and Phe297); the ‘hydrophobic subsite’ (Trp86, Tyr133, Tyr337 and Phe338); the cation–π interaction locus for charged moieties of substrates and other ligands at the active centre (Trp86); and the PAS [peripheral anionic site (Tyr72, Tyr124 and Trp286)].

Further examination of the functional architecture of the HuAChE active centre revealed that reactivity of the enzyme toward substrates and other ligands can also be affected through perturbation of functional domains which may include multiple subsites in the active centre. Thus, enhanced conformational mobility of the catalytic histidine residue was recently implicated in the activity differences between HuBChE (human butyrylcholinesterase) and the hexamutant HuAChE carrying aliphatic replacements of all the active-site gorge aromatic residues (Tyr72, Tyr124, Trp286, Phe295, Phe297 and Tyr337), distinguishing between the two enzymes [9,10]. Modulation of ligand interactions with the enzyme can also be effected through disruption of polar networks in the active centre. One of these may include residue Ser229 and the catalytic triad residue Glu334 [11].

Most of the AChE inhibitors approved for clinical use as AD drugs (Cognex, Aricept and Nivalin) are noncovalent inhibitors and hence their AChE complexes are amenable for crystallographic analysis [1214]. For the recently approved covalent AChE modifier, the carbamate rivastigmine (Exelon), such analysis can be carried out only on the carbamylated enzyme and is therefore relevant primarily to the enzyme regeneration step [15]. Yet the overall inhibition process by carbamates is determined by properties of both the carbamylated enzyme and the transient Michaelis complex. These two species determine the rates of decarbamylation and carbamylation respectively, and hence both contribute to the efficacy of the carbamate as a drug. Therefore, dissection of the affinity characteristics toward carbamates, through functional analysis of the carbamate–HuAChE Michaelis complexes, should provide information relevant to the design of more efficacious carbamate AD therapeutics [16]. In the past, we have shown that functional analysis of such Michaelis complexes can be carried out much in the same manner as for the noncovalent ligands [8].

In the present study, we examined the reactivity of HuAChE enzymes, modified at relevant binding subsites, toward the carbamates rivastigmine and analogues of physostigmine, which are currently used [17,18] or considered for use as AD drugs [1922]. Elements of the binding environment that determine the fate (carbamylation or dissociation; see 1) of the particular Michaelis complexes have been identified. We conclude that interactions of HuAChE enzymes with analogues of physostigmine are dominated by hydrophobic interactions of the tricyclic eseroline moiety, and therefore the properties of the corresponding Michaelis complexes are quite different from those of rivastigmine and pyridostigmine. Thus, functional analysis appears to be a tool of choice for analysis of molecular complexes, which are too unstable for structural studies and yet are important as templates for drug design.

Rate constants of progression of the carbamylation reactions

Scheme 1
Rate constants of progression of the carbamylation reactions

Under conditions where k−1k2Kd=k−1/k1 (dissociation constant of the Michaelis complex); ki=k2/Kd (bimolecular rate constant of carbamylation).

Scheme 1
Rate constants of progression of the carbamylation reactions

Under conditions where k−1k2Kd=k−1/k1 (dissociation constant of the Michaelis complex); ki=k2/Kd (bimolecular rate constant of carbamylation).

MATERIALS AND METHODS

Materials and enzymes

ATC (acetylthiocholine) iodide, DTNB [5,5′-dithiobis-(2-nitrobenzoic acid)], pyridostigmine bromide and (−)-physostigmine salicylate were purchased from Sigma, and rivastigmine was obtained from Teva Ltd. (+)-Physostigimine, enantiomers of physovenine, phenserine, as well as the enantiomeric pairs of cymserine, cymyl carbamate of physovenol and cymyl carbamate of tetrahydrofurobenzofurol, were synthesized according to published procedures [2326]. The chemical structures of the AChE inhibitors are shown in Figure 1, and full chemical characterization was performed to ensure chemical and chiral purity.

Chemical structures of the carbamates used in this study

Figure 1
Chemical structures of the carbamates used in this study

Only the (−)-3aS-enantiomers of the various physostigmine analogues are shown. The numbering shown for eseroline is applicable to all the other physostigmine analogues.

Figure 1
Chemical structures of the carbamates used in this study

Only the (−)-3aS-enantiomers of the various physostigmine analogues are shown. The numbering shown for eseroline is applicable to all the other physostigmine analogues.

Expression of recombinant enzymes, as well as the construction of all the HuAChE mutants, was as described previously [4,6,7,9,2731]. Construction of the double mutant W86A/Y337A was carried out by replacement of the appropriate DNA fragments of the AChE-w7 variant [4] with the respective fragments of the W86A and Y337A variants. Stable recombinant cell clones expressing high levels of each of the mutants were established according to the procedure described previously [27]. Enzymes were purified (over 90% purity) as described previously [27,32].

Determination of HuAChE activity and analysis of kinetic data

HuAChE activity was assayed according to the method of Ellman et al. [33] in the presence of 0.1 mg/ml BSA, 0.3 mM DTNB, 50 mM sodium phosphate buffer (pH 8.0) and various concentrations of ATC or TB (3,3-dimethylbutylthioacetate) at 27 °C and monitored with a Thermomax microplate reader (Molecular Devices).

The rate constants of progression of the carbamylation reactions (see 1) were estimated for at least four different concentrations (at least within a 10-fold range around the estimated value of Kd) of carbamate (CR), by adding substrate at various time intervals and measuring the enzyme residual activity (E) (enzyme concentration was approx. 1.0 nM). To avoid interference from regeneration of enzyme activity due to dissociation of enzyme carbamate conjugates, the initial velocity was used to determine kobs (V=kobs[E]) at each carbamate concentration. Thus, values of kobs were calculated from the slope of the straight lines obtained from the plots of 1/ln(E) against time of incubation prior to addition of substrate (Figures 2A and 2B, middle panels). Double reciprocal plots of 1/kobs against 1/[CR] were used to compute k2 from the intercept, ki from the slope and Kd from the ratio between the slope and the intercept according to 1 and eqn 1 [8] (Figures 2A and 2B, right panels):

 
formula
(1)

Derivation of inhibition rate and equilibrium constants of wild-type HuAChE and its mutant F295A/F338A by the carbamates physostigmine, phenserine and cymserine

Figure 2
Derivation of inhibition rate and equilibrium constants of wild-type HuAChE and its mutant F295A/F338A by the carbamates physostigmine, phenserine and cymserine

Left panels: inhibition time curve at indicated concentrations of the various carbamates (the actual curve was not calculated and serves as illustration). Middle panels: (A) and (B), the plots of ln(E) against time, allow for derivation of the observed first-order carbamylation rate constants (kobs) at indicated carbamate concentrations; (C) and (D), Lineweaver–Burk plots for the wild-type HuAChE and the F295A/F338A enzymes with or without the indicated carbamates. Right panels: (A) and (B), double reciprocal plots of kobs (derived from their respective middle panels) against the respect carbamate concentrations, from which k2, ki and Kd were derived (as described in the Materials and methods section); (C) and (D), Rs (relative slope) values as determine from the corresponding middle panels were plotted against the concentration of the carbamates. The values of Kd for cymserine and phenserine were obtained from the slope of these plots (see the Materials and methods section).

Figure 2
Derivation of inhibition rate and equilibrium constants of wild-type HuAChE and its mutant F295A/F338A by the carbamates physostigmine, phenserine and cymserine

Left panels: inhibition time curve at indicated concentrations of the various carbamates (the actual curve was not calculated and serves as illustration). Middle panels: (A) and (B), the plots of ln(E) against time, allow for derivation of the observed first-order carbamylation rate constants (kobs) at indicated carbamate concentrations; (C) and (D), Lineweaver–Burk plots for the wild-type HuAChE and the F295A/F338A enzymes with or without the indicated carbamates. Right panels: (A) and (B), double reciprocal plots of kobs (derived from their respective middle panels) against the respect carbamate concentrations, from which k2, ki and Kd were derived (as described in the Materials and methods section); (C) and (D), Rs (relative slope) values as determine from the corresponding middle panels were plotted against the concentration of the carbamates. The values of Kd for cymserine and phenserine were obtained from the slope of these plots (see the Materials and methods section).

In cases where steady state with respect to E was formed rapidly (within a few minutes, Figures 2C and 2D), and immediate recovery of full enzymatic activity was observed upon dilution (Figure 3), the inhibition was treated kinetically as reversible. Thus Lineweaver–Burk plots in the absence and in the presence of different carbamate concentrations (Figures 2C and 2D, middle panels) yielded values of relative slopes Rs {note that the Rs=(1+(1/Kd)[CR]) according to references [6,31]}. The Rs values were plotted against the carbamate concentration and the reciprocal of the slope provided the Kd values (Figures 2C and 2D, right panels).

Regeneration of enzymatic activity, following 50-fold dilution, from HuAChE phenserine and cymserine conjugates

Molecular modelling

Building and optimization of three-dimensional models of the HuAChE adducts with the various carbamates were performed on a Silicon Graphics workstation Octane2, using the SYBYL modelling software (Tripos Inc.). The initial models were constructed by manual docking of the ligands into the HuAChE active centre guided by interactions with residue Trp86, the active-site nucleophile (Ser203) and residues of the oxyanion hole. The initial models were optimized by molecular mechanics using the MAXMIN force field (and AMBER charge parameters for the enzyme) and zone-refined, including 122 amino acids [1.5 nm (15 Å) substructure sphere around γO-Ser203]. Initial optimization included restriction of the distances between the carbonyl oxygen and the amide nitrogen atoms of residues Gly121 and Gly122, and that between the carbonyl carbon and γO-Ser203 as well as positions of residues Cys69 and Cys96, the ends of the omega loop. Those constraints were relieved in the subsequent refinement [8].

RESULTS AND DISCUSSION

Reactivity of rivastigmine toward HuAChE enzymes modified at various binding subsites in the active centre

In a previous study, comparison of the reactivity characteristics of rivastigmine and pyridostigmine toward HuAChE enzymes suggested that accommodation of these carbamates in the active centre is analogous to that of noncovalent inhibitors like edrophonium [16]. Results presented in the present study indicate that, although pyridostigmine and rivastigmine share the same binding subsites in the HuAChE active centre, their distinct orientations with respect to the active site seem to influence the outcome of the carbamylation process. These results are summarized in Table 1, which includes HuAChE enzymes that carry replacements at the hydrophobic pocket, H-bond network, oxyanion hole, acyl pocket and the peripheral anionic site [68,10,11,30,31]. In addition, we report on reactivities of both carbamates toward HuAChEs, which were engineered to resemble the HuBChE active centre [9,11].

Table 1
Effects of mutations at the various subsites of the HuAChE active centre on reactivity toward pyridostigmine and rivastigmine

Values are means±S.D. for at least three independent measurements.

 Pyridostigmine Rivastigmine 
HuAChE type ki (×10−4 M−1·min−1k2 (min−1Kd (×107 M) ki (×10−4 M−1·min−1k2 (min−1Kd (×107 M) 
Wild-type 43.5±1.6 0.77±0.3 18±5 1.50±0.1 0.10±0.005 70±3 
Hydrophobic pocket       
 W86A 0.01±0.001 0.68±0.2 79000±2400 0.0007±0.0001 0.30±0.06 430000±80000 
 W86F 0.3±0.05 0.65±0.2 2300±500 0.60±0.02 0.70±0.01 1100±30 
 Y133A 0.02±0.001 1.80±0.3 94400±20000 0.0006±0.0002 0.60±0.2 1000000±300000 
 Y337A 7.6±1.0 0.80±0.1 105±27 0.50±0.05 0.10±0.01 160±10 
 Y337F 31.4±1.1 0.68±0.2 20±6 3.10±0.1 0.10±0.01 32±1 
 F338A 25.0±1.3 1.20±0.4 48±15 0.07±0.01 0.10±0.01 1430±70 
H-bond network       
 Y133F 1.4±0.1 0.20±0.04 120±30 0.07±0.01 0.10±0.01 1430±100 
 E202A 4.2±0.5 0.20±0.05 50±15 0.20±0.02 0.10±0.01 510±50 
 E202Q 2.5±0.1 1.00±0.2 410±80 – – 10000±100 
 E450A 1.3±0.2 0.15±0.02 120±34 – – 3700±40 
Oxyanion hole       
 G121A – – 2500±30 – – 160000±1000 
 G122A 0.76±0.2 0.12±0.02 154±2 0.09±0.01 0.10±0.01 1100±20 
Acyl pocket       
 F295A 62.0±3.4 0.17±0.02 2.7±0.5 44±2 0.50±0.05 11±1 
 F297A 5.1±1 0.64±0.1 125.0±40 0.55±0.03 0.10±0.01 190±10 
Peripheral anionic site       
 D74N 0.3±0.03 1.0±0.1 3600±730 0.60±0.03 0.60±0.1 1000±50 
 Y124A 13.8±0.4 0.1±0.01 6±0.8 1.30±0.03 0.08±0.01 63±2 
 W286A 14.0±0.6 1.8±0.5 124±35 3.30±0.1 0.70±0.1 200±15 
 Y341A 16.6±0.5 1.0±0.1 61±8 0.30±0.02 0.30±0.05 1000±70 
His447 ‘trapping’       
 F295A/F338A 0.8±0.5 0.5±0.4 625±500 – – 12±2 
 F295A/F338A/V407F 16.5±2.0 0.5±0.15 30.0±12.6 1.00±0.1 0.10±0.01 90±5 
Butyryl-like       
 Y72N/Y124Q/W286A 9.3±0.8 0.80±0.07 88±7 1.40±0.1 0.10±0.01 70±3 
 F295L/F297V/Y337A 2.8±0.1 0.55±0.09 195±30 3.70±0.1 0.30±0.06 80±8 
 Y72N/Y124Q/W286A/F295L/F297V/Y337A 0.6±0.1 0.40±0.1 665±150 1.10±0.05 0.10±0.01 91±7 
HuBChE wild-type 12.7±0.5 0.30±0.03 24±2 22±1.0 0.90±0.03 38±1 
 Pyridostigmine Rivastigmine 
HuAChE type ki (×10−4 M−1·min−1k2 (min−1Kd (×107 M) ki (×10−4 M−1·min−1k2 (min−1Kd (×107 M) 
Wild-type 43.5±1.6 0.77±0.3 18±5 1.50±0.1 0.10±0.005 70±3 
Hydrophobic pocket       
 W86A 0.01±0.001 0.68±0.2 79000±2400 0.0007±0.0001 0.30±0.06 430000±80000 
 W86F 0.3±0.05 0.65±0.2 2300±500 0.60±0.02 0.70±0.01 1100±30 
 Y133A 0.02±0.001 1.80±0.3 94400±20000 0.0006±0.0002 0.60±0.2 1000000±300000 
 Y337A 7.6±1.0 0.80±0.1 105±27 0.50±0.05 0.10±0.01 160±10 
 Y337F 31.4±1.1 0.68±0.2 20±6 3.10±0.1 0.10±0.01 32±1 
 F338A 25.0±1.3 1.20±0.4 48±15 0.07±0.01 0.10±0.01 1430±70 
H-bond network       
 Y133F 1.4±0.1 0.20±0.04 120±30 0.07±0.01 0.10±0.01 1430±100 
 E202A 4.2±0.5 0.20±0.05 50±15 0.20±0.02 0.10±0.01 510±50 
 E202Q 2.5±0.1 1.00±0.2 410±80 – – 10000±100 
 E450A 1.3±0.2 0.15±0.02 120±34 – – 3700±40 
Oxyanion hole       
 G121A – – 2500±30 – – 160000±1000 
 G122A 0.76±0.2 0.12±0.02 154±2 0.09±0.01 0.10±0.01 1100±20 
Acyl pocket       
 F295A 62.0±3.4 0.17±0.02 2.7±0.5 44±2 0.50±0.05 11±1 
 F297A 5.1±1 0.64±0.1 125.0±40 0.55±0.03 0.10±0.01 190±10 
Peripheral anionic site       
 D74N 0.3±0.03 1.0±0.1 3600±730 0.60±0.03 0.60±0.1 1000±50 
 Y124A 13.8±0.4 0.1±0.01 6±0.8 1.30±0.03 0.08±0.01 63±2 
 W286A 14.0±0.6 1.8±0.5 124±35 3.30±0.1 0.70±0.1 200±15 
 Y341A 16.6±0.5 1.0±0.1 61±8 0.30±0.02 0.30±0.05 1000±70 
His447 ‘trapping’       
 F295A/F338A 0.8±0.5 0.5±0.4 625±500 – – 12±2 
 F295A/F338A/V407F 16.5±2.0 0.5±0.15 30.0±12.6 1.00±0.1 0.10±0.01 90±5 
Butyryl-like       
 Y72N/Y124Q/W286A 9.3±0.8 0.80±0.07 88±7 1.40±0.1 0.10±0.01 70±3 
 F295L/F297V/Y337A 2.8±0.1 0.55±0.09 195±30 3.70±0.1 0.30±0.06 80±8 
 Y72N/Y124Q/W286A/F295L/F297V/Y337A 0.6±0.1 0.40±0.1 665±150 1.10±0.05 0.10±0.01 91±7 
HuBChE wild-type 12.7±0.5 0.30±0.03 24±2 22±1.0 0.90±0.03 38±1 

Replacements of aromatic residues comprising the HuAChE active centre hydrophobic pocket had a similar effect on the rates of carbamylation by rivastigmine and by pyridostigmine, implying that in both cases the positively charged moiety interacts with the cation-binding subsite, Trp86. The pronounced increase in the respective dissociation constants, due to replacement of Trp86 (4400- and 6150-fold for pyridostigmine and rivastigmine respectively), resembles that for all the charged active centre inhibitors [8,34,35]. Similar to pyridostigmine [8], replacement of Tyr133 by alanine but not by phenylalanine had a pronounced effect on the affinity of Y133A HuAChE toward rivastigmine. As noted previously, replacement of Tyr337 by alanine had little effect on interactions of cationic ligands, although corresponding crystal structures of AChE complexes [1214] and a molecular model of the HuAChE–pyridostigmine Michaelis complex [8] indicate close proximity of this residue to the ligand's charged moiety.

Perturbations of the H-bond network, through replacements of residues Tyr133, Glu202 and Glu450 [10], had a relatively uniform effect on the corresponding rates of carbamylation by pyridostigmine (see Table 1). Yet, for two of those enzymes, E202Q and E450A, interaction with rivastigmine did not lead to carbamylation but rather to a regular, albeit low affinity (for corresponding values of the dissociation constants, Kd, see Table 1), noncovalent inhibition. This observation suggests that the balance between the rates of carbamylation and of dissociation of the corresponding Michaelis complexes can be easily tipped away from the covalent reaction. This facet of carbamate reactivity toward HuAChEs will become even more evident for certain analogues of physostigmine.

Structural modification of the oxyanion hole through replacement of residue Gly121 by alanine [31] alters the reactivity characteristics of both carbamates, converting them into noncovalent inhibitors. We have already shown that interactions of the acyl oxygen (acetyl, carbamyl or phosphoryl) with the oxyanion hole are important for both stabilization of the Michelis complex and activation of the acyl moiety for nucleophilic attack by the catalytic Ser203 [31]. Thus dissociation of the G121A HuAChE–rivastigmine complex is probably much faster than that of the corresponding complex of the wild-type enzyme (the pronounced increase in the value of Kd is mostly due to increase of the dissociation rate constant k−1), while its conversion to the carbamylated enzyme is slower.

Replacements at the peripheral anionic site had only a limited effect on the carbamylation rate constants by rivastigmine. In particular, the corresponding value of ki for carbamylation of D74N HuAChe was 3-fold lower than that of the wild-type enzyme. On the other hand, carbamylation of this enzyme by pyridostigmine was nearly 150-fold slower, with the corresponding value of Kd being 200-fold higher than that of the wild-type HuAChE. It is already reported that this replacement resulted in a 50-fold increase in the dissociation constant for tacrine, while having only a small effect on the corresponding constant for edrophonium (5-fold increase) and no effect on huperzine A [8]. The reason for these uneven effects on affinities of the D74N enzyme toward the various charged (at the experimental pH) active centre ligands still remains elusive.

While replacement of acyl pocket residue Phe295 by alanine had little effect on the carbamylation rate by pyridostigmine, the corresponding rate for rivastigmine was 30-fold higher. This observation seems consistent with the size of the substituents on the carbamyl nitrogen. Namely, while interaction of rivastigmine with residue Phe295 of the wild-type HuAChE may perturb the ‘aromatic trap’ and affect the carbamylation step, such perturbation is avoided in binding to the F295A enzyme. Accordingly, analogous substitution of the second acyl pocket residue Phe297 had a similar effect on the carbamylation rates by both carbamates. Reactivity of rivastigmine toward HuBChE, in which the corresponding acyl pocket is lined by aliphatic residues, has been found to be 15-fold higher than toward HuAChE. Most of this difference was due to the 9-fold higher value of the carbamylated enzyme formation step rate constant k2 (see Table 1). Such reactivity enhancement was not observed for HuAChE enzymes in which the active centre was engineered to resemble that of HuBChE. For the ‘butyryl-like’ enzyme carrying replacements of aromatic residues vicinal to the active site (F295L/F297V/Y337A HuAChE) as well as for that substituted at both the active centre and the peripheral anionic site (Y72N/Y124Q/W286A/F295L/F297V/Y337A HuAChE) [9], carbamylation rates by rivastigmine resembled those of the wild-type enzyme. On the other hand, the corresponding rates of carbamylation by pyridostigmine were 15-fold and 73-fold lower respectively.

Analogues of physostigmine display distinct inhibition profiles toward HuAChE enzymes

Unlike the pronounced effects (over 4000-fold) of replacing residue Trp86 by alanine on the inhibitory activities of pyridostigmine and rivastigmine, the inhibition rate constant of physostigmine toward the W86A HuAChE was only less than 50-fold lower than toward the wild-type enzyme (see Table 2). Replacement of Tyr133 by alanine had a larger effect (700-fold), implying that steric congestion, rather than cation–π interaction, is the dominant factor in the accommodation of physostigmine in the hydrophobic pocket [28]. Perturbations of the H-bond network affect reactivities of the corresponding enzymes toward physostigmine to a similar extent as for pyridostigmine and rivastigmine. Modifications of the acyl pocket had a lower effect on reactivities toward physostigmine than toward rivastigmine (see Tables 1 and 2, and reference [8]).

Table 2
Effects of mutations at the various subsites of the HuAChE active centre on reactivity toward physostigmine, phenserine and cymserine

Values are means±S.D. for at least three independent measurements. ND, not determined.

 (−)-Physostigmine (−)-Phenserine (−)Cymserine 
HuAChE type ki (×10−4 M−1·min−1k2 (min−1Kd (×107 M) ki (×10−4 M−1·min−1k2 (min−1Kd (×107 M) Kd (×107 M) 
Wild-type 350±20 1.2±0.5 3.4±1.0 89±30 0.9±0.5 13.9±5.5 6.3±0.7 
Hydrophobic pocket        
 W86F 190±2 0.90±0.20 4.6±1.0 150±20 1.3±0.9 8.7±3.4 1.5±0.5 
 W86A 8.2±0.7 0.80±0.30 95±40 5±0.9 1.3±1.0 200±90 54±12 
 Y133A 0.5±0.1 0.40±0.02 820±150 0.5±0.09 0.4±0.06 760±150 220±100 
 Y337A 120±5 1.10±0.50 9.5±3.5 28±10 0.8±0.4 27.5±2.8 3.8±0.9 
 Y337F 360±50 1.90±0.40 5.2±1.5 171±49 0.9±0.07 2.8±0.3 3.0±0.3 
 W86A/Y337A 866±130 1.2±0.18 14000±2100 ND ND ND 470±127 
 F338A 100±3 0.40±0.20 4.2±1.0 1.6±0.5 0.7±0.1 413±95 40±18 
H-bond network        
 Y133F 19±0.2 0.13±0.01 6.9±1.0 4.2±0.8 0.3±0.09 76±19 37±13 
 E202A 7.7±1.0 0.24±0.05 32±4 2.2±0.6 0.2±0.1 91±36 120±50 
 E202Q 43±1.0 0.80±0.20 18±5 15±1.6 3.2±1 174±34 23±6 
 E450A 26±0.3 0.15±0.01 4.6±0.5 2.7±0.8 0.2±0.04 74±19 135±8 
Peripheral anionic site        
 D74N 140±4 2.40±0.50 17±4 21±5 0.7±0.01 34±6.1 8.7±3.5 
 Y72N 320±48 0.9±0.14 3.0±0.2 229±70 1.7±0.2 7.4±1.9 2.9±1.1 
 Y124A 150±3 6.0±1.8 0.9±0.3 ND ND ND ND 
 W286A 365±40 2.70±0.80 1.0±0.2 – – 1.6±0.3 7.2±2 
 Y341A 425±15 1.90±0.30 0.8±0.1 101±18 0.7±0.3 6.9±2.1 1.9±0.5 
Acyl pocket        
 F295A 1770±60 1.60±0.50 0.9±0.3 2300±480 2.1±1.2 0.9±0.4 0.4±0.1 
 F297A 160±15 0.60±0.10 3.8±1.0 35±6 1.0±0.5 2.9±1.0 7.5±1.4 
 F295L/F297V 90±9 2.0±0.4 23±5 377±170 3.0±1.8 8.0±3.6 1.0±0.05 
 F295A/F297A 55±17 0.6±0.2 11±3 305±90 8.5±2.5 28±8 1.6±0.3 
His447 ‘trapping’        
 F295A/F338A 78±19 4.5±0.9 58±14 – – 0.3±0.08 1.3±0.3 
 F295A/F338A/V407F 197±30 0.2±0.03 92±14 1.1±0.1 0.3±0.1 272±55 22±5 
Butyryl-like        
 F295L/F297V/Y337A 58±1 0.75±0.20 13±2 303±80 0.9±0.2 3±0.06 2.7±0.8 
 Y72N/Y124Q/W286A/F295L/F297V/Y337A 34±1.2 0.20±0.05 6.1±1.5 – – 1.2±0.3 2.3±0.5 
 Y72N/Y124Q/W286A/F295L/F297V/Y337A/V407F 18±3 1.25±0.25 71±17 – – 203±61 20±4 
HuBChE wild-type 1030±37 0.55±0.05 0.5±0.04 6.6±2.2 0.6±0.2 90±30 ki=20×104 M−1·min−1 
 (−)-Physostigmine (−)-Phenserine (−)Cymserine 
HuAChE type ki (×10−4 M−1·min−1k2 (min−1Kd (×107 M) ki (×10−4 M−1·min−1k2 (min−1Kd (×107 M) Kd (×107 M) 
Wild-type 350±20 1.2±0.5 3.4±1.0 89±30 0.9±0.5 13.9±5.5 6.3±0.7 
Hydrophobic pocket        
 W86F 190±2 0.90±0.20 4.6±1.0 150±20 1.3±0.9 8.7±3.4 1.5±0.5 
 W86A 8.2±0.7 0.80±0.30 95±40 5±0.9 1.3±1.0 200±90 54±12 
 Y133A 0.5±0.1 0.40±0.02 820±150 0.5±0.09 0.4±0.06 760±150 220±100 
 Y337A 120±5 1.10±0.50 9.5±3.5 28±10 0.8±0.4 27.5±2.8 3.8±0.9 
 Y337F 360±50 1.90±0.40 5.2±1.5 171±49 0.9±0.07 2.8±0.3 3.0±0.3 
 W86A/Y337A 866±130 1.2±0.18 14000±2100 ND ND ND 470±127 
 F338A 100±3 0.40±0.20 4.2±1.0 1.6±0.5 0.7±0.1 413±95 40±18 
H-bond network        
 Y133F 19±0.2 0.13±0.01 6.9±1.0 4.2±0.8 0.3±0.09 76±19 37±13 
 E202A 7.7±1.0 0.24±0.05 32±4 2.2±0.6 0.2±0.1 91±36 120±50 
 E202Q 43±1.0 0.80±0.20 18±5 15±1.6 3.2±1 174±34 23±6 
 E450A 26±0.3 0.15±0.01 4.6±0.5 2.7±0.8 0.2±0.04 74±19 135±8 
Peripheral anionic site        
 D74N 140±4 2.40±0.50 17±4 21±5 0.7±0.01 34±6.1 8.7±3.5 
 Y72N 320±48 0.9±0.14 3.0±0.2 229±70 1.7±0.2 7.4±1.9 2.9±1.1 
 Y124A 150±3 6.0±1.8 0.9±0.3 ND ND ND ND 
 W286A 365±40 2.70±0.80 1.0±0.2 – – 1.6±0.3 7.2±2 
 Y341A 425±15 1.90±0.30 0.8±0.1 101±18 0.7±0.3 6.9±2.1 1.9±0.5 
Acyl pocket        
 F295A 1770±60 1.60±0.50 0.9±0.3 2300±480 2.1±1.2 0.9±0.4 0.4±0.1 
 F297A 160±15 0.60±0.10 3.8±1.0 35±6 1.0±0.5 2.9±1.0 7.5±1.4 
 F295L/F297V 90±9 2.0±0.4 23±5 377±170 3.0±1.8 8.0±3.6 1.0±0.05 
 F295A/F297A 55±17 0.6±0.2 11±3 305±90 8.5±2.5 28±8 1.6±0.3 
His447 ‘trapping’        
 F295A/F338A 78±19 4.5±0.9 58±14 – – 0.3±0.08 1.3±0.3 
 F295A/F338A/V407F 197±30 0.2±0.03 92±14 1.1±0.1 0.3±0.1 272±55 22±5 
Butyryl-like        
 F295L/F297V/Y337A 58±1 0.75±0.20 13±2 303±80 0.9±0.2 3±0.06 2.7±0.8 
 Y72N/Y124Q/W286A/F295L/F297V/Y337A 34±1.2 0.20±0.05 6.1±1.5 – – 1.2±0.3 2.3±0.5 
 Y72N/Y124Q/W286A/F295L/F297V/Y337A/V407F 18±3 1.25±0.25 71±17 – – 203±61 20±4 
HuBChE wild-type 1030±37 0.55±0.05 0.5±0.04 6.6±2.2 0.6±0.2 90±30 ki=20×104 M−1·min−1 

To explore further the reactivity characteristics of HuAChE toward physostigmine, we now examine physostigmine analogues differing in substitution at the carbamyl nitrogen as well as analogues with a modified tricyclic moiety. Thus, physostigmine, phenserine and cymserine (see Figure 1) were expected to display similar accommodation in the HuAChE hydrophobic pocket while differing with respect to the acyl pocket and the peripheral anionic site. From the results exemplified in Figure 2 and the regeneration of HuAChE activity from the respective enzyme–inhibitor conjugate (Figure 3), it appears that, whereas the inhibition characteristics of phenserine toward the HuAChE enzymes resemble that of physostigmine, the reactivity of cymserine is that of a noncovalent inhibitor.

Notwithstanding the difference in the inhibitory activity of physostigmine and phenserine as compared with cymserine, it seems that the three compounds are similarly accommodated in the hydrophobic pocket. In all cases affinities were affected by replacements at residues 86 and 133 by alanine, while not being sensitive to substitutions of Tyr337. Substitution of residue 338 by alanine had some effect on the values of Kd for phenserine and cymserine (30-fold and 6-fold respectively) but not on the corresponding value for physostigmine (see Table 2). Thus it appears that the different reactivity characteristics of cymserine, as compared with physostigmine and phenserine, are not due to interactions of the respective eseroline moieties with the HuAChE hydrophobic pocket. Therefore, it seems reasonable to assume that in the HuAChE–cymserine Michaelis complexes, the ligand is sub-optimally oriented with respect to the catalytic machinery of the enzyme.

Substitutions at the H-bond network had similar effects on affinities toward phenserine and cymserine, as was the case for most of the replacements at the peripheral anionic site. The outstanding case was the failure of phenserine to carbamylate W286A HuAChE. Similar noncovalent inhibition was observed for interactions of phenserine with F295A/F338A and with the ‘butyryl-like’ HuAChEs (Table 2).

Perturbations of the acyl pocket had comparable effects on the affinities toward phenserine and cymserine, as well as minor effects on the carbamylation rate constant (k2) for phenserine. Thus it does not appear that the failure of cymserine to carbamylate HuAChE enzymes results from delocalization of the catalytic His447 due to perturbations of the acyl pocket [10]. Moreover, while the prototypical perturbation of the His447 positioning (F295A/F338A HuAChE [29]) abolished carbamylation by phenserine, the reactivity was restored by additional replacement at residue 407. Such a reactivity profile [29] could not be observed in the case of cymserine (Table 2).

In order to gain further insight into the unique inhibitory characteristics of cymserine toward all the HuAChE enzymes, molecular modelling experiments of the Michaelis complexes of wild-type HuAChE with physostigmine, phenserine and cymserine have been constructed (Figure 4). While in the model of phenserine, interactions of the N-aryl moiety with residues of the acyl pocket and with Phe338 could be observed, the disposition of the carbamyl moiety with respect to the active site residues resembled that of physostigmine. On the other hand, interactions of the 4-isopropyl aromatic substituent of cymserine with aromatic residues lining the mouth of the active-centre gorge forced an alternative conformation of the N-aryl moiety and consequently the removal of the carbamyl oxygen from the oxyanion hole. This finding seems consistent with the reversible noncovalent nature of cymserine interaction with wild-type HuAChE and any of its mutant derivatives.

Relative orientations of (−)-physostigmine and (−)-cymserine in models of their respective Michaelis complexes with HuAChE

Figure 4
Relative orientations of (−)-physostigmine and (−)-cymserine in models of their respective Michaelis complexes with HuAChE

Superposition of the carbamyl moieties is emphasized by the shaded area. Note that the carbamyl oxygen of physostigmine (cyan) is 2.997 Å and 2.387 Å from the peptidic NH groups of Gly121 and Gly122 respectively (shown with broken lines). Similar orientation of the carbamyl oxygen was observed also for phenserine (not shown for the sake of clarity). The corresponding distances for cymserine (grey) are 3.255 Å and 3.980 Å.

Figure 4
Relative orientations of (−)-physostigmine and (−)-cymserine in models of their respective Michaelis complexes with HuAChE

Superposition of the carbamyl moieties is emphasized by the shaded area. Note that the carbamyl oxygen of physostigmine (cyan) is 2.997 Å and 2.387 Å from the peptidic NH groups of Gly121 and Gly122 respectively (shown with broken lines). Similar orientation of the carbamyl oxygen was observed also for phenserine (not shown for the sake of clarity). The corresponding distances for cymserine (grey) are 3.255 Å and 3.980 Å.

The notion that cymserine fails to carbamylate HuAChEs (Figures 2 and 3) due to impaired polarization of the carbamyl oxygen is consistent with the carbamylation profile of the G121A enzyme with physostigmine [31] as well as with pyridostigmine and rivastigmine.

Accommodation of the tricyclic moiety in the hydrophobic pocket

Another manifestation of the tight accommodation of the tricyclic moiety in the hydrophobic pocket is the marked stereoselectivity toward the (−)-physostigmine (3aS-diastereomer) enantiomeric form [20]. Since the main difference between the diastereomers is in the eseroline moiety, stereoselectivity should originate from the asymmetric interactions in the hydrophobic pocket. It was therefore reasonable to assume that, through identifying the specific interactions leading to stereoselectivity toward (−)-physostigmine, a better understanding of its accommodation in the hydrophobic pocket would be achieved.

Replacement of the tricyclic eseroline group in physostigmine by the physovenyl moiety (see Figure 1) resulted in an analogous physovenine [24], with similar chirality due to asymmetric carbon at position −3a. Yet the overall inhibitory activities, and in particular the affinities of both diastereomers of physovenine toward HuAChE, were similar to that of (−)-physostigmine (see Table 3). It has already been proposed that the low affinity of AChE toward (+)-physostigmine is due to its N1-methyl group interfering with Trp84 (Trp86 in HuAChE) [36]. However, this residue is usually thought to interact with N-methyl groups, as in the case for its endogenous substrate, acetylcholine [4,6].

Table 3
Stereoselectivity of HuAChE enzymes mutated at the hydrophobic pocket and at the acyl pocket towards (+)- and (−)-enantiomers of physostigmine and physovenine

Values are means±S.D. for at least three independent measurements, numbers in brackets represent relative Kd (mutant/wild-type). Values of Kd were determined using linear regression of eqn (1) and plots similar to those presented in the middle and right panels of Figures 2(A) and 2(B). NI, no inhibition between 7.5×10−6 and 2.5×10−4 M. *Activity measured with TB.

 Kd (×107 M) 
HuAChE type (−)-Physostigmine (+)-Physostigmine (−)-Physovenine (+)-Physovenine 
Wild-type 3.4±1.0 (1) 1200±310 (1) 4.3±0.9 (1) 14±1.3 (1) 
Hydrophobic pocket     
 W86A 95±40 (28) 2000±460 (1.7) 1200±240 (279) 2800±285 (200) 
 Y133A 820±150 (241) NI NI NI 
 Y337A 9.5±3.5 (2.8) 482±120(0.4) 8.5±2 (2) 150±16 (10.7) 
 F338A 14±1.0 (4.1) 2300±810 (1.9) 25.6±6 (6) 7±1 (0.5) 
 W86A/Y337A* 14000±2100 (4100) 12000±2760 (10) 4130±850 (960) 2810±450 (201) 
Acyl pocket     
 F295A 0.9±0.3 (0.3) 90±26 (0.1) 1±0.27 (0.2) 0.9±0.25 (0.1) 
 F297A 3.8±1.0 (1.1) 3850±1150 (3.2) 6.3±0.4 (1.5) 10±3.5 (0.7) 
 F295A/F297A 11.4±5 (3.4) 7700±1850 (6.4) 2.3±0.7 (0.5) 2.8±0.8 (0.2) 
 Kd (×107 M) 
HuAChE type (−)-Physostigmine (+)-Physostigmine (−)-Physovenine (+)-Physovenine 
Wild-type 3.4±1.0 (1) 1200±310 (1) 4.3±0.9 (1) 14±1.3 (1) 
Hydrophobic pocket     
 W86A 95±40 (28) 2000±460 (1.7) 1200±240 (279) 2800±285 (200) 
 Y133A 820±150 (241) NI NI NI 
 Y337A 9.5±3.5 (2.8) 482±120(0.4) 8.5±2 (2) 150±16 (10.7) 
 F338A 14±1.0 (4.1) 2300±810 (1.9) 25.6±6 (6) 7±1 (0.5) 
 W86A/Y337A* 14000±2100 (4100) 12000±2760 (10) 4130±850 (960) 2810±450 (201) 
Acyl pocket     
 F295A 0.9±0.3 (0.3) 90±26 (0.1) 1±0.27 (0.2) 0.9±0.25 (0.1) 
 F297A 3.8±1.0 (1.1) 3850±1150 (3.2) 6.3±0.4 (1.5) 10±3.5 (0.7) 
 F295A/F297A 11.4±5 (3.4) 7700±1850 (6.4) 2.3±0.7 (0.5) 2.8±0.8 (0.2) 

As previously reported [8,11], replacement of Trp86 by alanine had a moderate effect (∼25-fold) on the affinity toward the natural compound [(−)-physostigmine]. Here we find that this modification has practically no effect on the affinity toward the (+)-diastereomer (see Table 3). Thus, the diminished stereo-selectivity exhibited by W86A HuAChE toward physostigmine diastereomers, as compared with the wild-type enzyme (20-fold, see Table 3), was solely due to a decrease in affinity toward (−)-physostigmine. This suggests that residue Trp86 does not participate at all in the interactions of the HuAChE hydrophobic pocket with (+)-physostigmine (see Figure 5). On the other hand, W86A HuAChE displayed a similar decrease in affinity toward both diastereomers of physovenine and, therefore, both the wild-type and the W86A HuAChEs show nearly no stereoselectivity toward the physovenines.

Gradual displacement of the cation-binding residue Trp86 from the hydrophobic pocket

Figure 5
Gradual displacement of the cation-binding residue Trp86 from the hydrophobic pocket

Models of HuAChE Michaelis complexes with pyridostigmine and physostigmine enantiomers are shown in (AC). (A) The positioning of residue Trp86 in the Michaelis complex of pyridostigmine is typical for complexes with charged noncovalent ligands and is shown here as a reference. (B) Docking of the (−)-physostigmine in the HuAChE hydrophobic pocket results in vertical displacement of the indole moiety of Trp86. Yet the conformation of the indole moiety relative to the ligand remains unchanged. (C) The modelled displacement of residue Trp86 (1.310 Å between the centroids of the indole moieties), in complex depicted in panel A (cyan) as compared to that shown in panel B (magenta), is facilitated by flexibility of the Cys69–Cys93 omega loop. (D) Steric interference due to the N-1 methyl substituent of (+)-physostigmine results in further displacement of residue Trp86 and to additional deformation of the Cys69–Cys93 omega loop. In this case, the indole moiety is too far removed from Tyr133 or from other residues stabilizing the overall structure of the omega loop [41], and therefore residue Trp86 is conformationally labile. (E) Superposition of the omega loop segments Phe80–Pro88 in the complexes of (−)-physostigmine (cyan) and (+)-physostigmine (red) shown in (B) and (D) respectively. The conformational change of Trp86 implies its practical removal from the hydrophobic pocket.

Figure 5
Gradual displacement of the cation-binding residue Trp86 from the hydrophobic pocket

Models of HuAChE Michaelis complexes with pyridostigmine and physostigmine enantiomers are shown in (AC). (A) The positioning of residue Trp86 in the Michaelis complex of pyridostigmine is typical for complexes with charged noncovalent ligands and is shown here as a reference. (B) Docking of the (−)-physostigmine in the HuAChE hydrophobic pocket results in vertical displacement of the indole moiety of Trp86. Yet the conformation of the indole moiety relative to the ligand remains unchanged. (C) The modelled displacement of residue Trp86 (1.310 Å between the centroids of the indole moieties), in complex depicted in panel A (cyan) as compared to that shown in panel B (magenta), is facilitated by flexibility of the Cys69–Cys93 omega loop. (D) Steric interference due to the N-1 methyl substituent of (+)-physostigmine results in further displacement of residue Trp86 and to additional deformation of the Cys69–Cys93 omega loop. In this case, the indole moiety is too far removed from Tyr133 or from other residues stabilizing the overall structure of the omega loop [41], and therefore residue Trp86 is conformationally labile. (E) Superposition of the omega loop segments Phe80–Pro88 in the complexes of (−)-physostigmine (cyan) and (+)-physostigmine (red) shown in (B) and (D) respectively. The conformational change of Trp86 implies its practical removal from the hydrophobic pocket.

Substitution of Tyr337 by alanine maintained the stereoselectivity toward physostigmines while inducing limited stereoselectivity toward the physovenines (18-fold, see Table 3). For both cases, stereoselectivity is completely abolished in the double mutant W86A/Y337A HuAChE. We note that while replacement of the aromatic residues Trp86 and Tyr337 led to a moderate decrease in affinity toward (−)-physostigmine (28- and 3-fold respectively), the corresponding effect for the double mutant is quite dramatic (4100-fold). In contrast, only a 10-fold decline in affinity of the W86/Y337 HuAChE toward the (+)-enantiomer has been observed, demonstrating that interactions with the hydrophobic pocket determine stereoselectivity toward physostigmines (see Table 3). For both physovenine enantiomers, affinities of the double mutant are comparable with those of the W86A enzyme. Thus, physostigmines and physovenines seem to be somewhat differently orientated with respect to the hydrophobic patch in the active centre [8]. Whereas residue Trp86 is essential in accommodation of (−)-physovenine, residues Trp86 and Tyr337 seem to compensate for one another in the case of (−)-physostigmine. Such compensation seems to account for the intriguing observation that removal of the aromatic moiety from position 86 had a larger effect on affinity toward the uncharged physovenines than toward the charged (−)-physostigmine.

The results described above seem consistent with the idea that steric congestion of Trp86 and the N1-methyl of (+)-physostigmine, indeed, interferes with accommodation of this diastereomer in the hydrophobic pocket. To examine further this hypothesis, affinities of HuAChE enzymes modified at the hydrophobic pocket, toward cymserine and cymyl carbamates of physovenol and of tetrahydrofurobenzofuran [26], have been compared. As for cymserine, all the cymyl analogues are noncovalent inhibitors. While HuAChE displayed ∼50-fold stereoselectivity toward (−)-cymserine, practically no stereoselectivity was observed toward the diastereomers of analogues bearing the physovenol and tetrahydrofurobenzofuran moieties. Replacement of Trp86 by alanine practically abolished stereoselectivity toward diastereomers of cymserine yet had only a limited effect on their binding affinities. Other cymyl carbamates were also little affected by residue replacements at the hydrophobic pocket (see Table 4).

Table 4
Stereoselectivity of HuAChE enzymes mutated at the hydrophobic pocket toward (+)- and (−)-enantiomers of cymserine and its analogues

Values are means±S.D. for at least three independent measurements. Values of Kd were determined from analysis of competition with ATC using plots similar to those presented in the middle and right panels of Figures 2(C) and 2(D). ND, not determined.

 Kd (×107 M) 
HuAChE type (−)-Cymserine (+)-Cymserine (−)-Cymyl carbamate of physovenol (+)-Cymyl carbamate of physovenol (−)-Cymyl carbamate of tetrahydrofurobenzofuran (+)-Cymyl carbamate of tetrahydrofurobenzofuran 
Wild-type 6.3±1.7 300±76 7.1±1.2 10±2 17±3.5 22±9 
 W86A 54±16 130±58 25±2 50±13 71±17 66±25 
 Y133A 220±57 4800±1900 ND ND ND ND 
 Y337A 3.8±1 50±13 27±10 22±7 200±45 123±50 
 F338A 40±11 135±14 400±120 571±150 1607±480 2900±870 
 Kd (×107 M) 
HuAChE type (−)-Cymserine (+)-Cymserine (−)-Cymyl carbamate of physovenol (+)-Cymyl carbamate of physovenol (−)-Cymyl carbamate of tetrahydrofurobenzofuran (+)-Cymyl carbamate of tetrahydrofurobenzofuran 
Wild-type 6.3±1.7 300±76 7.1±1.2 10±2 17±3.5 22±9 
 W86A 54±16 130±58 25±2 50±13 71±17 66±25 
 Y133A 220±57 4800±1900 ND ND ND ND 
 Y337A 3.8±1 50±13 27±10 22±7 200±45 123±50 
 F338A 40±11 135±14 400±120 571±150 1607±480 2900±870 

The notion that AChE stereoselectivity toward physostigmine is mainly due to interactions of the alkyl substituent at position −1 is also supported by previous studies on physostigmine analogues [3638]. In particular, it was interesting to examine the low AChE stereoselectivity toward analogues of 8-carbaphysostigmine, since these structures do contain the N(1)-alkyl substituent [39]. Examination of molecular models of the corresponding Michaelis complexes indicates that, due to bending of the tricyclic moiety at the sp3-C8, both enantiomers could be accommodated in the hydrophobic pocket without steric occlusion of Trp86. Thus the structural features of the eseroline moiety, that contribute to AChE stereoselectivity toward physostigmine, are the alkyl substituent at position −1 combined with the planar disposition of the tricyclic ring system.

Accommodation of carbamates in the HuAChE active centre

Carbamates are unique HuAChE inhibitors, binding both as covalent and noncovalent ligands to the different HuAChE enzymes. It appears that this property of carbamates originates from the particular dependence of the carbamyl moiety reactivity on its juxtaposition with the elements of the enzyme catalytic machinery. Carbamylation of AChEs involves nucleophilic attack on a relatively nonreactive carbonyl group, and therefore its rate depends critically upon the stability of the corresponding Michaelis complex, which manifests itself predominantly by variation in the values of the dissociation rate constant k−1 (under equilibrium conditions Kd=k−1/k1). Thus the efficiency of the carbamylation process depends mainly on the relative values of the rate constants k−1 and k2 (see 1), with the latter displaying rather limited variability [40]. Accommodation of the carbamylating agent in the AChE active centre is hence the most significant molecular event in the carbamylation process. The finding that the affinity of HuAChE toward the charged physostigmine is remarkably similar to that toward the structurally similar yet uncharged physovenine, indicates that both inhibitors are accommodated mainly through hydrophobic interactions.

Abbreviations

     
  • AChE

    acetylcholinesterase

  •  
  • AD

    Alzheimer's disease

  •  
  • ATC

    acetylthiocholine

  •  
  • DTNB

    5,5′-dithiobis-(2-nitrobenzoic acid)

  •  
  • HuAChE

    human AChE

  •  
  • HuBChE

    human butyrylcholinesterase

  •  
  • TB

    3,3-dimethylbutylthioacetate

FUNDING

This work was supported by the US Army Medical Research and Material Command [contract number DAMD17-00-C-0021 (to A. S.)] and the Intramural Research Program of the National Institute on Aging, National Institutes of Health.

References

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