(Received for publication, January 26, 1995; and in revised form, May 18, 1995)
From the
The acetylcholinesterase active site consists of a gorge 20
Å deep that is lined with aromatic residues. A serine residue
near the base of the gorge defines an acylation site where an
acyl enzyme intermediate is formed during the hydrolysis of ester
substrates. Residues near the entrance to the gorge comprise a peripheral site where inhibitors like propidium and fasciculin
2, a snake neurotoxin, bind and interfere with catalysis. We report
here the association and dissociation rate constants for fasciculin 2
interaction with the human enzyme in the presence of ligands that bind
to either the peripheral site or the acylation site. These kinetic data
confirmed that propidium is strictly competitive with fasciculin 2 for
binding to the peripheral site. In contrast, edrophonium, N-methylacridinium, and butyrylthiocholine bound to the
acylation site and formed ternary complexes with the fasciculin 2-bound
enzyme in which their affinities were reduced by about an order of
magnitude from their affinities in the free enzyme. Steady state
analysis of the inhibition of substrate hydrolysis by fasciculin 2
revealed that the ternary complexes had residual activity. For
acetylthiocholine and phenyl acetate, saturating amounts of the toxin
reduced the first-order rate constant k to
0.5-2% and the second-order rate constant k
/K
to 0.2-2% of
their values with the uninhibited enzyme. To address whether fasciculin
2 inhibition primarily involved steric blockade of the active site or
conformational interaction with the acylation site, deuterium oxide
isotope effects on these kinetic parameters were measured. The isotope
effect on k
/K
increased
for both substrates when fasciculin 2 was bound to the enzyme,
indicating that fasciculin 2 acts predominantly by altering the
conformation of the active site in the ternary complex so that steps
involving proton transfer during enzyme acylation are slowed.
Acetylcholinesterase (EC 3.1.1.7, AChE) ()hydrolyzes
its physiological substrate acetylcholine at a very high catalytic rate
(Rosenberry, 1975a, 1979). The unique features of AChE structure that
determine its catalytic power have been pursued for many years, and the
recent determination of the three-dimensional structure of torpedo AChE
(Sussman et al., 1991) offers opportunities for new insights.
A few key features of the AChE structure are diagrammed in Fig. 1. The active site is a narrow gorge lined with aromatic
residues that is about 20 Å deep and penetrates nearly to the
center of the 70-kDa catalytic subunit. At the base of the gorge is the
Ser
residue (torpedo sequence numbering) that is acylated
and deacylated during substrate turnover and His
and
Glu
residues that together with Ser
participate in a catalytic triad similar to those found in other
serine proteases and esterases (Glu-His-Ser). Because acetylcholine
occupies this site as acyl transfer is initiated, we shall refer to it
as the acylation site. Certain cationic inhibitors bind
selectively to this site. For example, crystal structure analysis shows
that edrophonium is bound with its hydroxyl group making hydrogen bonds
to both the N
atom of His
and the
O
atom of Ser
(Sussman et al.,
1992). Our earlier kinetic studies on eel AChE showed that the active
site has a high net negative charge that can electrostatically attract
cationic substrates and inhibitors (Nolte et al., 1980), and
molecular modeling calculations (Ripoll et al., 1993) from the
three-dimensional structure suggest that a number of negatively charged
residues in or near the active site gorge can provide such
electrostatic attraction. The three-dimensional structure confirms
several features of the active site that were inferred from kinetic
investigations of AChE catalysis. Early kinetic studies emphasized that
the enzyme active site is composed of several ``subsites.''
Anionic and esteratic subsites were proposed to accommodate the two
ends of the acetylcholine molecule (Nachmansohn and Wilson, 1951).
Model building of acetylcholine in the AChE active site (Sussman et
al., 1991) reveals the substrate trimethylammonium group in the
anionic subsite to be adjacent to Trp
, while in the
esteratic subsite the substrate carbonyl carbon is positioned to make a
tetrahedral bond with the O
of Ser
and
the acetoxy methyl group appears clamped in an ``acyl
pocket'' formed by Phe
and Phe
.
Site-directed mutagenesis has confirmed the importance of these 2
residues in the acyl pocket (Vellom et al., 1993; Ordentlich et al., 1993).
Figure 1: Schematic diagram of the sites for ligand binding in AChE.
The peripheral site was first
defined by cationic inhibitors like propidium that do not compete with
edrophonium in binding to the active site (Taylor and Lappi, 1975).
Studies involving affinity labeling (Weise et al., 1990;
Schalk et al., 1992) and site-specific mutants (Radic et
al., 1993; Barak et al., 1994) indicate that Trp on the rim of the active site gorge is a key component of the
peripheral site and that residues from at least two other polypeptide
loops at the gorge rim also contribute. Bisquaternary ligands like
decamethonium or BW284C51 compete with both propidium and edrophonium
for enzyme binding, and site-specific mutants indicate that these
ligands bridge the 12-15 Å distance between the acylation
and peripheral sites. Recently the fasciculins, a family of very
similar snake venom neurotoxins from mambas (genus Dendroaspis), have emerged as new probes of the AChE active
site. These 61-amino-acid polypeptides have three-dimensional
structures that are very similar to those of the short
-neurotoxins with four disulfide bonds that bind to nicotinic
acetylcholine receptors (le Du et al., 1992). They inhibit
AChE at subnanomolar concentrations with an apparent noncompetitive
inhibition pattern, and fasciculins and propidium interfere with each
other in binding to AChE (Karlsson et al., 1984; Marchot et al., 1993). Furthermore, site-specific mutants reveal that
residues at the gorge rim, particularly Trp
, are
essential for high affinity fasciculin binding (Radic et al.,
1994). These features suggest that fasciculins bind to the same
peripheral site on the gorge rim defined by propidium. We confirm this
point here by analyzing the kinetics of fasciculin 2 binding, and we
investigate the mechanism by which this binding alters AChE-catalyzed
substrate hydrolysis.
In , I binding to Eand EF is assumed to reach equilibrium instantaneously with
dissociation constants K and K
, respectively. Binding of F to Eand EI is much slower and occurs with
association rate constants k
and k
and dissociation rate constants k
and k
, respectively. Equilibrium dissociation
constants K
= k
/k
and K
= k
/k
. According to , the observed pseudo-first-order rate constant k for the approach to equilibrium is given by .
According to a and b, if F and I are competitive (i.e.the ternary complex EFI does not form),
then k=k
/(1+[I]/K
)
and k
= k
. If
F and I are noncompetitive, then at saturating concentrations of I, k
= k
and k
= k
.
Assay points v from association and dissociation reactions were fitted by the nonlinear regression analysis program Fig.P (BioSoft, version 6.0) to .
In , k is the observed pseudo-first-order
rate constant for the approach to equilibrium as given in and v and v
are the calculated values of v at time 0 and at
equilibrium, respectively.
In , the equilibrium dissociation constants K = k
/k
, where k
and k
are the respective
association and dissociation rate constants. It is also assumed that
substrate concentrations are low enough that ESS or EAS species leading to substrate inhibition are
negligible (see Rosenberry, 1975a). In the absence of fasciculin 2, the
steady state hydrolysis rate v = -d [S]/dt is given by the Michaelis-Menten
expression in .
In , V is given by k
[E]
, where k
= k
k
/(k
+ k
) and [E]
is the total concentration of all enzyme species. K
corresponds to K
k
/k
,
where K
= (k
+ k
)/k
. However,
as noted under the ``Appendix,'' formulation of an
appropriate equation for v when fasciculin 2 is present in is complicated by the fact that the general steady state
solution for this model is too complex for useful comparison to
experimental data. The conventional response to this problem has been
to assume virtual equilibrium of all ligand complexes in (e.g. [ES] =
[E] [S]/K
) by
requiring that k
k
; k
` ak
; k
k
and k
ak
; k
k
and k
bk
(see Bernhard, 1968). When
[S] and [F] are constant, the reciprocal of v is then given by (Krupka and Laidler, 1961; Barnett
and Rosenberry, 1977).
In , V and k
are unchanged from , but K
now is given by K
k
/k
.
Quantitative information about individual equilibrium constants is
obtained by first conducting reciprocal plots of 1/vversus 1/[S] at fixed inhibitor concentration
[F]. predicts that these plots will be linear,
and this is widely observed for a variety of inhibitors of AChE as long
as [S] remains low enough to avoid substrate inhibition (see ). Slopes of these plots are calculated by linear
regression analyses with appropriate weighting (here it is assumed that v has constant percent error), and a replot of these slopes
against [F] corresponds to .
Nonlinear regression analysis of (with slope
values weighted by the reciprocal of their variance) provides estimates
of K and the parameter
= aK
/K
. This approach has
been attractive, because and can account for
most reported experimental data on AChE. However, the inequalities
inherent in the virtual equilibrium assumption (noted above ) clearly are violated for a high affinity inhibitor like
fasciculin 2 with the small dissociation rate constants shown under
``Results,'' and no longer is necessarily valid.
A more appropriate expression for v in this situation is
outlined under ``Appendix,'' and conditions are identified
under which is valid.
Acetylthiocholine hydrolysis rates were determined by two methods.
1) After 30-min pre-equilibration, AChE (2 nM) and fasciculin
2 (500 nM) were mixed with acetylthiocholine iodide
(0.1-5 mM) and DTNB (0.10 mM) in 1.0 ml of pH 8
buffer (NaHPO
and Na
HPO
adjusted to 100 mM phosphate and pH 8.0, 0.02% Triton
X-100) and monitored as for butyrylthiocholine above. V
` and K
` (primes
indicate saturation with fasciculin 2; see ) were then
obtained by weighted linear regression analysis of the reciprocal plot
corresponding to . To compensate for substrate inhibition
in the absence of fasciculin 2 at these acetylthiocholine
concentrations, V
and K
were obtained by nonlinear regression analysis of ,
where K
is the substrate inhibition constant
(Hodge et al., 1992).
2) For better precision in comparing rates in HO and
deuterium oxide (D
O), v was measured at 0.5 mM acetylthiocholine and V
/K
or
V`/K
` was determined as
the constant j from the integrated form of at low
initial [S] = [S]
(12
µM, < 0.2 K
) as shown in .
To adjust j to 0.1 m
,
[E]
was maintained
15 nM in the presence of 500 nM fasciculin 2 and
30
pM in its absence. V
or V
` was then calculated from or , respectively.
Phenyl acetate hydrolysis rates were measured with pre-equilibrated
AChE (3-15 nM) and fasciculin 2 (500 nM) in
phenyl acetate (0.1-5 mM, 1% methanol final) or with
60-300 pM AChE in the absence of fasciculin 2 in 1.0 ml
of pH 8 buffer. Phenyl acetate reaction was monitored directly at 270
nm for 1-5 min (
A
= 1.40
mM
cm
; Rosenberry,
1975b). To improve the precision of isotope effect measurements,
multiple data sets were collected, K
and K
` values determined from were
averaged, and the data sets were subjected to a second cycle of
regression analysis with these average values fixed.
Reactions in DO were conducted by identical procedures
except that the pH 8 buffer was adjusted to pH 8.1.(
)AChE
dilutions into D
O were paired with dilutions into
H
O to maximize precision in comparing rates.
Figure 2:
Titration of AChE with fasciculin 2.
Identical AChE samples (active site concentration =
[E]) were incubated with the indicated
total concentrations of fasciculin 2 ([F]
) in
pH 7 buffer for 1 h, and small aliquots of acetylthiocholine and DTNB
were added for assay as described under ``Experimental
Procedures.'' Observed v were normalized to v
obtained in the absence of fasciculin
2 and fitted to the equation v/v
= (1
- R)(1 - [EF]/[E]
)+
Rwhere [EF] = [F]
when [F]
<
[E]
(-), [EF]
= [E]
when
[F]
> [E]
(-
- -), and R is the assay activity of the EF complex
relative to that of E. The lines intersect at
[E]
=
[F]
, corresponding to [F]
= 0.87 nM. This estimate is in reasonable
agreement with an [E]
of 0.64 nM calculated directly from v
.
Figure 3:
Kinetics of
fasciculin 2 reaction with AChE. Panel A, the association
reaction () was initiated by mixing fasciculin 2 and AChE, and
the dissociation reaction (
) was initiated by diluting these
components, to final concentrations of 36 and 4 pM,
respectively. At the indicated times, aliquots were taken for assay
with acetylthiocholine as outlined under ``Experimental
Procedures.'' Assay points were fitted by unweighted nonlinear
regression analysis to the exponential curve in to give
pseudo-first-order rate constants k of 0.073 ± 0.005
m
for the association reaction and 0.07 ±
0.02 m
for the dissociation reaction. Assay points
are shown after normalization to a control AChE activity (v
= 0.0178
A
/min) without fasciculin 2. Panel
B, values of k obtained from 11 association reactions
(
) and 1 dissociation reaction (
) as shown in panel A were plotted against the fasciculin 2 concentration according to . Points were weighted by the reciprocal of the observed
variance of the k values and fit to a linear plot with slope k
= k
and intercept k
= k
. Values
of these constants are given in Table 1.
Figure 4:
Kinetics of fasciculin 2 reaction with
AChE in the presence of propidium or edrophonium. Panel A,
values of k in the presence of 50 µM propidium
were determined as described in Fig. 3A and analyzed as
in Fig. 3B. Panel B, reciprocals of k obtained from the slopes of the plots in Fig. 3B and 4A and an additional plot at 20
µM propidium (data not shown) according to were plotted against the propidium concentration and
analyzed by a. The steep linear slope corresponded to K
= 0.31 ± 0.02 µM and
indicated that k
= 0. Panel C,
values of k in the presence of 10 µM edrophonium
were analyzed as in A. Panel D, reciprocals of k
obtained from the slopes of plots in Fig. 3B, 4C, and an additional plot at 5
µM edrophonium (data not shown) were analyzed as in B. The lack of dependence on edrophonium concentration
indicated that k
= k
.
In Panels A and C, the open and closed
symbols represent association and dissociation reactions,
respectively, as in Fig. 3B, and the dotted line corresponds to the plot without inhibitors in Fig. 3B. K
values obtained from
steady state inhibition of acetylthiocholine hydrolysis were 0.22
± 0.06 µM for edrophonium and 0.6 ± 0.2
µM for propidium.
In contrast to the
observations with propidium, saturating concentrations of edrophonium
had almost no effect on k for fasciculin 2 (Fig. 4, C and D and Table 1). This
indicates that the rate constant for fasciculin 2 binding to its
peripheral site is not significantly altered when edrophonium is bound
to the acylation site at the bottom of the active site gorge. However, k
for fasciculin 2 dissociation from the ternary
complex EFI with edrophonium and AChE did significantly
increase by 4-5-fold. The increase in k
suggests a modest conformational interaction between the sites in
the ternary complex.
Figure 5:
Kinetics of fasciculin 2 reaction with
AChE in the presence of butyrylthiocholine. Panel A,
measurements of k in the presence of 2 mM
butyrylthiocholine were analyzed as in Fig. 4A. The open and closed symbols represent association and
dissociation reactions, respectively, as in Fig. 3B,
and the dotted line corresponds to the plot without inhibitors
in Fig. 3B. Panel B, steady state inhibition
of butyrylthiocholine hydrolysis by fasciculin 2. Reciprocal plots of
1/vversus 1/[S] at fixed concentrations of
fasciculin 2 and one-tenth that concentration of AChE were analyzed by
weighted linear regression analyses as outlined under
``Experimental Procedures.'' Slopes and intercepts of these
plots were normalized to the slope and intercept of a paired control
data set obtained with the same AChE concentration but in the absence
of fasciculin 2. The slopes () were fit to by
weighted nonlinear regression analysis (solid line) to give
estimates of K
= 11 ± 2 pM and
= 0.030 ± 0.002. The intercepts (
)
were fit to an equation of the same form as (dotted
line) to establish a maximum increase of 5.6 ± 0.7 at
saturating concentrations of fasciculin 2. K
for butyrylthiocholine (assumed equal to K
in ) was 86 ± 5
µM.
Interpretation of
is complicated for a number of reasons. First, it includes
contributions from both the fasciculin 2 affinity in the ternary
complex (K
) and the relative acylation rate
constant in the ternary complex (a). Second, an estimate of a can be made only from k
` (a), and this parameter also includes potential
contributions from bk
, the deacylation
rate for the butyrylenzyme when fasiculin is bound. Third,
butyrylthiocholine is a relatively poor AChE substrate, and torpedo and
eel AChEs catalyze its hydrolysis with active site residues different
from those utilized with better substrates (Selwood et al.,
1993). However, a can be estimated if it is assumed that
acylation is slower than deacylation for butyrylthiocholine (i.e.k
< k
and ak
< bk
in ). No data directly support these assumptions,
but analysis of another poor AChE substrate (N-methyl-(7-dimethylcarbamoxy)quinolinium iodide) indicates
that fasciculin 2 has a much greater effect on acylation than
deacylation. (
)With this assumption, the relative increase
in reciprocal plot intercepts, also shown in Fig. 5B,
plateaus at a value of 1/a. Inserting values of K
and a from Fig. 5B into
the expression for
() leads to an estimate of 60
pM for K
(Table 1). K
should be identical to K
which was estimated to be 147 pM from k
/k
for butyrylthiocholine
in Table 1. The agreement within about a factor of two for these
independent estimates is reasonable given the accuracy of the data and
the assumptions used to estimate a.
As noted in Table 1, the ratio of k to k
in the absence of inhibitors is a measure of
the affinity of fasciculin 2 for the free enzyme. With saturating
concentrations of noncompetitive inhibitors, this ratio reflects the
affinity of fasciculin 2 in the ternary complex. From the data in Table 1, it is apparent that edrophonium and butyrylthiocholine
decreased the affinity of fasciculin 2 in the ternary complex
6-14-fold relative to the fasciculin 2 affinity for free AChE.
Thermodynamic considerations dictate that fasciculin 2 likewise
decrease the affinities of these ligands in the ternary complex by the
same amount. To investigate ligand affinity in the ternary complex by a
completely different technique, we conducted fluorescence titrations of
AChE with N-methylacridinium in the presence or absence of
saturating amounts of fasciculin 2. N-Methylacridinium binds
to the AChE acylation site and is noncompetitive with propidium (Taylor
and Lappi, 1975). In the ternary complex with fasciculin 2, N-methylacridinium affinity for AChE decreased about 13-fold (Table 1), a decrease comparable to that calculated from the
kinetic data for edrophonium and butyrylthiocholine.
Figure 6:
Steady state inhibition of AChE-catalyzed
hydrolysis of acetylthiocholine (panel A) and phenyl acetate (panel B) by a saturating amount of fasciculin 2. Hydrolysis
rates v (A/min) were obtained at pH 8 with
(
) or without (
) 500 nM fasciculin 2 as outlined
under ``Experimental Procedures.'' Total AChE concentrations
for the four data sets ranged from 20 pM to 3 nM, and v was normalized to 2 nM AChE for these graphs. The
data set for acetylthiocholine without fasciculin 2 was fit to by nonlinear regression analysis to obtain V
, K
= 78 ±
5 µM, and K
= 15 ± 2
mM. For the other three data sets, reciprocal plots of (1/vversus 1/[S]) were
analyzed by weighted linear regression analysis to obtain V
and K
or V
` and K
` (). Data are displayed as reciprocal plots with
calculated lines. Analysis of six similar data sets for phenyl acetate
in the absence of fasciculin 2 gave a mean K
= 1.74 ± 0.14 mM. Because of apparent
substrate activation in the presence of fasciculin 2 in panel
A, the three points at the highest [S] were deleted from
the linear regression analysis.
The kinetic analyses of fasciculin 2 binding presented here
indicate that this toxin and propidium bind to the same peripheral site
on the rim of the AChE active site gorge. The mechanism by which
fasciculin 2 binding to this site inhibits substrate hydrolysis by AChE
is an important focus of this paper. However, several precautions must
be taken for correct analysis of steady state substrate hydrolysis in
the presence of this inhibitor. 1) As in any measurement of K by steady state kinetics, accurate
determinations of K
for fasciculin 2 binding
require that incubation times be long enough to achieve equilibrium and
that fasciculin 2 be sufficiently in excess of AChE to permit the free
fasciculin 2 concentration to be approximated by its total
concentration. 2) Hydrolysis must be slow enough that substrate is not
significantly depleted over the time required for fasciculin 2 binding
to reach equilibrium. 3) A nonconventional solution to the steady state
rate equation must be considered because of the slow approach to
equilibrium fasciculin 2 binding. This point is amplified under
``Experimental Procedures'' and ``Appendix.'' We
chose to investigate the dependence of inhibition on fasciculin 2
concentration only with butyrylthiocholine because of constraints
imposed by points 2 and 3. For this substrate k
is only about 1% of that for acetylthiocholine or phenyl acetate
with human AChE (Gnagey et al., 1987), and the approach to
equilibrium fasciculin 2 binding could be measured without significant
substrate depletion. These measurements revealed that
butyrylthiocholine at concentrations up to 5 mM bound
exclusively to the AChE acylation site. The affinities of
butyrylthiocholine and of edrophonium and N-methylacridinium,
the other acylation site inhibitors employed in this study, in the
ternary complexes with fasciculin 2 and AChE were about an order of
magnitude lower than their affinities for the free enzyme. Steady state
analysis of the inhibition of butyrylthiocholine hydrolysis by
fasciculin 2 supported this conclusion and further revealed that
hydrolysis could still proceed in the ternary complex. Saturating
amounts of fasciculin 2 also failed to block completely the steady
state hydrolysis of acetylthiocholine and phenyl acetate. Values of k
` for the ternary complexes of these substrate
were about 0.5-2% of the k
values for the
corresponding binary enzyme-substrate complexes (Fig. 6). We
took advantage of the residual activity to obtain important mechanistic
information by investigating D
O effects on steady state
kinetic parameters.
The simplest mechanistic explanation for the
relative decreases in k`/K
` and k
` in Fig. 6is that fasciculin 2
inhibits largely noncompetitively by slowing formation of the acyl
enzyme in (i.e.a ≅ 0.01). To
examine more closely the ways in which fasciculin 2 binding could
interfere with substrate hydrolysis, it is useful to expand to consider additional intermediates on the catalytic
pathway as shown in .
makes explicit two additional intermediates, ES and ES
. ES
occurs prior to the general acid base catalysis step and may
involve an induced fit conformational change of the initial ES intermediate, while ES
occurs concomitant
with general acid-base catalysis and, with carboxylic acid esters, is
likely to involve formation of a tetrahedral intermediate. Solvent
isotope effects provide evidence for both of these additional
intermediates and indicate that their formation can be rate limiting
with certain substrates (Rosenberry, 1975b; Rao et al., 1993).
How could ligand binding to the peripheral site influence the reaction
pathway in ? In contrast to the anionic and esteratic
subsites that comprise the acylation site, the peripheral site is not
an obvious feature of AChE that would be predicted by simple
complementarity to acetylcholine. Its position at the rim of the active
site gorge suggests that acetylcholine could bind here transiently
before entering the acylation site, but it is unclear whether
conformational interaction between the two sites is an important part
of this process. An inhibitor bound to the peripheral site could act
sterically to block access of a second ligand to the acylation site,
but it could also alter the enzyme conformation in a way that would
reduce reactivity at the acylation site. To illustrate how these modes
of action can be distinguished with fasciculin 2, it is helpful to
consider explicitly the formulations of the second-order rate constants
for substrate hydrolysis in the presence () and absence () of saturating concentrations of fasciculin 2 as given by a and b.
These second-order rate constants are literally measures of the
rate-limiting step at low substrate concentrations in .
The rate constants k, k
, and k
from are combinations of the more explicit rate constants
involving the first four steps in . One useful
demarcation in analyzing these combinations is the first step in which
general acid base catalysis occurs. Because this step involves proton
transfer, its rate constant invariably shows a D
O isotope
effect (a decrease of 2-3-fold when D
O replaces
H
O as the solvent) and often shows a pH dependence. In and a, this step is represented by k
because it denotes general acid base-catalyzed
release of the alcohol from the ester substrate, although k
may in fact represent a combination of rate
constants from steps III and IV in . Likewise a combines rate constants in steps 1 and II of under the general ligand association and dissociation
constants k
and k
because these steps generally do not involve proton transfer. For
clarity we define a steric blockade of the active site as a
reduction of both k
and k
when fasciculin 2 is bound. In the extreme case of equal percent
reductions in these rate constants, fasciculin 2 binding at the
peripheral site would reduce the rates at which substrate could enter
and exit the active site without changing the equilibrium affinity of
the substrate in the ternary complex. However, steric blockade in
principle could be the sole mechanism of interaction between the sites
even if the substrate affinity did decrease, because the decrease could
be due to a reduction in the AChE negative electrostatic charge in the
binary and ternary complexes. This mechanism, for example, could
account in principle for the decreases in ligand affinities in the
ternary complex shown in Table 1. We define a conformational
interaction between AChE peripheral and active sites as a change
in any rate constant not compatible with a steric blockade when
fasciculin 2 is bound (i.e. in a change in k
or k
or an increase in k
or k
). These
definitions permit fasciculin 2 binding at the peripheral site to have
both steric and conformational effects on the AChE active site, and we
argue that a clear understanding of peripheral site function requires
insight into the relative contributions of these two effects.
It is
difficult to conduct experiments that measure direct steric blockade
due to fasciculin 2 binding. Most ligands that form ternary complexes
by binding to the acylation site equilibrate in less than a second even
when fasciculin 2 is bound, and no data on the rates of such
equilibration are presented here. However, analysis of DO
effects on the second-order rate constants in a and b
provides some insight into whether fasciculin 2 acts primarily through
a steric or a conformational blockade. Key relationships in these
constants are the ratios k
/k
= C and ak
/k
`
= C`, termed the commitments to catalysis (see Quinn,
1987). When C is small, ES is virtually in
equilibrium with Eand S, and k
is rate limiting for enzyme acylation. Conversely, when C is large, k
/K
= k
and the bimolecular reaction of Ewith S is rate limiting for acylation.
Saturation of the peripheral site with fasciculin 2 drastically
decreased k
` for both substrates in Fig. 6. This decrease suggests that fasciculin 2 binding has an
effect on enzyme conformation, but it does not indicate which step in
the catalytic pathway is affected. According to a, a small
value for C implies that k
/K
will include the k
term and show a D
O isotope effect,
typically in the range of 2.5. This is the usual case for substrate
hydrolysis by most enzymes. For AChE, however, k
/K
values for
acetylthiocholine, phenyl acetate, and certain other substrates show
only a small change in D
O (see Table 2), and this has
led to the conclusion that k
or some other step
prior to general acid base catalysis is rate limiting in these cases
(Rosenberry, 1975b; Quinn, 1987). The D
O isotope effects in Table 2provided a test of whether the decreased values of k
` and k
`/K
` in the complex of
fasciculin 2 with AChE had a predominantly steric or a conformational
basis. If the decreases resulted primarily from steric blockade, one
would expect C` to increase and the D
O isotope
effect on k
`/K
` to become
smaller than that on k
/K
.
Alternatively, if the decreased rates resulted primarily from a
conformational interaction, C` should decrease and the
D
O isotope effect on k
`/K
` should become
larger. Table 2shows that the D
O isotope effects on
this parameter for both substrates increased with fasciculin 2 binding,
indicating that fasciculin 2 acts predominantly to alter the
conformation of the active site in the ternary complex so that steps
involving proton transfer during enzyme acylation are slowed.
Fasciculin 2 binding also increased the DO isotope
effect on k
for acetylthiocholine in Table 2, suggesting greater involvement of proton transfer in the
transition state for acetylthiocholine cleavage in the ternary complex.
However, interpretation of this parameter is more complicated for
reasons noted under ``Results,'' and we do not attempt
further mechanistic conclusions. The D
O isotope effect on k
` for phenyl acetate was less clear because of
a larger error resulting from variation among AChE preparations. Three
different AChE preparations were used to obtain the data in Table 2, and these preparations showed 2-3-fold differences
in the maximal decreases in k
observed with
saturating fasciculin 2 for both substrates. These preparations showed
no differences in D
O isotope effects in the absence of
fasciculin 2 and only slight differences in its presence except for k
` for phenyl acetate.
Fig. 6indicates that the decrease in k` relative to k
was
substantial and of the same magnitude (about 100-fold) for both the
cationic substrate acetylthiocholine and the neutral substrate phenyl
acetate. This observation contrasts sharply with the effects of
saturating concentrations of other peripheral site ligands. Saturation
of AChE with propidium resulted in much larger decreases in k
for acetylthiocholine than for the neutral
substrate 7-acetoxy-4-methylcoumarin (Berman and Leonard, 1990) or for
phenyl acetate. (
)Pt(terpyridine)Cl forms a covalent complex
in human AChE with a histidine corresponding to residue 280. This
residue is located on the rim of the active site gorge adjacent to
Trp
, a residue noted above to be critical to the
peripheral site for propidium and fasciculin 2. With this covalent
conjugate, k
for acetylcholine was reduced to 9%
of that with the unmodified AChE control, but k
for phenyl acetate was increased to 150% of the control (Haas et al., 1992). Thus it is becoming clear that ligands which
bind competitively to the same peripheral site at the rim of the AChE
active site gorge nevertheless alter reactivity at the acylation site
in somewhat different ways. The conformational basis for these
differences remains to be explored.
The general solution for the steady state velocity v from involves many more parameters than the equilibrium solution in . Since most steady state inhibition data for AChE can be fitted to , it is useful to arrange the general solution in a form that resembles this equation. To condense these solutions, the key variables are normalized as follows:
In , for example, the reciprocal plot for
[F] = 0 reduces to y = 1 + x, and the plot involving competitive inhibition (I = I
= 0) reduces to y =
1 + x (1 + I
).
The general solution
for the initial steady state hydrolysis rate v from has been derived. ()A key point is to arrange
the solution in a form amenable to instructive simplifications, as the
general solution is too complex to be of practical use. The appropriate
simplification for fasciculin 2 binding to AChE is the case when
inhibitor equilibration is much slower than achievement of the
enzyme-substrate steady state: k
+ k
k
[F]; k
` + ak
k
; k
+ k
k
k
/k
`; k
` + ak
k
[F] k
`/k
; k
k
+ k
[F]; bk
k
+ k
[F]; and bk
k
+ bk
[F]. In this case, the ratios of
[E], [ES], and
[EA] approach those calculated in the virtual
equilibrium case, and the general solution reduces to .
Analyses of a complete range of fasciculin 2 concentrations in
this report is limited to the substrate butyrylthiocholine. The low
value of k for this substrate (about 10
s
; Gnagey et al., 1987) justifies the
approximation that Q ≅ 1. With this assumption, collapses to the identical expression given by the virtual
equilibrium model in and if C
= 0 (k
= k
). Even if C
is allowed to range up
to 0.5 (with Q = 1), computer simulations indicate that is an excellent approximation.
At saturating
concentrations of fasciculin 2 (both I and aQI
1), reduces to the form of the
Michaelis-Menten expression in , where K
is replaced by K
`; V
is replaced by V
` = k
` [E]
; and a and b hold.
This formulation is identical to the virtual equilibrium
expression at high [F] in if K` is substituted for K
`.