(Received for publication, March 31, 1995; and in revised form, May 3, 1995)
From the
The interaction of fasciculin 2 was examined with wild-type and
several mutant forms of acetylcholinesterase (AChE) where
Trp, which lies at the base of the active center gorge, is
replaced by Tyr, Phe, and Ala. The fasciculin family of peptides from
snake venom bind to a peripheral site near the rim of the gorge, but at
a position which still allows substrates and other inhibitors to enter
the gorge. The interaction of a series of charged and uncharged
carboxyl esters, alkyl phosphoryl esters, and substituted
trifluoroacetophenones were analyzed with the wild-type and mutant
AChEs in the presence and absence of fasciculin. We show that
Trp
is important for the alignment of carboxyl ester
substrates in the AChE active center. The most marked influence of
Trp
substitution in inhibiting catalysis is seen for
carboxyl esters that show rapid turnover. The extent of inhibition
achieved with bound fasciculin is also greatest for efficiently
catalyzed, charged substrates. When Ala is substituted for
Trp
, fasciculin becomes an allosteric activator instead of
an inhibitor for certain substrates. Analysis of the kinetics of
acylation by organophosphates and conjugation by
trifluoroacetophenones, along with deconstruction of the kinetic
constants for carboxyl esters, suggests that AChE inhibition by
fasciculin arises from reductions of both the commitment to catalysis
and diffusional entry of substrate into the gorge. The former is
reflected in the ratio of the rate constant for substrate acylation to
that for dissociation of the initial complex. The action of fasciculin
appears to be mediated allosterically from its binding site at the rim
of the gorge to affect the orientation of the side chain of Trp
which lies at the gorge base.
The fasciculins, 61 amino acid peptides found in venom of snakes
of the mamba or Dendroaspis family, are potent inhibitors of
most acetylcholinesterases (EC 3.1.1.7)
(AChEs)()(1) . Three fasciculins have been
identified to date with nearly identical amino acid sequences:
fasciculin 1 (FAS1), FAS2, and FAS3. FAS3 binds to rat brain AChE with
a dissociation constant one order of magnitude lower than that for FAS1
and FAS2(2) . The sequence and crystal structure of FAS1 (3) show that these toxins fall within a larger family of
``three finger'' Elapidae toxins which include
erabutoxin, cardiotoxins,
-bungarotoxin, and
-cobratoxin.
These latter toxins, however, do not inhibit AChE, whereas FAS at low
concentrations does not block acetylcholine receptor
function(4, 5) .
Torpedo and most mammalian AChEs are inhibited by FAS at picomolar concentrations, whereas the closely related butyrylcholinesterases (EC 3.1.1.8) (BuChE) require FAS concentrations approaching millimolar for inhibition(5, 6) . Three domains in AChE and butyrylcholinesterase appear responsible for their distinct substrate and inhibitor specificities(7, 8, 9, 10) . Two of them, the acyl pocket and choline-binding site of the active center, should be virtually inaccessible to FAS since they are located at the base of a narrow gorge leading to the active center of the cholinesterases. FAS, however, should access binding sites peripheral to the active center as evidenced by protection of the enzyme from FAS inhibition by micromolar concentrations of propidium and millimolar concentrations of acetylthiocholine (ATCh). This concentration range of ATCh results in substrate inhibition rather than maximal catalytic rates(2, 5) . Thus, FAS appears to be specific for a peripheral site on AChE. The high affinity of FAS is primarily a reflection of a slow dissociation rate yielding a half-time of several hours, while the rate of association between FAS and AChE is only one to two orders of magnitude slower than expected for a diffusion-controlled reaction between a peptide and a protein(2, 6) .
The mechanism of AChE inhibition by
FAS might involve physical occlusion of the entrance to the active
center gorge by the peptide, modification of the electrostatic field
affecting substrate entry into the gorge, and/or an allosteric
influence on the active center affecting the commitment to catalysis of
bound substrate. The capacity of the FASAChE complex to react
with diisopropyl fluorophosphate (2) reveals that the active
serine at the base of the gorge remains potentially accessible to
reaction with substrates. In this article we analyze the possible
mechanisms of inhibition by FAS by measuring kinetic constants for
inhibition of wild-type and several mutant cholinesterases. In
addition, we examine the capacity of FAS to affect AChE specificity for
carboxyl and alkylphosphoryl ester acylation and for conjugation with
substituted trifluoroacetophenones.
Figure ZI: Structure I.
where S denotes the substrate concentration, K and K
Michaelis-Menten and substrate inhibition constants, and b the productivity ratio of the ternary SES complex
to the ES complex (cf. 7).
and K is a reversible inhibition
constant for fasciculin, F, and is also equal to k
/k
. and were derived from :
with the constraints of F = 0 for , and KSK
and F
E for .
Rate constants for FAS and trifluoroacetophenone association and dissociation were determined as described earlier(6) . Rate constants for inhibition of the enzymes with paraoxon and echothiophate as well as rate constants of association of trifluoroacetophenones were determined by following the time course of the onset of inhibition using ATCh as substrate and three or more inhibitor concentrations as described earlier(16) .
Figure 1: Influence of varying FAS concentrations on the activity of wild-type and mutant mouse AChE. Enzyme activity was measured using ATCh (A) and pNPA (B) as substrates at concentrations denoted in Table 1. Theoretical curves were obtained by fitting experimental points to equation(2) . Trp at position 86 in the wild-type enzyme was replaced by Phe (F), Tyr (Y), and Ala (A). The recombinant DNA-derived enzymes were incubated overnight (14-16 h) with FAS prior to assay.
This behavior contrasts with the marked
increases in K and lack of effect on
that three substitutions at peripheral site residues,
Trp
Arg, Tyr
Asn, Tyr
Gln, have
on FAS interactions with AChE. It also distinguished from the virtual
lack of effect that substitutions at the acyl pocket and position 337
have on FAS inhibition(6) . With these mutants residual
activities of the FAS complexes measured with ATCh were equivalent to
those for wild-type enzyme. The residues at positions 286, 72, and 124
constitute part of the peripheral site and are located at the entrance
of the active center gorge. They are directly accessible to FAS and
might be expected to be in direct contact with FAS. Accordingly, their
replacement should influence the dissociation constant of FAS. The two
aromatic residues Trp
and Tyr
in the
choline-binding site are located deep within the active center gorge
and seem inaccessible for direct contact by FAS. Therefore, a minimal
effect of their replacement by Ala on K
of the FAS-AChE complex is not surprising. However, while
Tyr
Ala was almost totally inhibited by saturating FAS
concentrations (>99%), hydrolysis of ATCh by Trp
Ala was
only inhibited about 20% at saturating FAS concentrations. Catalytic
hydrolysis of pNPA was activated in the presence of FAS.
The small increase in K arising from
substitutions at Trp
is due to the increase in the
dissociation rate constant for FAS, k
(Table 2), as previously observed for the
Tyr
Gln and Asp
Asn mutants(6) . Rates
of FAS association are not affected by any of the mutations where the
rate constants are sufficiently slow for detection by conventional
measurement.
The presence of ATCh in concentrations up to 1
mM did not affect FAS association or dissociation rate
constants. FAS therefore binds with the same affinity to free enzyme
and enzymeATCh complex. Only high ATCh concentrations, sufficient
to cause substrate inhibition in wild-type AChE, decrease the rates of
FAS association with wild-type and mutant enzymes(6) . The
decrease may be a consequence of either a modified electric field in
AChE or, more likely, competition between occupation of ATCh and FAS at
peripheral sites.
Figure 2:
Influence of fasciculin on
acetylcholinesterase catalysis. A, representative plots of
activity versus substrate concentration for high (ATCh) and low (pNPA) turnover substrates in the
presence and absence of saturating (40-200 nM) FAS.
Curves were obtained by nonlinear regression of to fit the
experimental points. Fasciculin was incubated with the enzyme for at
least 1 h prior to the activity measurements. B, substrate
concentration dependence for catalysis of ATCh and pNPA by
wild-type AChEFAS complex. Data show enlarged ordinates from the top panels. The solid line was obtained
by a nonlinear regression of to fit the experimental
points. The dotted line shows the best fit to the data
assuming Michaelis-Menten kinetics.
Substitutions
of Phe, Tyr, or Ala for Trp at position 86 influence catalysis to the
largest extent with the most efficient substrate ATCh. Replacements of
the indole ring of Trp with other aromatic substituents reduce k/K
by an order of
magnitude, whereas deletion of the ring leaving the aliphatic side
chain in alanine reduces k
/K
by two orders of magnitude. The reductions in k
/K
are reflected
mainly in larger K
values except for the
Trp
Ala mutation where k
is reduced
50-fold. However, the Trp
Ala enzyme shows substrate
activation which will effectively increase catalysis at high substrate
concentrations.
In the case of PA, a neutral but relatively high
turnover substrate, k was decreased only
2-3-fold for the aromatic substituents, and 10-fold for the Ala
substitution. Since the K
values also
decrease slightly upon substitution of Phe or Tyr for Trp
, k
/K
is virtually
unaffected. pNPA is a relatively poor substrate whose k
/K
for the
wild-type enzyme is nearly three orders of magnitude less than that for
ATCh. All three substitutions for Trp
led to an increase
of k
/K
for pNPA; this is a cumulative effect of lowering K
and increasing k
.
Thus, the mutations reduced k in a similar manner for the two substrates of
rapid turnover, ATCh and PA, while two neutral substrates, PA and pNPA, have their K
values
similarly affected. k
/K
was affected the most for ATCh and pNPA, while for
PA k
was most affected.
The
binding of FAS to the wild-type and mutant enzymes completely abolished
substrate inhibition by ATCh. In fact, Fig. 2B shows
elements of substrate activation (b = 6.8) for
hydrolysis of ATCh by the complex of wild-type AChE with bound FAS.
This is similar to the activation observed for ATCh hydrolysis by the
TrpAla mutant in the absence of FAS. Substrate activation
should not arise as a result of FAS-ATCh competition since the FAS
dissociation rate is inherently slow (cf. Table 1).
The largest reduction of a second-order phosphorylation constant,
approaching three orders of magnitude, was produced by
TrpAla substitution in reaction with echothiophate (Table 4). With the same mutation, paraoxon inhibition was
affected only 3-fold. Substitution of Trp
with Phe
decreased the second-order inhibition constants for echothiophate and
paraoxon 7- and 3-fold, respectively, while the inhibition constant for
the Trp
Tyr mutant enzyme was not affected (Table 4).
The changes in inhibition parameters were mainly a consequence of an
increase in K
for echothiophate and a
decrease in maximal phosphorylation rate for paraoxon.
FAS
association had more effect on inhibition by echothiophate with its
cationic leaving group than by the neutral congener, paraoxon. The
second-order inhibition rate constant for the wild-type enzymeFAS
complex decreased 10-fold for echothiophate as a result of an increase
in its K
; paraoxon's overall
inhibition rate constant is largely unchanged, although both K
and k
were
decreased in a compensatory fashion in the presence of FAS. FAS binding
to the Trp
Phe and Trp
Tyr mutants only
modestly affects inhibition by paraoxon, whereas echothiophate
inhibition is decreased up to 10-fold as a consequence of a slower
phosphorylation rate. FAS had a minimal effect on inhibition of the
Trp
Ala mutant by paraoxon, but the second-order inhibition
constant for echothiophate was increased 100-fold. This mutant enzyme
shows both enhanced affinity and reactivity for the cationic
organophosphate in the presence of FAS.
Second-order inhibition rates for
trifluoroacetophenones reflect both formation of a reversible complex
and subsequent covalent bond formation. Since rates of conjugate
formation for both isosteres and all mutants showed a linear dependence
on concentration, formation of conjugates was treated as a simple
bimolecular association. When correlated for the ratio of non-hydrated
to hydrated species(12) , the measured second-order reaction
rate constants for the non-hydrated ketones fell well in the range of
rates for diffusion-limited reactions (10-10
M
min
). These
values are very similar to a rate of association of 4
10
M
min
determined
for the reversible inhibitor N-methylacridinium (18) and exceed k
/K
for ATCh. Even though NMR measurements for similar
trifluoroacetophenones (19) and the crystal structure of the
AChE
TFK
complex (17) show that
trifluoroacetophenones bind to the active serine covalently forming a
hemiketal, the rate of the TFK
reaction appears
limited by diffusion in the concentration range where measurements are
practical.
Bound FAS affects the reaction rates of
trifluoroacetophenones for both the wild-type and mutant enzymes. Rates
of conjugate formation with the wild-type and TrpPhe
enzymes decreased in the fasciculin complexes, and rates of conjugate
dissociation increased to a greater extent with the charged than with
neutral isostere. This results in a 10-fold greater increase in K
for the charged isostere. Bound FAS
also affected the interaction of both trifluoroacetophenones with
Trp
Tyr and Trp
Ala. While bound FAS increased K
for Trp
Tyr 10-fold, the K
for Trp
Ala mutant
decreased about 2-fold. It is interesting that a 1000-fold difference
in K
between charged and uncharged
isosteres for wild-type AChE is significantly reduced 5-100-fold
when FAS is bound to the mutant or wild-type enzymes.
Bound FAS
decreased rates of diffusion of the cationic TFK into
the enzyme gorge by an order of magnitude. Since bound FAS may restrict
diffusion-limited entry into the gorge, the reductions in rate of
TFK
association may reflect the gating influence of
the positively charged FAS molecule toward entry of cationic substrates
into the gorge. In addition, substitution of aromatic and aliphatic
residues at position 86 diminishes the stability of the hemiketal
conjugates formed between the substituted trifluoroacetophenones and
AChE. These factors are reflected in a larger k
and a smaller k
. As the overall k
diminishes, diffusion is no longer
rate-limiting. With the substitutions at position 86 which form the
less stable complexes, FAS has a greatly diminished influence on the
reaction. FAS affects rates of dissociation of the conjugate presumably
by allosterically influencing the site of TFK binding. The overall
effect of FAS binding is similar in magnitude to substitution of Ala
for Trp
.
In this study we have examined the influence of a peptidic
peripheral site inhibitor, FAS 2, on the catalytic parameters of AChE.
Substantial evidence has accumulated to show that FAS binds at a
peripheral site to regulate substrate
catalysis(1, 2, 5, 6) . We show here
by examining mutations in the choline binding subsite at the base of
the gorge that FAS, by acting near the rim of the gorge, controls the
overall configuration of the substrate-binding site. By comparing
charged and uncharged substrates, one approaches the question of
whether FAS affects initial entry of substrate into the gorge or the
subsequent steps of catalytic turnover of bound substrate. To this end
it becomes useful to deconstruct the catalytic parameters, k and K
, where
possible, into primary constants. For below:
we have examined three classes of substrates designated by AB: (a) the carboxylic acid esters: ATCh, pNPA, and PA.
These acetoxy esters differ about 600-fold in catalytic efficiency, k/K
, with the
following order ATCh>PA>pNPA; (b) the symmetric
organophosphates which only differ in their leaving groups: the
charged, diethoxyphosphoryl thiocholine (echothiophate), and uncharged
diethoxyphosphoryl p-nitrophenol (paraoxon). These two
compounds yield structurally identical covalent conjugates with the
active serine. Echothiophate and paraoxon have the same leaving groups
as ATCh and pNPA, respectively. In contrast to the carboxyl
esters, only rates of acylation require consideration in analyzing
substrate kinetics. For organophosphates K
(now defined as K
) is reduced
to (k
+ k
)/k
, and k
is reduced to k
; (c)
isosteric trifluoroacetophenones which contain charged and uncharged
moieties at the meta position. The conjugation reaction here
only involves nucleophilic addition by Ser
to form the
hemiketal without loss of a leaving group. Consequently K
reduces to the ratio of k
and k
. There is no
substrate turnover, and k
is zero.
Since the
AChEFAS complex remains catalytically active and susceptible to
phosphorylation by diisopropyl fluorophosphate(2) , or other
organophosphates (Table 4), FAS does not serve as a physical
barrier to block totally substrate entry into the AChE active center
gorge. Rather, binding of FAS to the enzyme appears to affect the
chemical acylation step of the catalytic reaction described by k
. This is indicated by pronounced reductions in k
and k
/K
for the three
carboxyl esters, as opposed to modest increases in their K
values. Values of k
and k
/K
for
hydrolysis of ATCh and PA by human AChE in the presence of FAS decrease
significantly in D
O buffers, while in the absence of FAS
the isotope effect of D
O is small(20) . These
findings indicate that FAS reduces the rate of ATCh and PA acylation
described by k
.
In addition, cationic ligands
may find their entry to the active center diminished due to the
electrostatic restrictions imposed by bound FAS. Positively charged
ATCh, unlike the other two neutral substrates, has a 5-fold greater K for the AChE
FAS complex compared
to AChE. Also, charged organophosphates and trifluoroacetophenones
react with AChE
FAS complex at rates an order of magnitude slower
than with AChE. The Trp
Ala enzyme is an exception where
FAS accelerates an inherently slow rate of echothiophate inhibition
while exerting little influence on the reaction with the neutral
organophosphates.
Substitution of Trp by two other
aromatic residues, Phe and Tyr, abolishes the capacity of FAS to reduce
acylation rates, whereas introduction of Ala at this position results
in bound FAS causing a slight increase of acylation rates for all
substrates. Thus, unlike the other residues in the choline binding site (i.e. Tyr
) and acyl pocket (i.e. Phe
, Phe
)(6) , the indole ring
of Trp
is linked to the mechanism of FAS inhibition. The
aromatic substitutions, however, slightly enhance the increase in K
induced by FAS for the two most
effective substrates ATCh and PA. This suggests that an aromatic
residue at position 86 influences k
or k
in the FAS
AChE complex. For
catalytic hydrolysis of both ATCh and PA, k
is likely to be increased by FAS, while we might also expect a
reduction of k
for the charged substrate ATCh.
Catalytic hydrolysis of pNPA is far slower (Table 3),
and it has been suggested, based on solvent isotope effects on
acylation rates, that it is rate limited by the chemical acylation
step(21, 22) .
Introduction of an aliphatic residue
at position 86 exerts a large reduction in the catalytic rates for both
fast substrates PhAc and ATCh. With the less efficient catalysis, the
influence of FAS is diminished so that the FAS complexes of these
mutant enzymes have catalytic constants which approach those of
wild-type enzymeFAS complex. The mild acceleration in catalytic
rate for the Trp
Ala AChE
FAS complex is likely to be
a consequence of an increase in the acylation rate constant k
, indicating an enhanced capacity to stabilize
acylation transition state upon FAS binding. This conclusion is
supported by the observation that the binding of FAS increases the
affinity of the Trp
Ala mutant for both charged and neutral
trifluoroacetophenones, substrate transition state analogs. Hydrolysis
of ATCh by the Trp to Ala mutant enzyme is enhanced upon binding of FAS
primarily through enhancing k
, suggesting an
improved fit of the ATCh transition state in the active center gorge.
Ordentlich and colleagues (23) previously examined the
TrpAla mutation on rates of catalytic hydrolysis of ATCh, pNPA, and other carboxyl esters. They also observed a dramatic
reduction on ATCh hydrolysis with the Trp
Ala mutation and
smaller effects on other substrates. They suggested that Trp
is not involved in the stabilization of uncharged substrates.
However, the Trp
to Ala mutation accompanies a large
volume change which must be accommodated by either collapse of the
peptide backbone or entry of several water molecules into the gorge.
Analysis of the kinetics through stepwise replacement of Trp by Tyr,
Phe, and then Ala suggests that subtle changes in alignment of the
associated carboxyl ester are sufficient to dramatically decrease
catalysis. Moreover, occupation of the peripheral site by FAS may
affect the alignment of the substrate through an allosteric influence
mediated between the lip of the gorge and its base. Support for this
linkage comes from previous studies of AChE structure involving
circular dichroism, fluorescence spectroscopy, and site-specific
mutagenesis when propidium is bound to the peripheral
site(24, 25, 26) . Efficient catalysis
requires an optimal alignment of substrate, and FAS exhibits its most
dramatic effects on inhibition parameters for the fast substrate. When
alignment is compromised through residue substitution at position 86,
FAS has a far smaller effect on catalysis and can, in some cases,
slightly increase k
/K
.
Since both
Trp mutations and FAS affect substrate inhibition
parameters, the binding of a second substrate molecule at the FAS site
may also mediate its effect on catalysis through
Trp
(6, 27) . Although FAS binding and
Trp
substitutions occur at two different locations, they
both influence the alignment of residues in the site of ATCh catalysis
and prevent the most productive binding orientation of substrate.
Substrate activation of the residual activity in the FAS-AChE complex (Fig. 2B), however, may be a consequence of ATCh
interaction with still another site on the enzyme.
The effect of
Trp substitutions on acylation by the organophosphates
resembles that seen for the carboxylic acid esters. The substrate pairs
of ATCh and of echothiophate and p-nitrophenyl acetate and
paraoxon share identical leaving groups, and their reaction rates show
similar sensitivities to Trp
substitution. This
underscores the importance of residue 86 in affecting the position of
the leaving group. However, FAS is a less effective inhibitor of
echothiophate acylation in the wild-type enzyme by at least two orders
of magnitude. Compared to ATCh, the thiocholine leaving group in
echothiophate is directed slightly farther away from Trp
and closer to the anionic residue of Asp
, due to the
tetrahedral geometry around the phosphorus in echothiophate (Fig. 3). (
)The difference in the leaving group
orientations in the transition state complexes with FAS-AChE may cause
Trp
substitutions to affect acylation rates in markedly
different manners since stabilizing forces contributed by Trp
decrease with the sixth power of distance(29) . This
conclusion is supported by the significantly greater acceleration of
echothiophate inhibition in Trp
Ala
FAS complex as
compared to the mild acceleration of ATCh hydrolysis.
Figure 3: Presumed orientation of the transition states for acylation of AChE by ATCh (black) and echothiophate (gray) obtained by molecular modeling of the acylation transition state for the reaction of the two compounds with AChE. Molecular dynamics modeling was performed as described in (28) .
The geometry
of the planar trifluoroacetophenones and the positioning of the
carbonyl oxygen toward the oxyanion hole should direct quaternary
ammonium or tertbutyl groups to a position equivalent to that of
choline in ATCh, in close apposition to Trp as confirmed
by crystal structure of the charged TFK
AChE complex(17) .
These tetrahedral adducts resemble the acylation transition state
analogues of ATCh hydrolysis. That FAS affects k
/K
for substrates
and K
for trifluoroacetophenones to
similar extents suggests that the major mode of FAS action is to affect
stabilization of the transition state for hydrolysis of substrates.
Correlations of the free energy of association of the TFKs with AChE
in the absence and presence of FAS with molar refractivities and
hydrophobicities of residues at position 86 (Fig. 4) support
such a mechanism. In the absence of FAS, the pK values for both TFKs increase with both molar refractivity
and hydrophobicity constants for the residues at position 86 indicating
that this residue is directly involved in stabilization of the
TFK
enzyme complex. In the presence of FAS, the contributions to
the energy of stabilization of the aromatic residues at position 86
remain unchanged and roughly equivalent to that of Ala. Hence FAS
association at a distant location on AChE appears to negate the
stabilization energy conferred by the electron-rich indole ring,
perhaps by allosterically influencing its alignment.
Figure 4:
Relationship between the inhibition
constant of trifluoromethyl acetophenones (TFK and TFK
) and the molar refractivity and
hydrophobicity of the side chain at residue 86 in the AChE choline
subsite. Molar refractivity and hydrophobicity indexes for amino acid
residues were taken from (30) and analyzed according to Nair et al.(29) . Measurements were made in the presence
and in the absence of 40 nM FAS following incubation with FAS
for at least 1 h.
Recently, it
was suggested that products of acetylcholine hydrolysis or perhaps
solvent molecules required for catalysis could exchange with bulk
solvent through an opening in the AChE gorge wall behind
Trp, termed the ``back
door''(31, 32) . A vectoral movement of substrate
and products would pose additional energetic requirements for acylation
and deacylation in catalysis. Mutagenesis of residues in the region of
the putative back door region has not yielded supportive evidence for
this hypothesis(33) , nor is there substantive evidence for
choline dissociation being a rate limitation. One might assume that
diminished FAS inhibition, arising from the smaller side chain at
position 86, might arise from increasing the opening at the putative
back door thereby increasing the rate of exchange of ligands between
bulk solvent and the active center gorge. The catalytic constants may,
however, point to the opposite conclusion. FAS most effectively blocks
acylation of rapidly reacting substrates and the most rapid catalysis
is achieved with an indole residue at position 86. Also, rates of
dissociation of positive and neutral trifluoroacetophenones increase
with small side chains at position 86. The binding of FAS further
increases these dissociation rates, whereas one might expect a decrease
if it directly or indirectly closed the back door.
While the
three-dimensional structure of AChEFAS complex is still unknown,
one may speculate on a possible sequence of events associated with FAS
binding and enzyme inhibition. Identification of interacting residues
on AChE through site-specific mutagenesis(6) , combined with an
computational analysis of the interacting forces between FAS and
AChE(34) , suggests that a likely area of their interaction is
the most amino-terminal disulfide loop in AChE encompassing residues
69-96 (Fig. 5). This loop covers the active center gorge
as a ``lid'' with Trp
residing at its tip.
Tyr
, one of the aromatic residues that form the peripheral
site, is located at the base of the loop, while the adjacent residues
Tyr
and Trp
, which also contribute to the
peripheral site, reside on the other two AChE loops. Binding of a
ligand to the peripheral site could therefore serve to partly close the
lid. A conformational change of a homologous loop in a lipase from Candida rugosa was shown from the crystal structure to be
essential for catalysis(35) , wherein an open site is found in
the active state. While there are no indications from crystallography
of mobility of the homologous loop in AChE(36) , flexibility of
such a loop may be required for efficient catalysis. Substitutions of
two aspartates at positions 92 and 93 in Torpedo californica AChE (equivalent to Asp
and Asp
in mouse
AChE) with neutral residues at the base of this loop yield inactive
enzyme (37) and are expected to break at least two salt
bridges.
Figure 5:
Stereo ribbon diagram of a model of the
fasciculinAChE complex(34) . The first disulfide AChE
loop (Cys
-Cys
) is represented by a bold
ribbon. Side chains of Trp
, active Ser
,
and peripheral site residues Tyr
, Tyr
, and
Trp
are displayed and labeled with their numbers. FAS
represented by the gray ribbon is sitting atop the disulfide
loop, while interacting with peripheral site
residues.
Hence FAS inhibition may arise from an influence on two
steps in the catalytic process. For cationic ligands where catalysis is
efficient, FAS may serve to partially gate their entry therein
diminishing k in . It also exerts an
allosteric influence on the alignment of all substrates in the active
center gorge. This occurs in the transition state where it apparently
affects the configuration of the leaving group in achieving productive
acylation. Capping a potentially mobile loop which extends between the
lip of the gorge, containing the peripheral site, and its base, where
Trp
resides, represents an attractive structural basis for
an allosteric linkage. This may serve to decrease the stability of the
initial complexes in the active center and/or diminish the commitment
to catalysis, represented by k
/k
(22) .