(Received for publication, September 6, 1994; and in revised form, November 18, 1994)
From the
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.
Acetylcholinesterase (AChE, EC 3.1.1.7) ()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) (
)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.
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,
respectively).
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 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
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
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), determined under pseudo-first order
conditions, were computed from the plot of slopes of ln(E) versus time at different inhibitor concentrations.
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 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
(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 (
), Y133F (
), and W86F (
) mutated HuAChEs. B, Y133A (
), W86E (
), and W86A (
) mutated
HuAChEs. Note that propidium concentrations in Panel B are
about 10-fold higher than those in Panel
A.
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 ( = -56.3 °;
= 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 (
= -65.9;
= 1.72 °). B, the displayed conformer of
Trp-86 side chain in Y133A (
= -57.7
°;
= 107.0 °) is more stable by 1.65
kcal/mol than the next stable conformer analogous to that displayed in Panel A (
= -68.5 °;
= 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, 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
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
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
, 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).
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 (
G = 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 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.
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 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.
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).
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, Mg
)
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.