Interaction Kinetics of Reversible Inhibitors and Substrates
with Acetylcholinesterase and Its Fasciculin 2 Complex*
Zoran
Radi
and
Palmer
Taylor
From the Department of Pharmacology, University of California San
Diego, La Jolla, California 92093-0636
Received for publication, July 31, 2000, and in revised form, October 13, 2000
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ABSTRACT |
Fasciculin 2 (Fas2), a three-fingered peptide of
61 amino acids, binds tightly to the peripheral site of
acetylcholinesterases (AChE; EC 3.1.1.7), occluding the entry portal
into the active center gorge of the enzyme and inhibiting its catalytic
activity. We investigated the mechanism of Fas2 inhibition by studying
hydrolysis of cationic and neutral substrates and by determining the
kinetics of interaction for fast equilibrating cationic and neutral
reversible inhibitors with the AChE·Fas2 complex and free
AChE. Catalytic parameters, derived by eliminating residual
Fas2-resistant activity, reveal that Fas2 reduces
kcat/Km up to
106-fold for cationic substrates and less than
103-fold for neutral substrates. Rate constants for
association of reversible inhibitors with the active center of the
AChE·Fas2 complex were reduced about 104-fold for both
cationic and neutral inhibitors, while dissociation rate constants were
reduced 102-to 103-fold, compared with AChE
alone. Rates of ligand association with both AChE and AChE·Fas2
complex were dependent on the protonation state of ionizable ligands
but were also markedly reduced by protonation of enzyme residue(s) with
pKa of 6.1-6.2. Linear free energy relationships
between the equilibrium constant and the kinetic constants show that
Fas2, presumably through an allosteric influence, markedly alters the
position of the transition state in the reaction pathway. Since Fas2
complexation introduces an energetic barrier for hydrolysis of
substrates that exceeds that found for association of reversible
ligands, Fas2 influences catalytic parameters by a more complex
mechanism than simple restriction of diffusional entry and exit from
the active center. Conformational flexibility appears critical for
facilitating ligand passage in the narrow active center gorge for both
AChE and the AChE·Fas2 complex.
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INTRODUCTION |
The interaction between acetylcholinesterase (EC 3.1.1.7)
(AChE)1 and fasciculin 2 (Fas2) is a well studied example of a tight binding peptide-protein
complex. The bound "three-fingered" peptide toxin, isolated from
venom of Elapidae snakes, resides at the opening of the AChE
active center gorge, thus sealing off the entry to the gorge and
resulting in virtually complete inhibition of hydrolysis of the
neurotransmitter acetylcholine. Static snapshots of the complex
structure revealed in crystals of mouse AChE·Fas2 (1), Torpedo
californica AChE·Fas2 (2) and human AChE·Fas2 (3) complexes
indicate that the "solvent-accessible" surfaces of two molecules
overlap in a nearly continuous area of more than 1000 Å2
around the entry to the AChE active center gorge. The complex, however,
retains residual catalytic activity whose magnitude was reported to
vary between species, being about 3% in human AChE (4) and about
0.09% in mouse AChE (5), when measured with acetylthiocholine (ATCh).
Inhibition of residual catalytic activity of both complexes by
conjugation of the active center serine by either organophosphate or
trifluoroacetophenone (TFK) inhibitors, showed only a 1-order of
magnitude reduction in reaction rates of cationic inhibitors for the
AChE·Fas2 complex when compared with uncomplexed AChE (5). Rates of
association and dissociation of reversible ligand
N-methylacridinium were found, however, to be reduced by
3-4 orders of magnitude in the AChE·Fas2 complex (6).
Recently, Mallender et al. (7) found that human AChE
contained Fas2-resistant esterase activity in purified preparations. Monitoring the phosphorylation rates of fluorescent organophosphates, which exhibit negligible dephosphorylation of the enzyme, enabled these
investigators to show that reactions of organophosphates with
AChE·Fas2 were 2-3 orders of magnitude slower than those for
uncomplexed AChE, a reduction greater than observed in earlier studies
(4). The predominant residual catalytic activity of the human
AChE·Fas2 complex appeared to be due to contaminating Fas2-resistant
activity (7). Although mouse AChE·Fas2 complex appeared to contain
only 0.09% activity resistant to Fas2 inhibition (5, 8), comparatively
high turnover rates of a fasciculin-resistant enzyme may also
complicate our analysis of ligand entry into the active center gorge.
By employing an irreversible inhibitor to inhibit selectively the
residual Fas2 resistant activity, we demonstrate here that mouse
AChE·Fas2 complex retains only about 0.004% catalytic activity at 1 mM ATCh instead of the previously reported 0.09% (5). Stopped-flow techniques with fluorescent detection enabled us to
determine rates of association and dissociation of several reversible
inhibitors that equilibrate with AChE in a subsecond time frame.
Furthermore, we have developed a competitive binding assay utilizing a
fluorescent carbamate, M7C, to measure binding rates of nonfluorescent
inhibitors to both mouse AChE and the AChE·Fas2 complex. Utilizing
these procedures, we present a quantitative characterization of
interaction of substrates and reversible inhibitors with the mouse
AChE·Fas2 complex. We find that the second order rate constants for
reaction of substrates (kcat/Km) and rate constants for association of reversible ligands are significantly reduced, and the transition state barrier is altered for the
AChE·Fas2 complex. While the rates of ligand association were reduced
around 4 orders of magnitude, catalytic throughput for substrate,
kcat/Km, was decreased between 2 and 6 orders of magnitude, thus enabling us to distinguish the rate-limiting
steps for catalysis by the AChE·Fas2 complex.
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MATERIALS AND METHODS |
Enzyme--
Wild-type mouse AChE was expressed in HEK-293 cells
stably transfected with cDNA encoding a monomeric form of the
enzyme truncated at its carboxyl terminus at position 548. The enzyme
secreted into the medium was purified to homogeneity on affinity
columns as described previously (9).
Inhibitors and Substrates--
Purified Fas2 was kindly provided
as lyophilized material by Dr. Pascale Marchot (CNRS, University of
Marseille, France). Concentrations of Fas2 stock solutions were
determined by absorbance (
276 = 4900 M
1 cm
1)
(20). m-Tertbutyl trifluoroacetophenone (TFK0)
and m-trimethylammonium trifluoroacetophenone
(TFK+) were kindly provided by Dr. Daniel Quinn (University
of Iowa, Iowa City, IA) (21). Reversible inhibitors BW286C51, tacrine, 9-aminoacridine, N-methyl acridinium, propidium, acridine,
and edrophonium were obtained from Sigma. (
)-Huperzine A was obtained from Calbiochem. M7C was obtained from Molecular Probes, Inc. (Eugene,
OR). The chiral organophosphonate enantiomer
SP-3,3-dimethylbutyl methylphosphonothiocholine
was kindly provided by Dr. Harvey Berman (State University of New York
at Buffalo, Buffalo, NY). The substrates, ATCh,
propionylthiocholine iodide (PTCh), butyrylthiocholine iodide (BTCh),
and p-nitrophenyl acetate (pNPAc), were obtained
from Sigma, while thiophenylacetate (S-PhAc) was a product of Aldrich.
Enzyme Activity--
Hydrolysis of ATCh, PTCh, BTCh, S-PhAc, and
pNPAc was measured spectrophotometrically at 412 nm for
released thiocholine (10) or at 405 nm for p-nitrophenol.
Activities of AChE·Fas2 complexes were measured following a 10-min
incubation of 640 nM AChE with 1.3 µM Fas2 to
form the complex and 30-min incubation of the complex with 530 nM SP-3,3-dimethylbutyl
methylphosphonothiocholine to inhibit hydrolytic activity not
originating from the complex. The very slow reaction of the charged
organophosphate with the active center of the Fas2·AChE complex gives
rise to selective inhibition. Kinetic constants for hydrolysis of the
above substrates by AChE and AChE·Fas2 complex were determined as
described previously (5).
Enzyme Inhibition--
The time course of AChE inhibition by
trifluoroacetophenones was monitored by measuring ATCh hydrolysis in
aliquots from the reaction mixture. Typically, 0.96-ml aliquots were
drawn from 10 ml of the reaction mixture and mixed with 30 µl of DTNB
(10 mM) and 10 µl of ATCh (100 mM) at
specified time intervals to stop inhibition and determine residual
enzyme activity. Both the inhibitory reaction and the assessment of
residual activity were conducted in 0.1 M phosphate buffer,
pH 7.0. First order rate constants for inhibition (k) were
obtained by fitting fractional enzyme inhibition to the following
equation,
|
(Eq. 1)
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where f, f0, and
f
are fractions of enzyme inhibited at times
t, t = 0, and t =
,
respectively. Second order inhibition rate constants were derived from
plotting the first order rate constants for inhibition against
inhibitor concentration and extrapolating to infinite dilution.
Stopped-flow Kinetic Assays--
Association and dissociation
rate constants for fast equilibrating fluorescent inhibitors were
determined using a SX.18 MV stopped-flow instrument equipped with
fluorescence detection (Applied Photophysics). Excitation wavelengths
were selected to achieve maximum signal amplitude. Acridine,
9-aminoacridine, and N-methyl acridinium were excited at
wavelengths between 250 and 260 nm, coumarin at 220 nm, and propidium
at 288 nm. For experiments with nonfluorescent inhibitors BW286C51,
edrophonium, and decamethonium, but also for selected fluorescent
ligands, AChE or AChE·Fas2 complex was excited at 276 nm (11).
Quenching of ligand or AChE fluorescence upon complex formation was
monitored using a 305-nm cut-off filter, and for 9-aminoacridine a
335-nm cut-off filter was used. Tacrine association was detected by
excitation at 323 nm, with a 335-nm cut-off filter for emission. Linear
plots of observed first order rate constants versus ligand
concentrations gave second order association rate constants from the
slope of the line and a first order dissociation rate constant from the
ordinate intercept (11, 12).
M7C Reaction Assay--
The time courses of TFK, (
)-huperzine,
and BW286C51 association with AChE and the AChE·Fas2 complex were
followed using N,N-dimethyl carbamoyl
N-methyl-7-hydroxy-quinolinium (M7C). This nonfluorescent dimethyl carbamoyl ester reacts covalently with the AChE active serine
releasing a fluorescent N-methyl-7-quinolinium leaving group
(M7H) (
excitation = 410 nm;
emission = 510 nm; Ref. 13), thus allowing one to monitor both the rate of M7C
reaction with the serine and the number of binding sites. The rate of
binding of nonfluorescent covalent inhibitors to AChEs was followed by monitoring a decrease in number of AChE binding sites available for M7C
reaction resulting from prior covalent reaction with the nonfluorescent
inhibitor. Forty µl of 1.0 mM M7C was added to 0.4-ml
aliquots drawn at different time intervals from 4 ml of reaction
mixture containing buffered solution of an AChE (10-70 nM)
with or without Fas2 (650 nM) and the nonfluorescent
inhibitor. Aliquots were quickly transferred into a spectrofluorometric
cuvette, and the fluorescence increase was followed at
emission = 510 nm (
excitation = 410 nm)
for 20 s in the absence of Fas2 or for 100 s in the presence
of Fas2. The fluorescence signal exponentially approached a maximum
value, which was diminished with increasing times of prior exposure to
the nonfluorescent inhibitor, indicating progress of its reaction. The
first order rate constant of binding of nonfluorescent inhibitors was
determined by fitting the time dependence of relative change in maximal
fluorescence to Equation 1.
Equilibrium Binding Measurements--
Equilibrium dissociation
constants for several fluorescent reversible ligands were determined by
titration with increasing concentrations of AChE or AChE·Fas2
complex, resulting in quenching of ligand fluorescence. Concentrations
of free fluorophores during titrations were determined from
corresponding calibration curves constructed for each fluorophore,
limiting fluorescence quenching in the bound state. Corrections were
made for small changes in reaction volume due to the addition of
titrant. Equilibrium dissociation constants (Kd)
were determined from the reciprocal of the slopes of Scatchard plots.
Determination of Ligand pKaValues--
UV-visible
spectra of fast equilibrating reversible AChE inhibitors with ionizable
functional groups were recorded in buffers of varying pH. Changes of
peak absorbances as a function of buffer pH were analyzed using the
following equations (rearranged from Ref. 14),
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(Eq. 2)
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or
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(Eq. 3)
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where Amin and
Amax are minimal and maximal peak absorbances
and Ka is the equilibrium proton dissociation
constant. The following buffers were used: 0.10 M citric
acid plus 0.20 M sodium phosphate (pH 2.6-7.8) and 0.050 M sodium phosphate plus sodium pyrophosphate (pH 5.0-12).
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RESULTS |
M7C Reaction Assay--
Time courses of the M7C reaction with the
AChE and the AChE·Fas2 complex are shown in Fig.
1. M7C added at a concentration of 100 µM binds to AChE at a rate too fast for measurement by conventional mixing and detection by spectrofluorometry. Hence, the
carbamoylation step is reflected in a rapid burst of fluorescence. The
reaction of M7C with the AChE·Fas2 complex was slower but was
virtually complete within 100 s. The final fluorescence
intensities upon carbamoylation and release of M7H were linearly
dependent on AChE and AChE·Fas2 concentrations (Fig. 1, B
and C).

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Fig. 1.
Monitoring of fluorescence of M7H following
reaction of M7C with AChE (open symbols)
and the AChE·Fas2 complex (filled
symbols). A, time course of reaction
of 100 µM M7C with no (circles), 10 nM (triangles), 20 nM
(squares), 40 nM (diamonds), 60 nM (inverted triangles), 80 nM (hexagons) AChE or AChE·Fas2, respectively.
M7H fluorescence was monitored at 510 nm with excitation at 410 nm.
Shown are plots of increases of maximal fluorescence in the
reaction as a function of concentration of mAChE (B) and
mAChE·Fas2 (C).
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The rapid and stoichiometric reaction of M7C with AChE or the
AChE·Fas2 complex enabled us to develop a fluorescent assay for
monitoring of binding rates for nonfluorescent ligands. The assay is
based on consecutive, rapid M7C titrations of residual catalytically
active AChE during the course of its reaction with nonfluorescent
ligands. The first order rate constants for reaction were determined by
fitting to Equation 1 the fluorescence intensities obtained in
subsequent titrations. Good agreement between rate constants for ligand
reaction measured from the decrement in the M7C reaction and ligand
inhibition of ATCh hydrolysis is evident in Table
I.
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Table I
Second order reaction (kr) and inhibition (ki) rate
constants for covalent inhibitors of mouse wild-type AChE
Comparison of reaction rate constants determined by M7C reaction assay
with inhibition constants determined previously in Refs. 5, 9, and 15
are shown as indicated. All assays were conducted in 0.1 M
phosphate buffer, pH 7.0, at 22 °C. Shown are means or averages from
2-4 experiments. The typical S.E. of determination was 30-40% of the
mean value.
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Hydrolysis of Substrates by AChE and AChE·Fas2
Complex--
Catalytic hydrolysis of three cationic (ATCh, PTCh, and
BTCh) and two neutral (S-PhAc and pNPAc) esters by AChE and
AChE·Fas2 was compared. Substrate concentration dependences of AChE
hydrolysis deviate from Michaelis kinetics at millimolar substrate
concentrations, with ATCh, PTCh, and BTCh inhibiting their own
hydrolysis. The kinetics of hydrolysis is characterized by substrate
inhibition constants (Kss) and relative activities
of enzyme-substrate ternary complexes (b), in addition to
Michaelis constants (Km) and maximum turnover rates
(kcat) (Table II;
Refs. 16 and 17). The three cationic substrates have similar Michaelis
constants (15-18), yet the commitment to catalysis (defined as a ratio
of the enzyme acylation rate constant and dissociation rate constant of
the Michaelis complex (cf. Ref. 19)) and the formation of the tetrahedral transition state, reflected in
kcat, appear severely restricted for BTCh and
other substrates containing a large acyl group (17, 19).
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Table II
The catalytic constants for hydrolysis of cationic and neutral
substrates by mouse AChE and by AChE · Fas2 complex
Michaelis constants (Km) and catalytic constants
(kcat) were determined by nonlinear regression of
the Michaelis equation, except for the constants indicated by an
asterisk where more complex kinetics was observed and the following
equation was used: v = (1 + bs/Kss) kcat/[(1 + s/Kss) (1 + Km/s)], where s is substrate
concentration, Kss is the substrate inhibition or
activation constant, and b is a measure of relative activity
of ternary enzyme-substrate complexes. ATCh, Kss = 15 mM, b = 0.23 (data of Radi
et al. (16)); PTCh, Kss = 1.3 mM, b = 0.21; BTCh, Kss = 7.1 mM, b = 0.48 (data of Hosea et
al. (17). Shown are means from 2-4 experiments. The typical S.E.
of determination was 30-40% of the mean value.
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Hydrolysis of neutral substrates by AChE obeyed Michaelis kinetics
(Table II), but with Km values up to 2 orders of
magnitude larger than for cationic substrates, thus emphasizing the
importance of electrostatic interactions in formation of Michaelis complexes. kcat of S-PhAc was similar to
kcat values for charged substrates and
significantly greater than kcat for
pNPAc. A similar relationship between catalytic constants
for PhAc and pNPAc has been well described for
Electrophorus electricus AChE (20).
Although the AChE·Fas2 complex hydrolyzed all substrates
significantly more slowly than AChE, cationic substrates with high turnover rates were most affected. Hydrolysis of both cationic and
neutral substrates by AChE·Fas2 obeyed Michaelis kinetics. Our
earlier published curve of ATCh hydrolysis by AChE·Fas2 was biphasic,
indicating substrate activation (5). The first phase, due to the
presence of residual, Fas2-resistant, esterase activity, was inhibited
selectively using the organophosphate,
SP-3,3-dimethylbutyl methylphosphonothiocholine. Hence with this correction, the ATCh hydrolysis profile for the Fas2·AChE complex shows typical Michaelis kinetics. kcat/Km values for ATCh and
PTCh were reduced by 5-6 orders of magnitude, whereas
kcat/Km for BTCh was reduced
by only 3 orders of magnitude. Neutral substrates S-PhAc and
pNPAc exhibited much smaller reductions in
kcat/Km (680 and 96 times,
respectively). The reduction in
kcat/Km for the five
substrates by AChE·Fas2 resulted in distinct clusters of values for
charged and neutral substrates. Values of 1.2-4.7 × 103 M
1
s
1 for neutral substrates approach the
kcat/Km value for benzoylthiocholine hydrolysis by AChE (1.1 × 104
M
1 s
1;
Ref. 17).
kcat/Km values for neutral substrates
with the AChE·Fas2 complex were 1-2 orders of magnitude greater than
for the charged substrates, suggesting that Fas2 association may impart a more restrictive barrier in the AChE·Fas2 complex to diffusional entry and acylation by cationic substrates. The reduction in values of
kcat/Km was reflected in both
an increase in Km and reduction in
kcat. Whereas for neutral substrates and free AChE, the large Km values were limiting, the
situation is reversed for the AChE·Fas2 complex, where large
Km values primarily limit hydrolysis of cationic
substrates. kcat values for the AChE·Fas2
complex were significantly reduced for all substrates. In addition,
kcat values for the AChE·Fas2 complex correlate with the size of the acyl moiety in the substrate, suggesting that the steric occlusion by acyl pocket residues also limits substrate
hydrolysis for the AChE·Fas2 complex. Coulombic repulsion from
arginines and lysines of bound Fas2 could also interfere with the
orientation of thiocholine in the transition state and its exit from
the gorge. Both the reversible binding steps and turnover of two
reactive cationic substrates, ATCh and PTCh, appeared to be reduced to
the greatest extent in the AChE·Fas2 complex, suggesting a similar
influence of the acyl pocket on the stability of the Michaelis
complexes and tetrahedral transition states. This was not the case for
neutral substrates, where kcat alone is affected.
Kinetics of Binding of Reversible Ligands to AChE--
The second
order association and the first order dissociation rate constants for
two substituted trifluoroacetophenones and 10 noncovalent reversible
ligands to AChE and AChE·Fas2 complex are listed in Table
III. The constants for slow equilibrating
inhibitors such as the trifluoroacetophenones and (
)-huperzine A were
determined using the M7C reaction assay, whereas for the
faster equilibrating inhibitors,
stopped-flow techniques with fluorescence monitoring were used (Figs.
2 and 3).
Rates of interaction for BW286C51 were analyzed by both techniques. The
association rate constants of the 10 ligands with AChE covered a narrow
range of less than 2 orders of magnitude (4.5 × 107
to 2.8 × 109 M
1
s
1), and only (
)-huperzine A had a
significantly slower association rate constant. Since several fast
equilibrating ligands from Table III bear ionizable groups, their
protonation states at pH 7.0 were analyzed by determining their
pKa values (Table IV). These pKa values indicate that, in addition to
quaternary ligands, tacrine and 9-aminoacridine will be positively
charged at pH 7.0; acridine will be neutral and coumarin exists as
approximately a 3:1 ratio of neutral to anionic species. The fastest
association rate constants were observed for quaternary ammonium
ligands bearing a permanent cation (k1 = 1.3 × 108 to 2.8 × 109
M
1 s
1).
Protonated cationic ligands, tacrine and 9-aminoacridine
(k1 = 1.3 × 108 to 1.5 × 108 M
1
s
1), approached the lower end of the range of
rates for quaternary compounds, while neutral ligands (acridine and
coumarin) had the slowest association rate constants
(k1 = 4.5 × 107 to 1.2 × 108 M
1
s
1). Association rate constants for
peripheral site ligands, propidium and coumarin, exhibited the
same rates as those ligands associating with the active center, despite
the greater surface accessibility of the peripheral site.
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Table III
Kinetic constants for interaction of reversible ligands with mouse AChE
and mouse AChE · Fas2 complex
Second order association rate constants (k1) and
first order dissociation rate constants (k 1) were
determined in 2-5 experiments (cf. Figs. 2 and 3) using
plots shown in Fig. 3. The typical S.E. was 30-40% of the mean value
of constants. The equilibrium dissociation constant
(Kd) was derived as the ratio of
k 1/k1. n.d., not determined due
to slow rates of dissociation (TFK+ and TFK0) or
dissociation rates too rapid for stopped-flow measurements (propidium).
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Fig. 2.
Kinetics of quenching of fluorescence upon
reaction of reversible ligands with AChE. A, quenching
of 9-aminoacridine (Ref. 500 nM) fluorescence in
reaction with AChE (500 nM). 9-Aminoacridine was
excited at 259 nm. B, quenching of intrinsic fluorescence of
AChE (1 µM) in reaction with edrophonium (1.0 µM). AChE fluorescence was excited at 276 nm.
C, quenching of intrinsic fluorescence of the AChE·Fas2
complex (100 nM) in reaction with edrophonium (300 µM). The AChE·Fas2 complex was excited at 276 nm.
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Fig. 3.
Relationship between the rates of reaction
(kobs) of fast equilibrating reversible
ligands with AChE and ligand concentration. Reaction rates were
determined from reaction traces similar to those shown in insets,
generated by fluorescence detection of the reaction components in a
stopped-flow spectrophotometer with fluorescence detection.
Inset, trace of AChE·Fas2 (100 nM)
fluorescence in reaction with decamethonium (200 mM).
A, N-methylacridinium (squares),
acridine (triangles), coumarin (circles),
propidium (black diamonds); B,
edrophonium (black circles), 9-aminoacridine
(black squares), BW286C51 (white
diamonds). Inset, trace of 9-aminoacridine (100 nM) fluorescence in reaction with AChE (10 nM).
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Table IV
pKa values for AChE inhibitors with ionizable functional groups
The values were determined from changes of peak absorbances in
UV-visible spectra of inhibitors as a function of pH in the given range
by nonlinear regression of Equation 2 or 3. Experiments were performed
in duplicate.
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The rate constants for dissociation of the 12 ligands extending over 7 orders of magnitude (1.8 × 10
5 to
4.2 × 102 s
1), spanned a
greater range than the association rate constants. Trifluoroacetophenones were the slowest dissociating ligands. These
slowly reversible ligands interact covalently at the esteratic site and
noncovalently in the choline binding site (12, 21). Comparison of
quaternary, protonated, and neutral ligands binding at the active
center reveals that apart from trifluoroacetophenones and
(
)-huperzine A, cationic bisquaternary BW286C51 and the cationic tertiary amines, tacrine and 9-aminoacridine, had the slowest dissociation rate constants.
Kinetics of Binding of Reversible Ligands to the AChE·Fas2
Complex--
Ligand interactions with the AChE·Fas2 complex reveal
marked reductions in both association and dissociation rate constants, compared with free AChE (Table III). The most affected should be the
peripheral site ligands, propidium and coumarin, whose binding is
competitive with Fas2 and the bisquaternary ligands, decamethonium and
BW286C51, which share a portion of the peripheral binding site with
Fas2. The 8-order of magnitude slower association rate constants of
BW286C51 and decamethonium accompanied with only 1-3-order of
magnitude slower dissociation constants indicate that these
bisquaternary ligands will form ternary complex with mAChE·Fas2.
By contrast, the interaction of coumarin with the AChE·Fas2 complex
was altered in a different manner than for propidium, BW286C51, and
decamethonium. The small reduction in the coumarin association rate
constant is characteristic of active center ligands. Coumarin's
dissociation rate constant was, however, reduced significantly more
than those for the above ligands. Taken together, the comparative kinetics indicate the existence of a secondary binding site for coumarin in the active center gorge of the AChE·Fas2 complex. Unlike
other peripheral site ligands coumarin appears small enough to enter
the active center gorge in AChE. Previous mutagenesis and molecular
modeling studies also suggest coumarin binding to the active center
when the peripheral site is occupied (22-24).
For the eight remaining active center ligands, the association rate
constants with AChE·Fas2 were about 4 orders of magnitude smaller
than the corresponding constants for AChE, irrespective of ligand
charge or size. Their dissociation rate constants were reduced by only
1-3 orders of magnitude. The greater reduction of association than
dissociation rate constants leads to an increase in
Kd for active center ligands in the AChE·Fas2
complex typically by 1 order of magnitude but in one case by 2 orders. The Kd for coumarin, with its presumed occupation of the active center in the AChE·Fas2 complex is actually slightly decreased.
Equilibrium Binding Measurements--
Equilibrium dissociation
constants for several reversible ligands,
determined also by fluorescence titrations (Fig. 4), are listed in
Table V and show good agreement with
corresponding constants derived from the ratios of dissociation to
association reaction rate constants (cf. Table III).

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Fig. 4.
Scatchard plots for fluorescence titrations
of 9-aminoacridine (circles) and tacrine
(triangles) by AChE (closed
symbols) and the AChE·Fas2 complex (open
symbols). Concentration of both ligands was 10 nM in titration with 5-500 nM AChE and was 50 nM (tacrine) or 100 nM (9-aminoacridine) in
titration with 25-3000 nM AChE·Fas2 complex.
Inset, decreases in fluorescent emission spectrum of
9-aminoacridine during titration with increasing concentrations of AChE
and AChE·Fas2 complex. Shown are 10 nM 9-aminoacridine
titrated with concentrations between 0 and 500 nM AChE and
100 nM 9-aminoacridine titrated with
concentrations between 0 and 3000 nM AChE·Fas2
complex.
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Table V
Equilibrium dissociation constants of fluorescent ligands with AChE and
the AChE · Fas2 complex
Constants were determined by titration of ligand fluorescence with AChE
and AChE · Fas2 in 0.10 M phosphate buffer, pH 7.0, as illustrated in Fig. 4. Average or mean values are shown from 2-4
experiments. S.E. of determination were about 40% of the mean. ND, not
determined.
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pH Dependence for Interaction of Reversible Ligands with AChE and
the AChE·Fas2 Complex--
AChE and several reversible ligands from
Table III bear ionizable groups, and their mutual interactions may
depend on pH of the reaction medium. Interaction of a reversible ligand
and enzyme, both bearing ionizable groups, can be described as follows.
It can be shown that for association between ligand and enzyme in
the above scheme, the following equation holds,
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(Eq. 4)
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where k1 and
k1max are second order association
rate constants for ligand and enzyme at a given pH and a maximal rate
constant of the reactive species, respectively. Factor b is
a measure of the difference in the second order rate constants for
reaction between enzyme and protonated versus nonprotonated
forms of ligand. The species EH+ is assumed not to bind
ligand. KaE and
KaL are equilibrium dissociation constants
for a proton from ionizable groups in the enzyme and ligand, respectively.
The pH dependence of reaction rate constants of four ligands with AChE
was studied in the range of pH 4.5 to 11.0 (Table
VI). Association rate constants
(k1) for all ligands decreased at pH values
below 6.0 by 1-3 orders of magnitude; the greatest reduction was
observed for quaternary cation edrophonium. Analysis of dependences of
association rate constants for edrophonium and 9-aminoacridine on pH,
using the model from Scheme 1 and the corresponding Equation 4, is
shown in Fig. 5A. Association
of both ligands in the low pH range depends on ionization of the same
group, presumably in AChE, with a pKaE
value of 6.1-6.2. The AChE group with pKa of 6.2 was implicated as critical for carbamoylation and phosphorylation of
AChE by inhibitors containing quaternary ammonium in the leaving group
(25). The reduction of association constants for edrophonium and
coumarin in the pH range above pH 7.0 is consistent with their deprotonation (cf. Table IV). The asymmetric shape of the pH
dependence curve for edrophonium, reflected in a value of b
larger than 0 (b = 0.14), may suggest that zwitterionic
edrophonium species formed by deprotonation of the hydroxyl group
associates with enzyme with k1max of
1.7 × 107 M
1
s
1, which is about 7-fold slower than
k1max for cationic edrophonium.
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Table VI
Effect of pH on association and dissociation rate constants for
selected reversible ligands and mouse AChE
Reactions were monitored in 0.1 M citrate-0.2 M
phosphate buffers (pH 4.5-7.0), 0.1 M phosphate buffers
(pH 5.5-8.0), and 0.050 M phosphate-pyrophosphate
buffers (pH 6.0-9.0). Values of constants measured in different
buffers of the same pH were similar and were therefore averaged.
Experiments were performed in duplicate.
|
|

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Fig. 5.
The pH dependence for association of
edrophonium ( ), and 9-aminoacridine ( ), with AChE
(panel A) and AChE·Fas2 complex
(panel B). The second order rate
constants of association were fitted to Equation 4 with the
following parameters. A,
k1max = (1.2 ± 0.3) × 108 M 1
s 1, pKaE = 6.2, pKaL = 7.6, b = 0.19 ± 0.03 (for edrophonium) and k1max = (1.4 ± 0.1) × 108
M 1 s 1,
pKaE = 6.1, pKaL = 10.3, b = 0 (for 9-aminoacridine). B,
k1max = (5.2 ± 0.2) × 104 M 1
s 1, pKaE = 6.1, pKaL = 11.9, b = 0 (for
9-aminoacridine).
|
|
On the other hand reduction of association constants of 9-aminoacridine
is reflected in a pKaL value of 10.3. This
value is close to both the pKa value of
9-aminoacridine (10.1) and the pKa value found for carbamoylation and phosphorylation of human AChE (10.25; Ref. 25) and
therefore could be either ligand- or enzyme-related. Minimal variations
of dissociation rate constants of cationic ligands (Table VI) in the pH
interval studied suggests an absence of a pH related conformational
change in the active center gorge. Protonation of acridine slowed the
dissociation rate 10-fold, emphasizing the role of electrostatic
interactions in stabilization of cationic ligands in the AChE active
center gorge.
The pH dependence of 9-aminoacridine association with AChE·Fas2
complex at low pH is very similar to the dependence observed for
association with AChE alone, and reflected in
pKaE value of 6.1 (Fig. 5B).
At the high pH, the association rate constants were only slightly
reduced, much less than observed for association with AChE alone. Thus,
both protonated and neutral 9-aminoacridine appear to associate with
AChE·Fas2 at similar association rates. In addition, protonation of a
basic AChE residue(s), with a presumed pKa of
~10.3 (Fig. 5A), may be affected by bound Fas2 or is no
longer a rate-limiting factor in ligand association. The
deviations of experimental points from the calculated curve observed in
Fig. 5B at intermediate and high pH appeared reproducible
and are possibly an indication of the interacting species being more
complex than described in Scheme 1. Rates of 9-aminoacridine
dissociation from AChE·Fas2 complex were largely independent on pH.
 |
DISCUSSION |
Our comparison of the interaction kinetics of five substrates and
12 reversible inhibitors between the AChE·Fas2 complex and free AChE
(Fig. 6) showed second order reaction
rate constants for substrates and association rate constants for
reversible ligands to be markedly reduced for the AChE·Fas2 complex.
When the contribution of the fasciculin-resistant enzyme species is
accounted for, the magnitude of the rate reductions approaches 4-6
orders of magnitude.

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Fig. 6.
Water-accessible Connolly surface of mouse
AChE (A) and mouse AChE·Fas2 complex
(B). The surface of Fas2 was made transparent to
reveal the peptide backbone of Fas2 represented by a solid
ribbon. Part of the AChE surface was removed to show the
position of the enzyme active center gorge. Side chains of the enzyme
peripheral site (Y72, D74, Y124, and
W286), acyl pocket (F295, F297), and
choline binding site (W86, Y337) are displayed in
the gorge. A, a molecule of decamethonium (black)
stabilized in the AChE gorge was superimposed from the crystal
structure of mouse AChE (1maa.pdb; Ref. 26) into the crystal structure
1mah.pdb (1), upon deletion of Fas2. B, a molecule of
edrophonium (black) stabilized in the gorge of the
AChE·Fas2 complex, superimposed from the crystal structure of
edrophonium with Torpedo AChE (1ax9.pdb; Ref. 27) into the
crystal structure 1mah.pdb.
|
|
Fas2-resistant Esterase--
A Fas2-resistant esterase in
quantities consisting of 0.09% of the total activity is retained and
eluted from the affinity columns. Characterization of this species is
problematic due to its comparatively small quantities. Its specificity
for cationic organophosphates differs from mouse butyrylcholinesterase
and suggests that it may be a proteolysis product or a spontaneous modification of AChE. Allowing for the contaminant, we now estimate the
catalytic efficiency for ATCh with the AChE·Fas2 complex to be
0.004% of uncomplexed AChE activity instead of the previously reported
0.09% (5). Mallender et al. (7) found that a large fraction
of catalytic activity of the human AChE·Fas2 complex, that
represented 2-31% of uncomplexed human AChE activity, was in fact
contaminating, Fas2-resistant esterase activity. Their findings
prompted us to develop multiple approaches to examine whether mouse
AChE, despite its low residual activity in the presence of Fas2, also
contained a Fas2-resistant component.
Development of a novel competitive binding assay based on fluorescent
product of the carbamoyl ester, M7C, enabled us to determine rate
constants for binding of nonfluorescent ligands to mouse AChE·Fas2
complex and to unliganded mouse AChE. The carbamoylation reaction
weights each active center equivalently and avoids amplification from
the high turnover by residual enzyme not inhibited by Fas2. In
addition, incubation of a mixture of the AChE·Fas2 complex and
Fas2-resistant esterase with a bulky organophosphate selectively inhibits the latter, enabling one to account for the disparity of
findings with previous studies. Finally, employing stopped-flow kinetics with direct fluorescent detection of the complexes allowed us
to separate association and dissociation reaction rate constants for
binding of several rapidly equilibrating reversible inhibitors to both
free AChE and the AChE·Fas2 complex.
Influence of Fasciculin on Hydrolysis of Substrates--
Analysis
of the kinetics indicates that inhibition of AChE activity by Fas2 is
not due solely to a diminished rate of substrate entry into the active
center in the AChE·Fas2 complex. The commonly considered reaction
steps in substrate hydrolysis by AChE (14, 28, 29) are shown in Scheme
2,
where enzyme (E) and substrate (S) associate to form a
Michaelis complex (ES). In turn, the complex is converted to
an unstable acyl enzyme (EA) with loss of the substrate
leaving group (P1). EA is hydrolyzed to free
enzyme and acyl group of substrate (P2). The corresponding
catalytic parameters, Km,
kcat, and kcat/Km are described as
follows.
|
(Eq. 5)
|
|
(Eq. 6)
|
|
(Eq. 7)
|
Second order rate constants for reaction of cationic
substrates with AChE·Fas2, reflected in
kcat/Km values between 60 and
100 M
1
s
1, are slower than rate constants for entry
of reversible ligands into the AChE·Fas2 complex. The exceptionally
slow association rates of BW286C51 and decamethonium are most easily
explained by partial overlap of their binding site with the site of
Fas2 residence due to their extended conformation (cf. Fig.
6). Thus, additional conformational constraints apply to their
association rates. Furthermore, the reduction of nearly 6 orders of
magnitude in kcat/Km for ATCh
and PTCh exceeds the reduction in association rate constants for the
reversible cationic inhibitors with the AChE·Fas2 complex. Catalyzed
hydrolysis by AChE·Fas2 for various substrates yielded sets of
limiting values for kcat and for
kcat/Km. The similarity of
kcat values for charged (ATCh) and two neutral
acetate esters (S-PhAc and pNPAc) suggests that the rate of
deacylation of EA (k3) may be
involved in rate limitation for the chemical part of the catalytic
reaction under the influence of bound Fas2.
Parallel reductions in k2 by Fas2 are also
likely, since the inhibition rates of several organophosphates were
shown to be reduced in the AChE·Fas2 complex by several orders of
magnitude more than the rates of association of reversible inhibitors
(6).2 Furthermore, a second
clustering of kcat/Km values
for hydrolysis of charged acyl thiocholines by AChE·Fas2 (~83
M
1 s
1)
as opposed to neutral acetates (~3000
M
1 s
1)
indicates that the corresponding k
1 and
k2 constants are of the same magnitude and,
consequently, together with k1 both influence
values of kcat/Km. This
follows because the reduction of
kcat/Km for ATCh, imposed by
Fas2, is larger than reductions observed in rates of association of
reversible inhibitors. Charge on the leaving group influences both
reactions, described by k1 and
k2, which both participate in limiting the rates
of hydrolysis by the AChE·Fas2 complex. The absence of the selective
steric constraints of the acyl pocket in the AChE·Fas2 complex (Table
II) further suggests that the leaving group of the substrate is
limiting for catalytic throughput
(kcat/Km) in the AChE·Fas2
complex. Thus, in addition to the pronounced effects of bound Fas2 on
rates of ligand entry into AChE, reflected in
k1, an allosteric interaction between the Fas2
binding site and the active center appears to affect the efficiency of
AChE catalysis primarily through reduction of both
k3 and k2.
Influence of Fasciculin on the Binding of Reversible
Ligands--
Our compilation of reaction rate constants for diverse
reversible inhibitors of AChE permits one to dissect the factors
controlling ligand entry to the gorge and steric constraints within the
gorge. The affinities of these inhibitors are governed by two factors. First, the association rate constants for all but one ligand studied approach the diffusion-limited range, ensuring rapid formation of
inhibitory complexes. Similar rates of association for ligands binding
at the peripheral site and active center reflect minimal steric
constraints for entry into the narrow, 18-20 Å in depth active center
gorge (cf. Fig. 6) for those ligands not exceeding a
critical volume (propidium) or shape ((
)-huperzine A).
Second, the residence times of ligands in the stabilized complexes with
AChE varied over 7 orders of magnitude. Previously reported rate
constants for ligand association with AChE, limited to slow
dissociating fasciculins, huprines, trifluoroacetophenones, huperzine
A, ambenonium, and bisquaternary o-CIB-BQ (Table
VII), and only three fast equilibrating
ligands, N-methylacridinium, 7-hydroxyquinolinium, and
propidium, were in good agreement with the constants reported here
(cf. Tables III and VII). To date,
N-methylacridinium is the only reversible inhibitor
characterized for interaction with the AChE·Fas2 complex
(k1 = 1.0 × 105
M
1 s
1,
k
1 = 0.40 s
1 for
human AChE; Ref. 6).
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Table VII
Literature values of kinetic constants for interaction of reversible
inhibitors with acetylcholinesterase
Second order association rate constants (k1) and
first order dissociation rate constants (k 1) were
determined under the following experimental conditions: 50 mM sodium phosphate buffer, pH 7.2, with ~100
mM NaCl and 25 µM Triton X-100 (12), 100 mM phosphate buffer, pH 7.0, with 0.01% bovine serum
albumin (5, 31), 10 mM Tris/HCl buffer, pH 7.5, with 100 mM NaCl and 0.1 mg/ml bovine serum albumin (30), 20 mM phosphate buffer, pH 7.0, with 0.02% Triton X-100 (4,
32), 100 mM phosphate buffer, pH 7.0, with 1.0% Triton
X-100 (33), 100 mM Tris/HCl buffer, pH 8.0, with 100 mM NaCl and 40 mM MgCl2
(o-CIB-BQ; Ref. 11), 1 mM Tris/HCl buffer, pH
8.0 (propidium; Ref. 11), 50 mM Tris/HCl buffer, pH 8.0 (35), 100 mM phosphate buffer, pH 8.0 (36), 20 mM phosphate buffer, pH 7.0, with 0.1% Triton X-100 (6).
|
|
The binding of several ligands, such as edrophonium and decamethonium,
that lack a spectral overlap with tryptophan fluorescence emission
nonetheless results in diminished AChE fluorescence in the
ligand-enzyme complex. Since fluorescence resonance energy transfer is
not possible in this situation, the quantum yields of several
tryptophans have been affected by ligand occupation of the enzyme
active center. This observation not only increases the range of ligands
whose association with the enzyme can be measured directly but also
indicates that the microenvironments of several aromatic residues are
perturbed in the complex. The implications of this finding will be
addressed in subsequent studies.
Linear Free Energy Relationships--
Reduced association and
dissociation rate constants for ligands interacting with the
AChE·Fas2 complex reveal significant steric hindrance for entry of
ligands into the gorge. The different magnitude of the steric
intervention on association and dissociation steps, however, suggests
that Fas2 alters the position of the transition state relative to the
free and bound species. This is evident from the strikingly different
linear free energy relationships between association or dissociation
rate constants of the ligands and their equilibrium dissociation
constants for AChE and AChE·Fas2 complex (Fig.
7).

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Fig. 7.
Linear free energy relationships for
interaction of small reversible inhibitors with AChE (open
symbols) and the AChE·Fas2 complex
(closed symbols). The
numbers on the symbols indicate their order in
Table III. Quaternary ammonium, cationic inhibitors are noted by
circles, protonated cationic inhibitors by
diamonds, and uncharged inhibitors by squares.
Lines were generated by linear regression of Equations 8 and
9. ( )-Huperzine A (inhibitor number 4) was not included in the
calculation. A, linear free energy relationship for
dissociation from AChE and AChE·Fas2. Calculated slopes were
(1.0 ± 0.1) for AChE, and (0.074 ± 0.13) for AChE·Fas2.
B, linear free energy relationship for association with AChE
and AChE·Fas2. Calculated slopes were (0.093 ± 0.056) for
AChE, and (0.93 ± 0.13) for AChE·Fas2.
|
|
Relating free energy changes in the transition state to those in the
ground states for the various ligands enables one to better describe
the energetic pathway for ligand entry and exit from the gorge for AChE
and the AChE·Fas2 complex (37-39). Thus, for a series of ligands,
L, the following linear free energy relationships can be developed.
|
(Eq. 8)
|
|
(Eq. 9)
|
G
A,L and
G
D,L are the activation free
energies for association and dissociation,
G0L is the respective equilibrium
free energy for each ligand, and
G
0 is a constant, intrinsic
activation barrier. The slope of the relationships is related to
,
an estimate of the position of the transition state for ligand
association, which can vary between 0 and 1. Analysis of the data in
Fig. 7 shows a
value of 0.093 for ligand association with AChE and
0.93 for ligand association with AChE·Fas2. For dissociation, 1
is 0.91 for AChE and 0.07 for the AChE·Fas2 complex. In
each case, huperzine was not included in the analysis, since it was an
obvious exception to the correlations, which may relate to its slow
association rate. The slow rate of trifluoroacetophenone dissociation
and the mutually exclusive binding between propidium and Fas2 preclude
the analysis of their interactions with the AChE·Fas2 complex.
The capacity of Fas2 to alter the relationship between the transition
and ground states points to the role of the Fas2 in influencing the
energetics of the pathway for small ligand entry into the gorge. In the
absence of Fas2, the activation barrier for formation of ligand-AChE
complexes correlates with the stabilization energy of the complex as
ligand structure is varied. In the presence of Fas2, the transition
barrier energetics correlate with the energies of the free species.
Hence, diffusional entry of the ligand to the gorge requires bypassing
the cationic bound Fas2, and this becomes the new energetic barrier
limiting for the interaction. In the ligand-bound AChE·Fas2 complex,
escape of the ligand from its residence site deep in the gorge no
longer is the rate limitation for dissociation. This altered energetic
requirement arises from not simply the charge and steric barriers
imparted by the Fas2 molecule per se (6) but also the
capacity of Fas2 to restrict conformational flexibility of the ligand
binding site, deep in the AChE gorge.
Factors Controlling Kinetics of Ligand Interaction--
By using
stopped-flow instrumentation, we examined interaction kinetics for
rapid and slow equilibrating ligands with selectivity for the active
and the peripheral sites. It has been suggested (7, 40-42) that
binding of cationic substrates to peripheral site of mammalian, fish,
or insect AChE effectively increases the local substrate concentration
available for binding to the active center and thus enhances catalytic
efficiency. Such a mechanism seems plausible where substrates would
have to traverse an energy barrier imposed by a narrow gorge to reach
to the site of catalysis in the active center. As would be expected,
the kinetic barriers imposed by the pre-Michaelis complex are minimal,
since active center ligands and peripheral site ligands associate with
AChE at comparable rates. The modular analysis of molecular traffic through the AChE active center (41) suggested distinct limits for
diffusion of neutral and cationic ligands, differing by about 1 order
of magnitude. Our determined average second order association rate
constants of 8.9 × 108
M
1 s
1
(for cationic ligands) and 8.2 × 107
M
1 s
1
(for neutral ligands) are in accord with the prediction.
Protonation of neutral ligands (9-aminoacridine, tacrine) only modestly
affects association rates with AChE and the AChE·Fas2 complex,
perhaps reflecting a balance between rate acceleration by the addition
of a charge and deceleration from the increased hydrated molecular
volume. The hydrated cation structure, however, decreases the
dissociation rates, as evidenced by a comparison of
N-methylacridinium and acridine with 9-aminoacridine and
tacrine as well as by pH-dependent decrease of the
dissociation rate of acridine. Net neutralization of charged
edrophonium by turning it into zwitterion, as well as the addition of
negative charge to neutral coumarin markedly reduced their association
rate constants, as reported for 1-methyl 7-hydroxyquinolinium (36). We
find that protonation of enzyme group(s) of pKa
6.1-6.2 critically decreases association rates of positively charged
ligands with both AChE and the AChE·Fas2 complex in addition to
reduction of phosphorylation and carbamoylation rates of the AChE
active serine (25).
Rapid conformational isomerizations may control the opening times of
the active center gorge to admit ligands (43). In addition to pathway
restrictions imposed by charge and steric constraints of Fas2, these
isomerizations could be far less frequent in AChE·Fas2 due to
restrictions in the breathing motions of domains of AChE immobilized by
Fas2. Fas2 at the gorge entry can also be expected to influence
allosterically the conformation of residues controlling catalysis and
ligand dissociation at the base of the gorge.
Comparative analysis of the kinetics of ligand entry and exit from the
active center gorge for free AChE and AChE·Fas2 complex reveals that
the steric influences of Fas2 at the gorge entry are insufficient to
explain the differences in linear free energy relationships between the
kinetic and equilibrium constants for free AChE and the Fas2·AChE
complex (Fig. 7).
 |
FOOTNOTES |
*
This work was supported by United States Public Health
Service Grants GM18360 and DAMD 17-1-8014 (to P. T.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
858-534-1366; Fax: 858-534-6833; E-mail: pwtaylor@ucsd.edu.
Published, JBC Papers in Press, October 17, 2000, DOI 10.1074/jbc.M006855200
2
Radi
and Taylor, manuscript in preparation.
 |
ABBREVIATIONS |
The abbreviations used are:
AChE, acetylcholinesterase;
Fas2, fasciculin 2;
ATCh, acetylthiocholine
iodide;
BTCh, butyrylthiocholine iodide;
PTCh, propionylthiocholine
iodide;
TFK, trifluoroacetophenone;
S-PhAc, thiophenylacetate;
pNPAc, p-nitrophenyl acetate;
pyridostigmine, 3-[[(dimethylamino)-carbonyl]oxy]-1-methylpyridinium bromide;
eserine, 1'-methylpyrrolidino(2',3',2,3)1,3-dimethylindolin-7-yl
N-methylcarbamate;
echothiophate, O,O'-diethyl S-ethyltrimethylamine
phosphorothiolate iodide;
paraoxon, diethyl p-nitrophenyl
phosphate;
haloxon, O,O-di(2-chloroethyl)-O-(3-chloro- 4-methylcoumarin-7-yl)phosphate;
M7C, N,N-dimethyl carbamoyl
N-methyl-7-quinolinium;
M7H, N-methyl-7-hydroxy-quinolinium.
 |
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