©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Contribution of Aromatic Moieties of Tyrosine 133 and of the Anionic Subsite Tryptophan 86 to Catalytic Efficiency and Allosteric Modulation of Acetylcholinesterase (*)

(Received for publication, September 6, 1994; and in revised form, November 18, 1994)

Arie Ordentlich (1) Dov Barak (2) Chanoch Kronman (1) Naomi Ariel (1) Yoffi Segall (2) Baruch Velan (1) Avigdor Shafferman (1)(§)

From the  (1)Departments of Biochemistry and (2)Organic Chemistry, Israel Institute for Biological Research, Ness-Ziona, 70450, Israel

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Substitution of Trp-86, in the active center of human acetylcholinesterase (HuAChE), by aliphatic but not by aromatic residues resulted in a several thousandfold decrease in reactivity toward charged substrate and inhibitors but only a severalfold decrease for noncharged substrate and inhibitors. The W86A and W86E HuAChE enzymes exhibit at least a 100-fold increase in the Michaelis-Menten constant or 100-10,000-fold increase in inhibition constants toward various charged inhibitors, as compared to W86F HuAChE or the wild type enzyme. On the other hand, replacement of Glu-202, the only acidic residue proximal to the catalytic site, by glutamine resulted in a nonselective decrease in reactivity toward charged and noncharged substrates or inhibitors. Thus, the quaternary nitrogen groups of substrates and other active center ligands, are stabilized by cation-aromatic interaction with Trp-86 rather than by ionic interactions, while noncharged ligands appear to bind to distinct site(s) in HuAChE. Analysis of the Y133F and Y133A HuAChE mutated enzymes suggests that the highly conserved Tyr-133 plays a dual role in the active center: (a) its hydroxyl appears to maintain the functional orientation of Glu-202 by hydrogen bonding and (b) its aromatic moiety maintains the functional orientation of the anionic subsite Trp-86. In the absence of aromatic interactions between Tyr-133 and Trp-86, the tryptophan acquires a conformation that obstructs the active site leading, in the Y133A enzyme, to several hundredfold decrease in rates of catalysis, phosphorylation, or in affinity to reversible active site inhibitors. It is proposed that allosteric modulation of acetylcholinesterase activity, induced by binding to the peripheral anionic sites, proceeds through such conformational change of Trp-86 from a functional anionic subsite state to one that restricts access of substrates to the active center.


INTRODUCTION

Acetylcholinesterase (AChE, EC 3.1.1.7) (^1)is a serine hydrolase whose function at the cholinergic synapse, is the rapid hydrolysis of the neurotransmitter acetylcholine (ACh). The recently resolved three-dimensional structure of Torpedo californica AChE (TcAChE) revealed a deep and narrow ``gorge,'' which penetrates halfway into the enzyme and contains the catalytic site at about 4 Å from its base (Sussman et al., 1991). In addition, the structure reveals two remarkable features of the enzyme which may bear upon its catalytic efficiency. One of these is the uneven overall distribution of negative charge giving rise to a large electrostatic dipole, aligned with the axis of the active site gorge, that could draw the positively charged substrate down the gorge to the active center (Ripoll et al., 1993). However, it was recently shown, by septuple replacement of negatively charged amino acids, that electrostatic attraction does not contribute to the catalytic rate of the enzyme (Shafferman et al., 1994). The second striking feature of the enzyme is related to the 14 aromatic residues that contribute to the lining of the active site gorge. Most of these residues are highly conserved in enzymes from different species (for sequence compilation, see Gentry and Doctor(1991) and Massoulie et al.(1993)). This complex array of aromatic residues was hypothesized to provide a guidance mechanism facilitating a two-dimensional diffusion of ACh to the active site (Sussman et al., 1991), to be involved in substrate accommodation and to participate in allosteric modulation of catalysis (Shafferman et al., 1992b; Ordentlich et al., 1993a; Barak et al., 1994). Chemical affinity labeling (Weise et al., 1990), x-ray structure of TcAChE and of TcAChE-ligand complexes (Sussman et al., 1991; Harel et al., 1993), site-directed mutagenesis, and molecular modeling (Barak et al., 1992; Shafferman et al., 1992a, 1992b; Ordentlich et al., 1993a; Vellom et al., 1993; Radic et al., 1993; Barak et al., 1994; Gnat et al., 1994; Taylor and Radic, 1994) elucidated some aspects of the functional role of 9 of the 14 aromatic amino acids. Residues Phe-295(288) (^2)and Phe-297(290) determine specificity for phosphylating agents (Barak et al., 1992; Fournier et al., 1993) and for the acyl moiety of the substrate (Harel et al., 1993; Ordentlich et al., 1993a; Vellom et al., 1993). The hydrophobic site for the alcoholic portion of the covalent adduct (tetrahedral intermediate) includes residues Trp-86(84), Tyr-337(330) and Phe-338(331), which operate through nonpolar and/or stacking interactions, depending on the substrate (Ordentlich et al., 1993a). Residues Tyr-72(70), Tyr-124(121), Trp-286(279) and Tyr-341(334) are localized at or near the rim of the active center gorge and together with Asp-74(72) constitute the peripheral anionic subsite(s) in AChE (Weise et al., 1990; Shafferman et al., 1992a; Radic et al., 1993, 1994; Barak et al., 1994). Binding of ligands to the peripheral sites was suggested to modulate the AChE catalytic activity through conformational changes in the active center (Changeux,1966; Shafferman et al., 1992b; Ordentlich et al., 1993a).

Residue Trp-86(84) was recently shown to be essential for interaction of AChE with the quaternary ammonium moiety of choline as well as of active center inhibitors (Weise et al., 1990; Sussman et al., 1991; Shafferman et al., 1992a, 1992b; Ordentlich et al., 1993a; Harel et al., 1993) and was therefore suggested to be a main element of the classical anionic subsite of the enzyme. The structure of this subsite and the nature of its interactions with quaternary ammonium groups is a matter of a longstanding controversy. One opinion, argued on the basis of the alleged presence of multiple negative charges in the active center, that the anionic subsite is a true anionic locus (Quinn, 1987). The opposite view based on the structure-activity studies with charged and noncharged substrates and inhibitors suggested that the anionic subsite is in fact a trimethyl site, binding the ligands through hydrophobic interactions (Hassan et al., 1980; Cohen et al., 1984) or dispersive forces (Nair et al., 1994).

In this study we investigate in depth the constitution and function of the anionic subsite of HuAChE through replacement of residue Trp-86 by aromatic or charged amino acids and through substitution of additional residues projecting into the gorge cavity such as Tyr-337 and the highly conserved Glu-202 and Tyr-133. Guided by the catalytic properties of the mutated enzymes, their interactions with reversible and irreversible inhibitors and by molecular modeling, we conclude that the stabilization of charged ligands at the active center does not appear to be mediated by true ionic interactions but rather through cation-aromatic interactions with the residue at position 86. The functional conformation of the anionic subsite Trp-86, which is critical to the overall catalytic efficiency, is achieved through aromatic-aromatic interactions with residue Tyr-133. We also provide evidence, consistent with the notion, that the hydroxyl group of Tyr-133 participates, through hydrogen bonding, in maintaining the functional integrity of the active center.


EXPERIMENTAL PROCEDURES

Mutagenesis of Recombinant HuAChE and Construction of Expression Vectors

Mutagenesis of AChE was performed by DNA cassette replacement into a series of HuAChE sequence variants which conserve the wild type (Soreq et al., 1990) coding specificity, but carry new unique restriction sites (Shafferman et al., 1992a). Generation of mutants W86A(W84), Y337A(F330), and Y337F was described previously (Shafferman et al., 1992b). The W86E(W84) and W86F(W84) mutated HuAChEs were formed by replacement of the AccI-NarI DNA fragment of the pAChEw4 (Shafferman et al., 1992a) variant with synthetic duplexes carrying the mutated codons GAG(Glu) or TTC(Phe). Generation of mutations Y133A(Y130) and Y133F(130) was carried out by replacement of the NarI-XhoI DNA fragment of the pAChEw4 variant with synthetic DNA duplexes carrying the mutated codons GCC(Ala) or TTC(Phe). All the synthetic DNA oligodeoxynucleotides were prepared using the automatic Applied Biosystems DNA synthesizer. The sequences of all new clones were verified by the dideoxy sequencing method (U. S. Biochemical Corp. Sequenase kit). The recombinant HuAChE cDNA mutants were expressed in tripartite vectors which allow expression of the cat reporter gene and the neo selection marker (Kronman et al., 1992; Shafferman et al., 1992a).

Transient Transfection, Preparation, and Quantitation of AChE and Its Mutants

Human embryonal kidney 293 cells were transfected with various purified plasmids using the calcium phosphate method. Transient transfection was carried out as described previously (Velan et al., 1991; Shafferman et al., 1992a, 1992b) and efficiency of transfection was normalized by levels of co-expressed chloramphenicol acetyltransferase activity. The various AChE polypeptides secreted into the medium were quantified by AChE-protein determination relying on the various specific enzyme linked immunosorbent assays (Shafferman et al., 1992a).

Substrates and Inhibitors

Acetylthiocholine iodide (ATC), 5,5`-dithiobis(2-nitrobenzoic acid), ethyl(m-hydroxyphenyl)dimethylammonium chloride (edrophonium), 3,8 diamino-5-3`-(trimethylammonium) propyl-6-phenylphenanthridinium iodide (propidium), 1,10-bis(trimethylammonium)decane (decamethonium), and diisopropyl phosphorofluoridate (DFP) were all purchased from Sigma. 3,3-Dimethylbutylthioacetate (TB) was synthesized and purified as described previously (Ordentlich et al., 1993a). Structures of the substrates and inhibitors used in this study are shown in Fig. 1.


Figure 1: Substrates and inhibitors used in this study. The computed volumes of the trimethyl ammonium group of ATC and the t-butyl group of TB are almost identical (71.2 and 72.0 Å^3, respectively).



Determination of HuAChE Activity and Analysis of Kinetic Data

Catalytic activity of the recombinant HuAChE and its mutant derivatives collected from transiently or stably transfected cells were assayed according to Ellman et al. (1961). Assays were performed in the presence of 0.1 mg/ml BSA, 0.3 mM 5,5`-dithiobis(2-nitrobenzoic acid) in 5 mM or 50 mM sodium-phosphate buffer, pH 8.0, and varying ATC (0.01-0.6 mM) concentrations. The assays were carried out at 27 °C and monitored by a Thermomax microplate reader (Molecular Devices). Michaelis-Menten constant (K(m)) and the apparent first order rate constant (k) values, were determined as described before (Shafferman et al., 1992a, 1992b). The apparent bimolecular rate constants (k) were calculated from the ratio k/K(m).

Kinetic data for inhibition by edrophonium, decamethonium and propidium were analyzed as described previously (Ordentlich et al., 1993a) according to the kinetic treatment developed by Barnet and Rosenberry(1977) and Berman and Leonard(1990) for reaction Fig. S1. This scheme is consistent with the linear mixed inhibition patterns observed for all the cases reported here. K(i) is the competitive inhibition constant and K the noncompetitive inhibition constant. A kinetic solution for the dependence of the reciprocal rate on the inverse concentration of substrate is provided in . The slopes of the double reciprocal plots of rate versus substrate concentration allow derivation of K(i) while the intercepts the calculation of K. This is accomplished by reploting the relative slopes and intercepts, in the presence or absence of inhibitor, against the inhibitor concentration. The reciprocal of the slopes of these replots yield the values of K(i) and K.


Scheme 1:


Phosphorylation experiments were carried out using at least four different concentrations of DFP with two concentrations of enzyme, and the residual enzymatic activity (E) at various times was monitored. The apparent bimolecular phosphorylation constants (k(i)), determined under pseudo-first order conditions, were computed from the plot of slopes of ln(E) versus time at different inhibitor concentrations.

Structure Analysis and Molecular Graphics

Building and analysis of the three-dimensional models was performed on a Silicon Graphics workstation IRIS 70/GT using SYBYL modeling software (Tripos Inc.). Construction of models for the HuAChE and the mutated enzymes was based upon the model structure of the enzyme obtained by comparative modeling (Barak et al., 1992) from the x-ray structure of TcAChE (Sussman et al., 1991). Examination of the conformational mobility of residue Trp-86(84) side chain in the wild type enzyme included an initial search within the acceptable values of (1) and (2) followed by structural optimization of the most probable conformers. The two lowest energy structures were considered as representing the alternative conformational states of Trp-86(84). Similar search was carried out for models of Y133F(Y130) and Y133A(130) mutants. Energy optimizations were carried out by zone refinement including amino acids that either line the active site gorge or are vicinal to residue Tyr-133 (33 residues).


RESULTS

The Functional Consequences of Replacement of Residue Trp-86 in HuAChE by Aromatic and Aliphatic Amino Acids

The significance of residue Trp-86 in HuAChE for interaction with the charged substrate was already demonstrated by the over 660-fold increase in the K(m) value for ATC in the W86A mutant as well as by the over 100-fold increase of the inhibition constant (K(i)) for the active center inhibitor edrophonium (Ordentlich et al., 1993a). On the basis of these observations we proposed that the noncovalent complex of the charged substrate is stabilized through cation-aromatic interactions. To further examine this hypothesis additional modifications of the side chain at position 86 were made by replacement of Trp-86 by phenylalanine and by glutamic acid. If our hypothesis is valid, then the replacement of the indole moiety at position 86 by another aromatic group, phenyl should have a minor effect on the Michaelis-Menten constant (K(m)) for ATC and none for its noncharged isostere TB (Fig. 1). In addition, the values of k for both substrates should not be affected by this mutation. Indeed, results presented in Table 1show that the kinetic values for hydrolysis of ATC and TB by the W86F enzyme are similar to those of the wild type enzyme except for the small increase in the K(m) value for ATC (6-fold). On the other hand replacement of Trp-86 by a charged aliphatic residue (W86E) resulted in over 600-fold increase of the K(m) value for ATC but had no significant effect on the corresponding value for TB. Like the replacement of Trp-86 by alanine, substitution by glutamate results in a moderate decrease in k for both ATC and TB (10- and 8-fold, respectively). The moderate effects of the W86A and W86E mutations on the turnover numbers (k) suggests that residue Trp-86 is not essential for the acylation- deacylation steps of the catalytic reaction. The differential effect of residue at position 86 on the reactivity toward charged and noncharged ligands was corroborated also by testing the various Trp-86 mutants with the irreversible noncharged inhibitor DFP and with the two classical reversible quaternary ligands edrophonium and decamethonium (Fig. 1). Like in the case of TB, the bimolecular rate constants of phosphorylation of W86F, W86E, and W86A enzymes are similar to the corresponding rate constant of the wild type enzyme exhibiting at most a 6-fold decrease (Table 2). On the other hand, for the charged inhibitors, edrophonium and decamethonium (Fig. 1), the substitution of Trp-86 by aliphatic residues results in a substantial decrease in affinity (Table 2). Thus, for concentration of edrophonium as high as 45 mM no inhibition could be observed. This suggests that replacement of the indole moiety by either acidic or neutral aliphatic side chain brings about an estimated 100,000-fold decrease in binding affinity of edrophonium. The same pattern is observed for the bisquaternary ligand decamethonium, however the magnitude of the change is somewhat lower: 10,000-fold higher than wild type: from a K(i) value of 6 µM in the wild type enzyme to 50 and 90 mM for the W86E and W86A mutated HuAChEs, respectively. The noncompetitive component of inhibition (K) of these enzymes by decamethonium appears to change in a similar manner to that observed for the competitive component: an increase in K from 5.7 µM in the wild type HuAChE to 120 mM and 30 mM for W86E and W86A enzymes, respectively. We note that substitution at position 86 of the indole group by a phenyl moiety results in a relatively moderate decrease in affinity (60-80-fold) for the charged reversible inhibitors (Table 2). These results demonstrate that the classical anionic subsite has a predominantly aromatic character. Such conclusion is also supported by the finding that replacement of Glu-202, which is the only acidic residue proximal to the active site, by glutamine resulted in moderate and nonselective decrease of catalytic activity toward ATC and TB (14- and 4-fold, respectively). Furthermore, the consequence of the mutation E202Q, on the rate of phosphorylation (k(i)) by DFP, is by far more pronounced than that resulting from any replacement of Trp-86 (Table 2). The about 5-fold decrease in the value of k(i) due to replacement of Trp-86 by an aliphatic residue is consistent with the conclusion that Trp-86 does not interact with neutral agents reacting at the active site Ser-203. On the other hand, the 50-fold decrease in the DFP phosphorylation rate, due to replacement of Glu-202, signifies the involvement of this residue in the hydrolytic reaction as has been also reported for mouse AChE (Radic et al., 1992).





A further indication of the distinct characteristics of HuAChE enzymes, carrying aliphatic residues at position 86, is the marked decrease in affinity of the W86A and W86E mutants toward the peripheral site inhibitor propidium ( Fig. 2and Table 2). This inhibitor, which binds to amino acids at the entrance to the gorge, is too short to interact with residues at position 86 at the active center (in the propidium-AChE complex the tetraalkyl ammonium group of propidium is 9 Å away from Trp-86; Barak et al., 1994). Yet, replacements of Trp-86 by aliphatic residues generate enzymes which are highly resistant to inhibition by propidium (420- and 1450-fold increase in the competitive inhibition constants for W86E and W86A HuAChE enzymes, respectively, relative to the wild type enzyme) ( Fig. 2and Table 2). On the other hand, the W86F enzyme shows only a 5-fold increase of K(i) for propidium relative to the wild type enzyme (Table 2). The relative increase of the noncompetitive inhibition component (K) of W86E enzyme by propidium (see Table 2) resembles that observed for the value of k(i) (Ordentlich et al., 1993a). The behavior of the W86A and W86E HuAChEs toward propidium may provide some clues to yet another role of aromatic residues at position 86 such as the previously proposed cross-talk between the periphery and the active center (Ordentlich et al., 1993a; Barak et al., 1994) (see ``Discussion'').


Figure 2: Dependence of relative slopes (Rs) on propidium concentrations used for inhibition of ATC hydrolysis by the various HuAChE enzymes. The values of Rs = 1+(1/K)[I] were determined from the slopes of the double reciprocal plots according to (see ``Experimental Procedures'') utilizing 0.02-25 mM of ATC. A, wild type HuAChE (circle), Y133F (up triangle), and W86F (box) mutated HuAChEs. B, Y133A (), W86E (), and W86A (bullet) mutated HuAChEs. Note that propidium concentrations in Panel B are about 10-fold higher than those in Panel A.



Effects of Substitutions of the Active Center Residue Tyr-133 on Reactivity of the Resulting HuAChE Enzymes

The possible functional importance of Tyr-133 is suggested both by virtue of the evolutionary constraint on the conservation of this residue in all ChEs (Gentry and Doctor, 1991; Massoulie et al., 1993) and by its unique location proximal to residue Trp-86 ( Fig. 3and 4A). In addition, molecular modeling suggests that Tyr-133 may participate in hydrogen bond interactions in the active center (Ordentlich et al., 1993b) (Fig. 3). To evaluate the possible role of the hydroxyl moiety in such interactions as well as the potential structural role of the aromatic moiety of Tyr-133, we replaced the tyrosine by phenylalanine and by alanine, and analyzed the effects of these substitutions on the catalytic activity. For comparative purposes, we also included in this study the Y337F and Y337A mutated HuAChEs ( Table 1and Table 2). The Tyr-337 residue is located on the opposite side of to Trp-86, relative to Tyr-133 (Fig. 4A) and like the latter its side chain projects into the gorge cavity. In contrast to the minor effect of replacement of Tyr-337 by phenylalanine, an analogous replacement at position 133 yielded an enzyme that is 7-fold less efficient (k) than the wild type HuAChE in hydrolysis of either ATC or TB (Table 1). The affinity of the Y133F enzyme toward the peripheral site-specific ligand propidium is comparable to that of the wild type HuAChE (Table 2) as could be expected from the location of its binding locus at the entrance to the gorge (more than 10 Å away from Tyr-133 (Barak et al., 1994)). Similarly the interaction of the Y133F mutated enzyme with the bisquaternary ligand decamethonium, which bridges the peripheral and the active center binding sites, shows a minimal change (less than 2-fold) in the value of K(i) as compared to the wild type enzyme (Table 2). It was therefore quite surprising that the specific active center inhibitor edrophonium exhibited a 30-fold decrease in affinity relative to the wild type enzyme (Table 2). The molecular modeling analysis of the Y133F enzyme complexes, described below, provides a rationale for the differential behavior of the two quaternary ligands decamethonium and edrophonium, on the basis of the effect of this mutation on the orientation of Glu-202. This explanation is consistent with the proposed involvement of the hydroxyl group of Tyr-133 in the hydrogen bond network (Fig. 3) (Ordentlich et al., 1993b) and could also account for the decrease in k for hydrolysis of ATC and TB or for the 10-fold decrease in the phosphorylation rate constant of the Y133F enzyme by DFP (Table 2).


Figure 3: Stereo view of the HuAChE active center demonstrating the hydrogen bond interaction between residues Tyr-133 and Glu-202 through a water molecule. The position of water molecule W1 in the model of HuAChE is assumed to be similar to that of water-4 (HETATM-4297; PDB1ACE.ENT) in the x-ray structure of TcAChE (distances: O(Tyr-133)-O(W1) 2.50 Å; O(Glu-202)-O(W1) 3.01 Å). This interaction is a part of a hydrogen bond network spanning the cross section of the active site gorge of HuAChE (Ordentlich et al., 1993b). Other residues depicted in the figure are: the catalytic triad Ser-203, His-447, and Glu-334; Trp-86 (shown in heavy line), Tyr-337, and the peripheral anionic subsite residues Tyr-72 and Trp-286 at the entrance to the active site gorge.




Figure 4: Stereo view of the most stable positions of the indole moiety of Trp-86 in the active center of wild type and Y133A HuAChEs. The most stable conformer of the Trp-86 side chain is depicted for each type of enzyme. Proximity of the side chains, of residues at positions 86 and 133, is illustrated by van der Waals surfaces. The position of the quaternary ammonium group of the substrate ATC, relative to the aromatic ring of Trp-86 is depicted in its noncovalent complex with the wild type HuAChE in Panel A. A, The displayed conformer of Trp-86 side chain in wild type HuAChE ((1) = -56.3 °; (2) = 108.2 °) is more stable by 0.48 kcal/mol than the second most stable conformer which is analogous to that shown in Panel B ((1) = -65.9; (2) = 1.72 °). B, the displayed conformer of Trp-86 side chain in Y133A ((1) = -57.7 °; (2) = 107.0 °) is more stable by 1.65 kcal/mol than the next stable conformer analogous to that displayed in Panel A ((1) = -68.5 °; (2) = 4.6 °). Note that in the conformation displayed in Panel B the indole moiety of Trp-86 should interfere with binding at the active center.



The outcome of the replacement of Tyr-133 by alanine is by far more dramatic than that resulting from its replacement by phenylalanine. For the Y133A HuAChE, we observed a 90-fold increase in K(m), relative to the wild type enzyme, for ATC (Table 1). To date, out of the 80 HuAChE residues analyzed by mutagenesis, the effect of Tyr-133 substitution on the K(m) is second only to that of Trp-86 replacement by alanine or glutamate. In addition, replacement of Tyr-133 by alanine resulted in 8-fold reduction of k, similar to the 5-fold decrease in the corresponding rate observed for W86A enzyme. The combined effects on K(m) and k are reflected in a 760-fold reduction in k for ATC in the HuAChE Y133A enzyme, comparable to the 3400-fold decrease for hydrolysis of the same substrate in the W86A HuAChE (Table 1). The observed similarity in kinetic parameters for ATC hydrolysis, and especially in K(m), of W86A and of Y133A HuAChE enzymes (but not for the W86F or the Y133F HuAChEs) may imply that these two aromatic residues, are involved in stabilization of the enzyme-substrate complex. However unlike the differential effect of mutation W86A on the hydrolysis of ATC versus TB, mutation Y133A results in a marked reduction in catalytic activity for both substrates (Table 1). It therefore appears that replacement of Tyr-133 by alanine has more profound consequences on the integrity of the active center than the analogous replacement of Trp-86. This assumption is also supported by comparison of the reactivities of the Y133A enzyme toward the noncharged and charged inhibitors. As can be seen in Table 2, substitution of Tyr-133 by alanine results in almost 400-fold reduction in the phosphorylation rate by DFP, similar to the decrease in k for ATC. It is also noteworthy that the change in the apparent bimolecular rate constants for phosphorylation by DFP usually parallels that of the k for hydrolysis of TB by any of the mutated HuAChEs tested ( Table 1and Table 2). If a similar reduction in reaction rate is assumed for catalysis of TB by the Y133A enzyme the expected value of k should be below the background readings in our system which explains our inability to measure hydrolysis of TB by the Y133A mutant. Like the replacements of Trp-86 by aliphatic residues also substitution of Tyr-133 by alanine, but not phenylalanine, results in marked resistance to inhibition by peripheral ligands such as decamethonium or propidium ( Table 2and Fig. 2).

The limited effect of substitution of Tyr-337 by alanine or phenylalanine, relative to the wild type enzyme, is in accordance with the observation that of the 3 residues studied Trp-86, Tyr-133, and Tyr-337, the latter is the least conserved among ChEs and is replaced by alanine and phenylalanine in butyrylcholinesterase and TcAChE, respectively (Gentry and Doctor, 1991; Massoulie et al., 1993).

Molecular Modeling of the Various Mutants and Their Complexes with ATC and with Reversible Quaternary Ligands

According to the crystal structures of TcAChE complexes with edrophonium and decamethonium (Harel et al., 1993) and the models of the corresponding HuAChE complexes (Barak et al., 1994) the positions of the quaternary groups of the two ligands, adjacent to the indole moiety of Trp-86, are practically equivalent. The positional equivalence of the quaternary groups, in the complexes of both ligands implies that nearly optimal juxtaposition with the Trp-86 indole moiety has been achieved. Thus, modeling of complexes of edrophonium with HuAChE enzymes mutated at positions 86 and 133, together with the measured reactivities of the corresponding mutants toward ATC and edrophonium, may provide insight as to the particular roles of Trp-86 and Tyr-133 in catalysis. Moreover, the HuAChE-edrophonium complex may serve a template for modeling of the Michaelis-Menten complex of ATC since the position of the quaternary group for the latter appears to be strongly dependent upon the presence of Trp-86.

Modeling of the W86F and Y133F HuAChE Enzymes and Their Edrophonium and Decamethonium Complexes

Models of the W86F and Y133F HuAChE enzymes were obtained from the HuAChE model (Barak et al., 1992) by replacing the appropriate side chains, adjusting their geometry and optimizing the resulting structure as described before (Ordentlich et al., 1993a; Barak et al., 1994). The resulting model of W86F mutated enzyme is very similar to that of the wild type enzyme, including the overlapping positions of the aromatic moieties at position 86. Only minor shifts in residues Tyr-419 and Trp-430, adjacent to residue at position 86, could be observed. The only notable change in the model of Y133F mutant, relative to that of the wild type, is a moderate conformational modification of the side chain of Glu-202 and a shift of the water positioned originally between Tyr-133, Glu-202, and Gly-120.

Edrophonium and decamethonium were docked into the active center of W86F and Y133F enzymes according to their positions in the complexes with the wild type enzyme (Barak et al., 1994). In the optimized structures of the enzyme complexes, the positions of the quaternary groups of the ligands, relative to phenylalanine 86 in W86F enzyme (see Fig. 5B) and to tryptophan 86 in Y133F enzyme (not shown) are very similar and correspond closely to the wild type complexes (see Fig. 5A for HuAChE-edrophonium complex). Thus, the cation- interaction of the quaternary ammonium group with the aromatic substituent at position 86 is maintained by phenylalanine, although the interaction energy is lower than that for tryptophan. The aromatic group of the latter is -excessive and contains an extended -electron system which is therefore more negative and polarizable than that of benzene (Remers, 1971) (the SYBYL force field reproduces a more favorable interactions of tetramethyl ammonium ion with indole than with phenyl moieties; however, it is not specifically parametrized to allow for quantitative estimations of such differences). The more favorable cation- interaction of edrophonium or decamethonium with indole than with phenyl at position 86, may account for the 60- and 80-fold increase of the inhibition constant (DeltaDeltaG = 2.44 kcal/mol and 2.65 kcal/mol, respectively), for these ligands in W86F HuAChE relative to the wild type enzyme.


Figure 5: Stereo views of edrophonium complexes with the wild type HuAChE and the W86F enzyme. The ligand, residues Tyr-133, Glu-202 and amino acid at position 86 are marked by a heavy line. The volume of the ligand is marked by dot surface. Note the distinct interactions of each of the three structural moieties of the ligand: the quaternary group is facing the aromatic ring of residue at position 86; the edge of the ligand aromatic moiety is wedged between the carboxylate oxygens of Glu-202; the hydroxyl group is within hydrogen bond distance from O-Ser-203 and N-His-447. A, HuAChE-edrophonium complex; B, W86F HuAChE-edrophonium complex.



The equivalent positions of the quaternary groups in Y133F HuAChE and in the wild type enzyme complexes indicate that the ligands do not interact directly with the aryl moiety of Y133 (Fig. 4A and 5A). Therefore, the 30-fold increase of the K(i) value for inhibition of Y133F by edrophonium, relative to the wild type enzyme, is probably due to an indirect effect of Tyr-133 replacement, affecting the conformation of other active center residues. We note that the carboxylate of Glu-202 in the wild type enzyme is in a more favorable position to interact with the aromatic group of edrophonium. In the model of Y133F-decamethonium complex (not shown) no appreciable differences with respect to the model of the wild type enzyme complex (Barak et al., 1994) could be observed.

Modeling of ATC Complexes with HuAChE and W86F Mutant

Construction of the HuAChE-ACh noncovalent (Michaelis-Menten) complex started with superposition of the ACh quaternary ammonium group on the equivalent moiety in the HuAChE-edrophonium complex. In the all trans conformation of ACh the carbonyl oxygen was positioned within interaction distance from the amide nitrogens of the oxyanion hole (Gly-121, Gly-122), bringing the terminal methyl group into the acyl pocket (Phe-295, Phe-297). In the final structure the O-Ser-203 is within hydrogen bond distance (2.95 Å) from the alkoxy oxygen of ACh (Fig. 4A). Similar docking of ACh into the model of W86F HuAChE results in a structure which is equivalent to that of the complex with the wild type enzyme.

Modeling of the Y133A HuAChE Enzyme

As already mentioned, residue Tyr-133 does not appear to interact directly with the ligands in HuAChE complexes with ATC, edrophonium or decamethonium. Therefore, it is quite striking that the Y133A mutation had such dramatic effects on the various aspects of the enzyme chemical reactivity including catalytic activity toward charged and noncharged substrates as well as covalent and noncovalent inhibitors. Molecular modeling provides insight into these surprising results by investigating the effect of elimination of aromatic residue from position 133 on the conformation of other residues at the active center. Examination of molecular models shows that replacement of residue Tyr-133 by alanine induces an altered conformation of Trp-86 in which the indole moiety is rotated by over 100 ° (around (2)) from its position in the wild type enzyme (Fig. 5B). On the other hand, replacement of Tyr-133 by phenylalanine had only a small effect on the expected conformational mobility of residue Trp-86 and resembles that of wild type. The conformational transition introduced by replacement of Tyr-133 by alanine places the Trp-86 indole group across the gorge obstructing the access to the active site. Therefore, this energetically favored conformation (1.6 kcal/mol) for the Y133A HuAChE mutant (Fig. 4B) should interfere with any ligand interacting with the active center.


DISCUSSION

How Is the Quaternary Ammonium Moiety of AChE Ligands Stabilized in the Active Center?

Early studies of the rate of hydrolysis of various charged and noncharged substrates as well as the observed ability of tetraalkylammonium derivatives to inhibit AChE in a pH-dependent manner, have led to the hypothesis that the active center contains a quaternary ammonium binding subsite (for reviews, see Rosenberry(1975), Quinn(1987), and Massoulie et al. (1993)). Wilson(1952) postulated that this subsite is predominantly involved in ionic interactions and therefore should be regarded as the anionic subsite. An alternative view negated the ionic character of this site and suggested it to be essentially nonpolar in character, accommodating the charged ammonium groups through either ion-induced dipole or through van der Waals interactions (O'Brien, 1971; Hassan et al., 1980; Nair et al., 1994).

These opposing views regarding the nature of the classical anionic subsite which were based mainly on structural and mechanistic assumptions may now be reevaluated relying on the molecular structure of AChE and on modern techniques for site directed manipulation of the enzyme structure. Examination of the x-ray structure of TcAChE (Sussman et al., 1991) and the derived model of HuAChE (Barak et al., 1992), reveals that the only negatively charged residue vicinal to the catalytic serine is Glu-202. The two other acidic residues: Asp-74 and Glu-450, located within the active site gorge, are 15.1 and 8.9 Å away from residue Ser-203, respectively (measured from O-Glu-450 or O-Asp-74 to O-Ser-203) and are therefore unlikely to participate in interactions of the anionic subsite. From the kinetic data in Table 1it is evident that substitution of Glu-202 by the neutral residue glutamine has a comparable effect on catalysis for both ATC and its noncharged isostere TB, suggesting that residue Glu-202 has no specific role in stabilizing positively charged substrates and therefore is not a part of the anionic subsite. On the other hand, recent kinetic data (reviewed by Taylor and Radic(1994)) indicates that the negative charge at position 202 plays an important role in the acylation step of the catalytic reaction (Radic et al., 1992; Shafferman et al., 1992b) as well as in phosphylation, carbamoylation (Radic et al., 1992; Ordentlich et al., 1993b), aging (Ordentlich et al., 1993b; Saxena et al., 1993) and for interactions with noncovalent ligands (Radic et al., 1992; Shafferman et al., 1992b).

Unlike the indiscriminate effect on catalysis due to replacement of Glu-202, substitutions at position 86 affect differentially the hydrolytic activity toward charged and noncharged substrates (Table 1). While the wild type enzyme is 20-fold more active toward ATC than toward TB, the HuAChE mutants carrying aliphatic residues at position 86 show 50-fold higher reactivity for TB. This reversal of selectivity toward the sterically identical noncharged substrate, and the fact that kinetic parameters for TB are only marginally affected by the various mutations, is a clear manifestation of the existence of a functional anionic subsite and of the role of residue Trp-86 in this subsite. The effect of substitution of Trp-86 by nonaromatic residues suggests a role for this position in stabilizing the Michaelis-Menten complexes of HuAChE with charged substrates. Such conclusion is also supported by: (a) the lack of measurable affinity of W86A and W86E mutants toward the charged active center inhibitor edrophonium; (b) the 8,500- and 15,000-fold increase, relative to the wild type enzyme, in K(i) value for decamethonium in the W86A and W86E enzymes respectively; (c) the hundredfold higher affinity of W86F HuAChE toward edrophonium or decamethonium, compared to either the W86A or the W86E enzymes (Table 2). In marked contrast, the nature of residue at position 86 has only a marginal contribution to the activity of HuAChE toward noncharged substrates like TB or noncharged inhibitors like DFP. Taken together these observations imply also that in the Michaelis-Menten complexes, the orientations of the trimethyl ammonium and the 3,3-dimethylbutyl groups, of ATC and TB, respectively, are not equivalent relative to residue Trp-86. The topographical distinction between trimethyl and trimethylammonium sites, was also suggested by Berman and Decker(1986) on the basis of the differential affinity of the covalent adducts of AChE with isosteric charged and noncharged methylphosphonates, toward the fluorescent ligand decidium. The contrasting suggestions, regarding the anionic subsite as a common trimethyl binding site (Cohen et al. 1984, 1987) or a common locus for dispersion interactions (Nair et al., 1994), cannot be reconciled with either the differential effects of substitutions at position 86 of HuAChE or the results of Berman and Decker(1986).

Accommodation of the quaternary ammonium groups of AChE ligands by the indole moiety of residue Trp-86 is an additional example of interactions between organic cations and protein aromatic residues, the importance of which is recently becoming recognized as major contributors to molecular recognition. The stabilizing interaction involves the positive charge and the electron-rich face of an aromatic ring. Such cation- interactions were investigated both theoretically (Gao et al., 1993; Kearney et al., 1993) and experimentally, using several synthetic host molecules (Dougherty and Stauffer 1990; McCurdy et al., 1992; Garel et al., 1993). The nature of the interactions is predominantly electrostatic involving ion-dipole, ion-quadrupole, and ion-induced dipole (Schneider, 1991; McCurdy et al., 1992). Analysis of protein structures shows that protonated amines interact favorably with aromatic groups (Burley and Petsko, 1988). The crystal structures of TcAChE complexes with edrophonium and decamethonium (Harel et al., 1993) and that of the Fab McPC603-phosphocholine complex (Satow et al., 1986) demonstrate interactions of quaternary ammonium moieties with tryptophan and tyrosine residues. Mutagenesis, NMR and fluorescence binding studies indicate that aromatic moieties are important determinants in binding of quaternary amines to nicotinic and muscarinic ACh receptors (Galzi et al., 1991; Wess, 1993; Fraenkel et al., 1990) as well as to the peripheral anionic sites of AChE (Ordentlich et al., 1993a; Radic et al., 1993; Barak et al., 1994).

In conclusion, results from site directed mutagenesis of HuAChE and from x-ray crystallography of TcAChE (Sussman et al., 1991) and its complexes (Harel et al., 1993) provide a compelling evidence for the presence of a specific anionic subsite locus. This site stabilizes the quaternary ammonium groups of substrates and other ligands through cation-aromatic interactions mainly with residue Trp-86, rather than through ionic interactions.

The Dual Role of Tyr-133 in Maintaining the Functional Integrity of the Active Center

Although molecular model of the mutant Y133F enzyme suggests only minor changes, as compared to the wild type enzyme, mainly in orientation of Glu-202, kinetic results indicate that removal of the hydroxyl group, at position 133, affects both hydrolytic activity and affinity toward edrophonium. Such effects are compatible with the proposed participation of the hydroxyl group of Tyr-133 in a hydrogen-bond network (Ordentlich et al., 1993b) that maintains the proper orientation of the carboxylate of Glu-202. Indeed, substitution of Tyr-133 by phenylalanine affects catalysis almost to the same extent as replacement of Glu-202 by glutamine (Table 1). This parallelism, in behavior of the Y133F and E202Q enzymes, is not limited to hydrolytic activity toward substrates but is observed also for phosphorylation of the respective mutants by DFP or with their comparable decrease in affinity toward the active center inhibitors edrophonium and decamethonium (Table 2). In the HuAChE-edrophonium complex the edge of the aromatic moiety of the inhibitor is wedged between the two negatively charged O-Glu-202 (Fig. 5A) and thus can participate in charge-quadruple interactions (Burley and Petsko, 1988). The nature of these interactions obviously depends on the exact positioning of the carboxylate with respect to the ligand. It is worth noting that in decamethonium, which unlike edrophonium cannot participate in such interactions, the Y133F and E202Q enzymes show only 2-5-fold decrease of affinity relative to the wild type enzyme. Thus, it appears that the main role of the hydroxyl group of Tyr-133 is in providing the exact juxtaposition of the Glu-202 carboxylate, relative to the elements of the active site. Such positioning may be important in stabilizing the evolving transition states of acylation and other covalent reactions of the catalytic serine like phosphylation and carbamoylation.

Replacement of residue Tyr-133 by alanine produces enzyme severely impaired in its reactivity toward substrates and inhibitors ( Table 1and Table 2). This may be interpreted to suggest that Tyr-133 is a key element in stabilization of the complexes with the various ligands; however, the corresponding models show that residue at position 133 does not interact directly with any of the substrates or inhibitors examined here. Therefore, the altered reactivity of Y133A probably originates from modification of the active center induced by the replacement at position 133. On the other hand such modification affects mainly the noncovalent binding of substrates and inhibitors since Y133A mutant is still a very efficient catalyst of ATC hydrolysis with k only 8-fold lower than that of the wild type enzyme, suggesting that the catalytic machinery has been only slightly affected. A possible characterization of the modification of the active center in the Y133A enzyme is provided by the molecular model according to which removal of the aromatic group from position 133 induces rotation of the Trp-86 side chain placing the indole moiety across the active site gorge (Fig. 4B). In this conformation the indole is stabilized by interactions with other residues lining the wall of the gorge and consequently obstructing the access of substrates and other ligands to the active site of the enzyme. Finally, the particular way of maintaining the proper geometry of the quaternary ammonium binding subsite, through aromatic-aromatic interactions, may not be unique to AChE. Clusters of aromatic residues have been implicated, by site-directed mutagenesis and molecular modeling, in the putative binding sites of receptors for ACh and other monoamine neurotransmitters, suggesting that aromatic-aromatic interactions can be instrumental in binding of ammonium ions in a variety of macromolecular systems (Hibert et al., 1993).

Possible Functional Significance of Two Conformational States of Trp-86

Molecular models of the wild type, Y133F, and Y133A enzymes reveal that the side chain of residue Trp-86 can occupy two conformational states: one that is functional as the anionic subsite while the other occludes the active center and should reduce catalytic efficiency (Fig. 6). This effect on catalytic activity, due to the alternation between the two conformational states of a single residue, raises the intriguing possibility that the enzyme actually utilizes this structural flexibility to adjust its catalytic efficiency.


Figure 6: Proposed accessibility of the HuAChE active center in the two conformational states of residue Trp-86. A side view cross-section of the active site gorge is represented by volume contours of the amino acids lining its walls. The volume of Trp-86 side chain is marked by dot surface and the side chain of Ser-203 marks the position of the active site. A, the functional conformation of Trp-86. The substrate ACh can access the active site and orient its quaternary ammonium for favorable interaction with the indole moiety (anionic site). B, the blocking conformation of Trp-86. The side chain is positioned across the gorge obstructing access of substrates and other ligands to the active site. Transition between state A and B is proposed to be induced in response to binding of ligands to the peripheral anionic site at the entrance to the active site gorge.



One of the more elusive features of AChE reactivity is the allosteric modulation (Changeux, 1966) of the catalytic activity following ligand binding to the peripheral sites on the enzyme surface. In the past, evidence was presented that such peripheral site ligands, including the natural substrate, or other chemical stimuli present in the synaptic cleft such as bivalent cations (Ca^2, Mg^2) affect the conformation of the active center (Radic et al., 1991; Berman and Nowak, 1992). Furthermore, allosteric modulation of AChE activity was demonstrated only for charged substrates and inhibitors. Since we have demonstrated the role of Trp-86 in the binding of charged substrate and its potential conformational mobility, it is possible that motion of this residue, induced by an allosteric signal on the surface, abolishes the anionic subsite and at the same time blocks the access to the active site (Fig. 6). Such mechanism of allosteric modulation of AChE provides a ready explanation for the baffling inhibition patterns of W86E, W86A, and Y133A HuAChE enzymes, by the peripheral site ligand propidium compared to the marginal effects in the W86F and Y133F HuAChEs. Accordingly, the resistance of W86A and W86E enzymes to inhibition is simply due to the absence of a bulky residue, suitable for blocking the access to the active site, in these mutated HuAChEs. In the Y133A enzyme (but not in the Y133F HuAChE) the conformation of Trp-86 blocking the active center (Fig. 4B) is present prior to the exposure to propidium and therefore only a minor inhibitory effect due to addition of propidium can be expected. In both cases propidium binding to the peripheral anionic site, at the entrance to the active site gorge, should be hardly affected. Indeed, preliminary results from fluorescence binding studies of propidium with purified W86A mutated HuAChE appear to support this prediction.

Although the conformational flexibility of Trp-86 and its effects on the catalytic activity provide a possible mechanism for the cross-talk between the peripheral sites and the active center, the relay path of the allosteric signal is still unclear. Clues to a possible way for signaling the incidence of binding at the periphery to the active center and for inducing motion of Trp-86, can be found in the fact that the central binding element of the peripheral site, Asp-74, which was also implicated in the cross-talk mechanism (Shafferman et al., 1992b), as well as Trp-86 are part of the sequence comprising the small cysteine loop (Cys-69-Cys-96). It is conceivable that external stimuli affecting Asp-74 might alter somewhat the position of this loop relative to the rest of the structure, separating the indole moiety of Trp-86 from the aromatic ring of Tyr-133 and thus inducing the conformational transition of the side chain of Trp-86 that obstructs the active site. The potential flexibility of the TcAChE small cysteine loop was indeed suggested by molecular dynamics studies (Axelsen et al., 1994). Accordingly, one may design selective structural modifications within this loop, concomitant with other changes of residues in the active center gorge, and utilize various molecular probes to further explore this putative mechanism of signal transduction that modulates the enzymatic activity of AChE.


FOOTNOTES

*
This work was supported by the U.S. Army Research and Development Command, Contract DAMD17-93-C-3042 (to A. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 972-8-381518; Fax: 972-8-401404.

(^1)
The abbreviations and trivial names used are: AChE, acetylcholinesterase; HuAChE, human acetylcholine esterase; TcAChE, Torpedo californica acetylcholinesterase; ACh, acetylcholine; ATC, acetylthiocholine; TB, 3,3-dimethylbutylthioacetate; DFP, diisopropyl phosphorofluoridate; edrophonium, ethyl(m-hydroxyphenyl)dimethylammonium chloride; propidium, 3,8-diamino-5-3`-(trimethylammonium)propyl-6-phenylphenanthridinium iodide; decamethonium, 1,10-bis (trimethylammonium)decane.

(^2)
Amino acids and numbers refer to HuAChE, the numbers in parentheses refer to the position of analogous residues in TcAChE, according to the recommended nomenclature (Massoulie et al., 1992).


ACKNOWLEDGEMENTS

We thank Dana Stein, Tamar Sery, and Nehama Zeliger for their excellent technical assistance.


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