Organophosphorylation of Acetylcholinesterase in the Presence of
Peripheral Site Ligands
DISTINCT EFFECTS OF PROPIDIUM AND FASCICULIN*
William D.
Mallender
,
Tivadar
Szegletes, and
Terrone L.
Rosenberry§
From the Department of Pharmacology, Mayo Foundation for Medical
Education and Research, and the Department of Research, Mayo Clinic
Jacksonville, Jacksonville, Florida 32224
 |
ABSTRACT |
Structural analysis of
acetylcholinesterase (AChE) has revealed two sites of ligand
interaction in the active site gorge: an acylation site at the base of
the gorge and a peripheral site at its mouth. A goal of our studies is
to understand how ligand binding to the peripheral site alters the
reactivity of substrates and organophosphates at the acylation site.
Kinetic rate constants were determined for the phosphorylation of AChE
by two fluorogenic organophosphates,
7-[(diethoxyphosphoryl)oxy]-1-methylquinolinium iodide
(DEPQ) and 7-[(methylethoxyphosphonyl)oxy]-4-methylcoumarin (EMPC), by monitoring release of the fluorescent leaving group. Rate
constants obtained with human erythrocyte AChE were in good agreement
with those obtained for recombinant human AChE produced from a high
level Drosophila S2 cell expression system. First-order rate constants kOP were 1,600 ± 300 min
1 for DEPQ and 150 ± 11 min
1 for
EMPC, and second-order rate constants
kOP/KOP were 193 ± 13 µM
1 min
1 for DEPQ and
0.7-1.0 ± 0.1 µM
1 min
1
for EMPC. Binding of the small ligand propidium to the AChE peripheral site decreased kOP/KOP
by factors of 2-20 for these organophosphates. Such modest inhibitory
effects are consistent with our recently proposed steric blockade model
(Szegletes, T., Mallender, W. D., and Rosenberry, T. L. (1998)
Biochemistry 37, 4206-4216). Moreover, the binding of
propidium resulted in a clear increase in kOP
for EMPC, suggesting that molecular or electronic strain caused by the
proximity of propidium to EMPC in the ternary complex may promote
phosphorylation. In contrast, the binding of the polypeptide neurotoxin
fasciculin to the peripheral site of AChE dramatically decreased
phosphorylation rate constants. Values of
kOP/KOP were decreased
by factors of 103 to 105, and
kOP was decreased by factors of 300-4,000.
Such pronounced inhibition suggested a conformational change in the
acylation site induced by fasciculin binding. As a note of caution to
other investigators, measurements of phosphorylation of the
fasciculin-AChE complex by AChE inactivation gave misleading rate
constants because a small fraction of the AChE was resistant to
inhibition by fasciculin.
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INTRODUCTION |
Acetylcholinesterase
(AChE)1 terminates
neurotransmission by catalyzing hydrolysis of the neurotransmitter
acetylcholine at rates near that of a diffusion-controlled process (1).
The x-ray crystal structure of AChE reveals that despite the impressive turnover rate of the enzyme, substrate molecules must penetrate 20 Å into a deep active site gorge to be hydrolyzed (2-4). This gorge
contains two sites of ligand interaction: a peripheral site at the
surface of the enzyme and an acylation site at the base of the gorge
where the substrate acyl group is first transferred to residue
Ser200 (Torpedo californica AChE sequence
numbering) and then hydrolyzed. In the acylation site, a catalytic
triad consisting of residues Ser200, His440,
and Glu327 promotes the acyl transfers, and
Trp84 binds the acetylcholine trimethylammonium group,
positioning the substrate for hydrolysis. Certain ligands can bind
selectively to either the acylation site or the peripheral site, and
ternary complexes can be formed in which ligands are bound to
both sites simultaneously (5, 6). Ligands specific for the peripheral site include the small aromatic compound propidium and the snake venom
neurotoxin fasciculin, both of which are potent inhibitors of the
hydrolysis of the chromogenic acetylcholine analog, acetylthiocholine.
The AChE peripheral site is an attractive target for the design of new
classes of therapeutic agents, so it is important to understand how
ligand binding to the peripheral site affects substrate hydrolysis. We
recently provided evidence for a steric blockade model which proposes
that small peripheral site ligands like propidium inhibit substrate
hydrolysis by decreasing the association and dissociation rate
constants for an acylation site ligand without significantly altering
their ratio, the ligand equilibrium constant (7, 8). Cationic
substrates like acetylthiocholine also were shown to bind to the
peripheral site as the first step in their catalytic pathway, and
steric blockade arising from this substrate binding accounted for the
well known phenomenon of substrate inhibition for AChE at very high
concentrations of substrate (8). A key feature of the steric blockade
model is that ligand binding to the peripheral site results in
significant inhibition only if substrate fails to reach equilibrium
binding prior to reaction at the acylation site. Substrates that are
thought to form equilibrium complexes at the acylation site can be
examined to test this prediction. Among these substrates are the
organophosphates (OPs), a class of compounds that inactivate
cholinesterases because they are poor substrates (9-11). OPs readily
phosphorylate the active site serine of AChE, but very slow hydrolysis
of this phosphoryl enzyme results in essentially irreversible
inactivation of the enzyme (12). In this paper we examine the effects
of ligand binding to the peripheral site on OP phosphorylation of AChE
in the context of the steric blockade model. Rate constants of
phosphorylation are measured in two ways. The classical method involves
periodic measurements of AChE activity toward substrates as the enzyme becomes inactivated. The second method involves continuous assay of the
phosphorylation reactions either with mixtures of acetic acid ester
substrates and OPs (13, 14) or by monitoring loss of a fluorogenic OP
leaving group (15, 16). This method can be adapted to stopped-flow
kinetic techniques to allow determination of both first- and
second-order phosphorylation rate constants (13, 14). Here we monitor
the reactions of two fluorogenic OPs, EMPC and DEPQ (Fig.
1), with erythrocyte and recombinant human AChE. Phosphorylation rate constants obtained directly by fluorescence measurement of their AChE-mediated hydrolysis products 7HMC and 7HMQ, respectively (Fig. 1), are compared with those obtained
by enzyme inactivation.
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EXPERIMENTAL PROCEDURES |
Materials--
Human erythrocyte AChE was purified as outlined
previously, and active site concentrations were determined by assuming
410 units/nmol (17, 18).2
DEPQ (15, 19) and EMPC were synthesized by established procedures (see
Ref. 16). [(Methylethoxyphosphonyl)oxy]chloride was reacted with
7HMC (Molecular Probes, Inc.) to give EMPC, and
[(diethoxyphosphoryl)oxy]chloride was linked to 7-hydroxyquinoline
(Acros Organics) and quaternized by methylation to form DEPQ.
Structures were confirmed by 1H NMR and 31P
NMR, and stock concentrations were determined by absorbance (
310 nm = 11.0 mM
1
cm
1 for EMPC, and
317 nm = 8.3 mM
1 cm
1 for DEPQ). Fasciculin
was the fasciculin 2 form obtained from Dr. Carlos Cervenansky at the
Instituto de Investigaciones Biologicas, Clemente Estable, Montevideo,
Uraguay (6). Propidium iodide was purchased from Calbiochem.
Recombinant Human AChE--
The full-length cDNA for human
G4 AChE was obtained from Dr. Avigdor Shafferman in the
vector pACHE10 (20). To obtain a secreted dimeric form of human AChE, a
96-base pair truncation sequence including a stop codon was synthesized
and inserted just downstream from the exon 4/5 boundary (see Ref. 21).
Insertion of the modified exon 4/5 sequence (corresponding to
544ASEAPSTC-DGDSS-stop, human AChE sequence numbering)
resulted in a partial duplication of the 3'-end of the exon 4 region of
the gene. To remove this duplicated segment, the
NotI-NheI 3'- segment of the gene was cloned into
NotI-NheI-digested pCIneo (Promega Corp.). This
construct, pCIneo3'-AChE, contained an EspI site in both the
original and modified sections of exon 4. The unwanted duplicated gene
segment was removed by digestion with EspI followed by
cloning of the resolved NotI-NheI fragment back
into the AChE gene cassette. The final gene construct was confirmed by
DNA sequencing carried out at the Mayo Clinic Rochester Molecular
Biology Core Facility. The modified human AChE cDNA was moved into
the pPac vector for transfection into and expression from
Drosophila S2 cells in tissue culture (21). S2 cells were
maintained in Schneider's Drosophila medium (Life
Technologies, Inc.) with 10% fetal bovine serum and appropriate
antibiotics at 28 °C. S2 cells were cotransfected with pPac carrying
the hygromycin phosphotransferase gene for selection of cells with
hygromycin B. After selection with 0.2 mg/ml hygromycin B, monoclonal
cell lines were isolated from colonies formed using a modified soft
agar cloning protocol (21). Briefly, 104 to 105
selected cells were suspended in complete medium with 0.3% low melting
temperature agarose. This mixture was plated onto a base layer of
solidified 1.5% low melting temperature agarose (in complete medium
with 0.15 M NaCl) in 12-well tissue culture plates. After cell/medium layer solidification, a layer of complete medium was placed
on top of the agarose. Colonies (>2 mm) were picked and grown in
24-well plates until confluence. At this point clones were assayed for
AChE activity, and lines with high activity were kept for large scale
culturing and long term propagation. AChE was purified from culture
medium by two cycles of affinity chromatography on acridinium resin
(17). Purified recombinant AChE samples analyzed by SDS-polyacrylamide
gel electrophoresis (22) showed no contaminants. In the absence of
disulfide reducing agents, a prominent band of 140-kDa dimer and a
minor band of 70-kDa monomer were apparent, whereas in the presence of
reducing agents a single 70-kDa band was observed. Comparison of the
recombinant AChE with purified human erythrocyte AChE showed no
differences in kcat, Kapp, or KSS for
acetylthiocholine hydrolysis (8), in KI for propidium inhibition, in kon, the fasciculin
association rate constant (8), or in phosphorylation rate constants
(see Table I below).
AChE Phosphorylation Determined with Fluorogenic OPs--
Direct
reaction of AChE with EMPC or DEPQ was followed by formation of their
respective fluorescent leaving groups 7HMC or 7HMQ on a Perkin-Elmer
LS-50B luminescence spectrometer in 20 mM sodium phosphate
buffer and 0.02% Triton X-100 at 23 °C. Because these groups are
fluorescent only when their 7-OH substituents are deprotonated (7HMC,
pKa = 7.8 (catalog from Molecular Probes, Inc.);
7HMQ, pKa = 5.9 (23)), measurements were conducted
at pH 8.0 for EMPC and pH 7.0 for DEPQ. Ratios of OP to AChE
concentrations were adjusted to at least 20 for EMPC and 9 for DEPQ in
all cases to prevent significant depletion of OP during the course of
the reaction. Formation of 7HMC (
360 nm = 19.0 mM
1 cm
1 at pH 9.0; catalog of
Molecular Probes, Inc.) was monitored with excitation at 360 nm and
emission at 450 nm, and 7HMQ formation was monitored with excitation at
400 nm and emission at 500 nm (for 7HMQ,
406 nm = 10.0 mM
1 cm
1 at pH 9.0). For
reactions that were completed in less than 1 min, a Hi-Tech SFA 20 stopped-flow apparatus was used to mix equal volumes (300 µl) of AChE
(or AChE with inhibitor) and OP solutions rapidly, and fluorescence was
recorded at fixed intervals as short as 20 ms. Formation of 7HMC or
7HMQ did not follow a simple exponential time course. Nonenzymatic
hydrolysis of EMPC under all conditions and of DEPQ in the presence of
propidium or fasciculin as inhibitors was significant, and under these
conditions data were fitted by nonlinear regression analysis (Fig. P
version 6.0, BioSoft, Inc.) to Equation 1.
|
(Eq. 1)
|
In Equation 1, finitial is the
fluorescence at time zero,
f is the fluorescence change
corresponding to an amount of fluorescent product equal to the AChE
concentration (23), C is the nonenzymatic hydrolysis rate,
and k is the rate constant for the approach to the
steady-state level of phosphorylation. With DEPQ in the absence of
inhibitors the release of 7HMQ occurred in two phases, a large rapid
phase and a small slower phase (see "Results") for both erythrocyte
and recombinant AChE. These data were fitted to Equation 2, where
f2 and k2 were the
respective amplitude and the rate constant for the slower phase, and
the other parameters were as defined in Equation 1.
|
(Eq. 2)
|
The rate constants k were analyzed according to the
catalytic pathway in Scheme 1. In this
Scheme, OPX is the intact OP with leaving group X; EOPX is
the initial complex of the OP with AChE, characterized by the
equilibrium dissociation constant KS = k
S/kS; and
EOP is the phosphorylated enzyme. The inhibitor I can bind to the peripheral site in each of the enzyme species (as denoted by the
subscript P). This scheme is identical to a general pathway
for substrate hydrolysis by AChE considered elsewhere (7). Kinetic
analysis of this scheme was simplified here in two ways. First,
dephosphorylation rate constants (k3 in Scheme 1), which appeared consistent with a value of 2-4 × 10
4 min
1 reported previously for
diethoxyphosphorylated eel AChE (24, 15; data not shown), were assumed
to be negligible in all our experiments. Second, OPs were assumed to
equilibrate with the AChE acylation site even when inhibitors were
bound to the peripheral site. This assumption was supported by
nonequilibrium kinetic simulations with the program SCoP, as noted
under
"Results,"3
and it obviated the need for more complex mechanistic schemes that
apply to substrates that do not equilibrate with AChE (8). With these
assumptions, the dependence of k evaluated from either Equation 1 or Equation 2 on the OP concentration was analyzed by
weighted nonlinear regression analysis (assuming constant percent error
in k) according to Equation 3 to give
kOP and
kOP/KOP, the respective
first- and second-order rate constants of phosphorylation.
|
(Eq. 3)
|
These rate constants are related to the intrinsic rate constants
in Scheme 1 as shown Equations 4 and 5.
|
(Eq. 4)
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(Eq. 5)
|
In the absence of I, kOP = k2 and KOP = KS. In the presence of I,
KOP is given by Equation 6.
|
(Eq. 6)
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Assays with the peripheral site inhibitors propidium (30 µM) or fasciculin (0.5-10 µM) were
conducted at inhibitor concentrations at least 30 times their
respective KI values to ensure that most of the
AChE was complexed with inhibitor. Values of KI
were taken as 1.0 ± 0.1 µM for propidium (7),
11 ± 0.2 pM for fasciculin (6), and 100 pM for fasciculin in the presence of DTNB and acetylthiocholine in the standard assay (6, 8). Measurements that
included fasciculin were modified in several ways. First, the enzyme
was incubated with fasciculin for 5-10 min to generate equilibrated
complex before the addition of OP. Second, the reaction buffer with
EMPC was adjusted to pH 7.0 to reduce nonenzymatic hydrolysis of the
OP. Finally, a four-cell cuvette changer was employed (except where
noted) for reaction times as long as 4 h. This device minimized
fluorophore photobleaching because the sample was cycled in and out of
the light path. Each reaction was measured in parallel with cuvettes
corresponding to an air blank and a nonenzymatic hydrolysis control
devoid of AChE.
AChE Phosphorylation Determined by OP Inactivation of
AChE-catalyzed Substrate Hydrolysis--
AChE activity was monitored
by a modified acetylthiocholine assay (25). Standard assays were
conducted in 3.0 ml of 20 mM sodium phosphate, 0.02%
Triton X-100, 0.33 mM DTNB, and 0.5 mM acetylthiocholine (pH 7.0) at 25 °C. Enzyme hydrolysis was monitored by formation of the thiolate dianion of DTNB at 412 nm (
412
nm = 14.15 mM
1 cm
1 (26))
for 1-5 min on a Varian Cary 3A
spectrophotometer.4 The
inactivation of AChE by an OP was initiated by mixing AChE and OP at
23 °C in 20 mM phosphate buffer and 0.02% Triton X-100 (pH = 7.0). At various times a 1.0-ml aliquot was removed to a cuvette, 40 µl of acetylthiocholine and DTNB were added to final concentrations of 0.5 mM and 0.33 mM,
respectively, and a continuous assay trace was recorded immediately at
412 nm. Background hydrolysis rates in the absence of AChE were
subtracted. To assess the effects of peripheral site inhibitors on OP
inactivation rates, propidium (30 µM) or fasciculin
(50-250 nM) was incubated with AChE for at least 10-30
min prior to the addition of the OP. In some cases, DEPQ was also added
(to 10-60 nM) for 60-120 min after incubation of
fasciculin with AChE to eliminate a minor population of AChE which was
refractory to normal fasciculin inhibition (see "Results"). Titrations of AChE activity with substoichiometric amounts of DEPQ were
conducted by procedures similar to those in other inactivation measurements except that initial incubation mixtures contained higher
concentrations of AChE (28-260 nM) and fasciculin (0-2 µM) and that after 90-120 min small aliquots of the
mixtures (15-20 µl) were diluted into the standard acetylthiocholine
assay solution.
OP inactivation reactions were measured under pseudo first-order
conditions in which the ratio of OP to AChE concentrations was adjusted
to at least 5. Assay rates v during inactivation were
divided by the control assay rate in the absence of OP to give a
normalized value v(N), and these values were
fitted by nonlinear regression analysis (Fig. P) to Equation 7, where
v(N)initial and v(N)final
are the calculated values of v(N) at time zero
and in the final steady state, respectively.
|
(Eq. 7)
|
OP concentrations also were sufficiently low that the observed
inactivation rate constant k was proportional to [OP]. The second-order rate constant for inactivation
kOP/KOP was fitted by
weighted linear regression analysis of the relationship
k = (kOP/KOP)[OP] (see
Equation 3), assuming a constant percent error in k.
 |
RESULTS |
Direct Fluorometric Measurement of AChE Phosphorylation by OPs in
the Presence and Absence of Peripheral Site Ligands--
The rapid
reactions of EMPC and DEPQ with AChE require the use of stopped-flow
methods if both first- and second-order phosphorylation rate constants
are to be measured. Fig. 2A
illustrates the measurement of an individual k value for the
reaction of DEPQ with AChE. The release of the fluorescent product 7HMQ
occurred largely with a single rapid exponential time course, but a
slower phase corresponding to about 10% of the overall reaction also
was apparent. The amplitude, or amount of product released, in the
predominant faster reaction phase equaled the AChE concentration
(Equations 1 and 2) and thus corresponded to a fluorescence titration
of the enzyme normality which reacted rapidly with DEPQ (15, 23).
Fitted k values for the rapid phase were analyzed according
to Equation 3 (Fig. 2B) to obtain the first-order
phosphorylation rate constant kOP and the
second-order rate constant
kOP/KOP. A second, slower
phase was not apparent in reactions of EMPC with AChE or in reactions of either OP in the presence of the peripheral site inhibitors propidium or fasciculin. These reaction time courses, however, were
superimposed upon significant nonenzymatic OP hydrolysis rates that
were incorporated into the curve fitting of the k values. Estimates of kOP and
kOP/KOP were obtained
from these k values by analyses similar to that in Fig.
2B (Table I). Purified
recombinant human AChE expressed in Drosophila S2 cells gave
rate constants for both OPs and relative amplitudes for DEPQ which were
in good agreement with those for purified human erythrocyte AChE.
Furthermore, our kOP/KOP
value for DEPQ (1.9-2.1 × 108
M
1 min
1) agreed with previous
estimates of this second-order phosphorylation rate constant determined
by inactivation of eel AChE (15, 27; see below and Table I). No
previous estimates of kOP for either DEPQ or
EMPC or of kOP/KOP for
EMPC have been reported. We observe that
kOP/KOP is about 200-300
times larger for DEPQ than for EMPC and that kOP
is about 10 times larger for DEPQ than for EMPC. These differences are
consistent with previous expectations that the cationic nature of DEPQ
and the lower pKa of its leaving group relative to
neutral EMPC should result in higher rates of AChE phosphorylation (14,
27, 28).

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Fig. 2.
Reaction of DEPQ with recombinant AChE.
Panel A, equal volumes of DEPQ and AChE were mixed rapidly
with the stopped-flow accessory to final concentrations of 2 µM and 100 nM, respectively, and generation
of 7HMQ was monitored by spectrofluorometry as outlined under
"Experimental Procedures." Prior to the reaction, the stopped-flow
cuvette was washed extensively with the DEPQ stock solution alone;
fluorescence in this wash solution results from about 5% contamination
of the stock DEPQ with 7HMQ. In this recording, data collection on the
spectrometer was triggered manually at time zero, and mixing was
initiated at 1.6 s. Points observed for the reaction from 1.7 to
19 s ( and dotted line) were fitted to Equation 2
(solid line), with approximately 90% of the reaction
amplitude corresponding to a reaction rate constant of 356 ± 13 min 1 and 10% to a rate constant of 24 ± 2 min 1. Fluorescence units were converted to 7HMQ product
formed (nM) by comparison with a 7HMQ standard solution.
Panel B, rate constants for the faster phase of the reaction
of DEPQ with AChE obtained as in panel A were plotted
against the DEPQ concentration according to Equation 3 to obtain first-
and second-order rate constants of 1,600 ± 200 min 1
and 205 ± 11 µM 1 min 1,
respectively (Table I).
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Table I
Rate constants for the phosphorylation of AChE by OPs
Rate constants were calculated from the dependence of k on
[OP] as outlined under "Experimental Procedures."
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The effects of the small peripheral site ligand propidium on
phosphorylation of AChE by OPs have not been widely studied. In Fig.
3A we show that propidium
decreased kOP/KOP but
increased kOP for the phosphorylation of AChE by
EMPC. The decrease in
kOP/KOP was small (2.6- and 1.5-fold, for the erythrocyte and recombinant enzymes, respectively
(Table I)) and consistent with those reported previously for neutral
OPs with T. californica AChE (29). This small
decrease appears consistent with the prediction of our steric blockade
model that small peripheral site inhibitors like propidium will have
little effect on the reaction of substrates that equilibrate with AChE
(7; see Footnote 3 and Discussion). The extent of the increase in
kOP for EMPC when propidium is bound was less
clear because the very slight curvature of the plot with propidium in Fig. 3A made extrapolation to kOP
problematic. Inclusion of 30 µM propidium increased the
KOP for EMPC by factors of 15 ± 9 and 6 ± 2 for the two AChEs to a value greater than 1 mM,
and increased signal to noise prevented us from extending the EMPC
concentration into the mM range to improve the precision of
these factors. Insertion of these factors into Equation 6 and
calculation from Equation 5 indicated that both
KSI/KI and a
in Scheme 1 were on the order of 10 (Table I), indicating both lower
affinity and higher reactivity of EMPC in the ternary complex with AChE
and propidium. Propidium (30 µM) had a more pronounced
effect on kOP/KOP for
DEPQ, producing 18- and 14-fold decreases for the two AChEs (Table I).
These factors again are consistent with those reported previously for cationic OPs with T. californica AChE (29). As observed for EMPC, bound propidium also increased KOP for
DEPQ to the point where it became technically difficult to measure the
corresponding kOP. It appeared that
kOP for DEPQ with propidium was at least as
large as kOP for DEPQ alone (Table I), but more
precise estimates were not possible.

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Fig. 3.
Phosphorylation of erythrocyte AChE by EMPC
in the presence and absence of peripheral site ligands. Rate
constants k, measured by spectrofluorometric detection of
7HMC as outlined under "Experimental Procedures" (see also Fig.
2A), were plotted against the EMPC concentration and fitted
to Equation 3 (lines) to obtain kOP
and kOP/KOP (Table I).
Panel A, indicates no inhibitor;
kOP = k2 = 149 ± 9 min 1 and
kOP/KOP = k2/KS = 0.95 ± 0.04 µM 1 min 1. indicates plus
30 µM propidium; kOP = 900 ± 500 min 1 and
kOP/KOP = 0.37 ± 0.02 µM 1 min 1. Points for
[EMPC] 200 µM are shown as means of two to nine
k measurements. Panel B, indicates plus 1.3 µM fasciculin; kOP = 0.46 ± 0.09 min 1 and
kOP/KOP = 0.0013 ± 0.0001 µM 1 min 1.
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Unlike propidium, the binding of fasciculin to the AChE peripheral site
had a drastic effect on the phosphorylation of AChE by OPs (Fig.
3B). At saturating fasciculin concentrations
(104 to 106 times greater than its
KI),
kOP/KOP was decreased
about 700-fold for EMPC and by about 105 for DEPQ. Bound
fasciculin also decreased kOP for both OPs by factors of 300-4,000 (Table I). The amplitudes of the OP reactions with the fasciculin-AChE complex again were consistent with the AChE
normality, indicating that most of the enzyme was involved in the
slowly reacting complex. A previous report of the effects of bound
fasciculin on the reaction of AChE with the OPs echothiophate and
paraoxon found only modest decreases of less than an order of magnitude
for either kOP/KOP or
kOP (30). These relative changes in rate
constants, however, were determined from rates of enzyme inactivation
monitored by progressive reductions in the residual activity of the
fasciculin-AChE complex, not from the release of the OP leaving group.
To assess whether there were discrepancies between these methods, we
repeated measurements of EMPC and DEPQ reaction rate constants by
following enzyme inactivation.
Measurement of AChE Phosphorylation by OPs by Enzyme
Inactivation--
We measured AChE inactivation by EMPC and DEPQ
without the use of stopped-flow kinetic methods, and our determinations
were limited to the second-order phosphorylation rate constants
kOP/KOP. With either OP
alone or in the presence of propidium, values of kOP/KOP determined by
inactivation were in agreement with those from spectrofluorometric
assays within about a factor of 2 (Table I). However, when the reaction
of either OP was measured in the presence of fasciculin, a striking
discrepancy between the two methods became apparent. Bound fasciculin
decreased kOP/KOP
determined by inactivation only 2-5-fold (Table I), in agreement with
the above report by Radic et al. (30) but in contrast to the
decreases of up to 105-fold determined by
spectrofluorometry (Table I). The discrepancy raised a concern that the
enzyme activity observed during inactivation by OPs did not arise from
the fasciculin-AChE complex. Because fasciculin binding reduces the
activity of human AChE preparations to a residual 0.1-1% of the
activity of the free enzyme (6), a tiny fraction of the AChE
preparation resistant to fasciculin inhibition for any reason could
become the dominant activity during the OP inactivation measurements.
To examine this possibility, we altered the ratio of the OP to the AChE
concentrations for the inactivation reaction in the presence of
saturating fasciculin. When the DEPQ/AChE ratio was a typical value of
8, about 80% of the residual fasciculin-AChE activity was inactivated
with a rate constant k consistent with the
kOP/KOP of 6.2 × 107 M
1 min
1 in
Table I (lower trace, Fig. 4).
The inactivation reaction was then repeated at the same concentration
of DEPQ but with 50 times as much AChE (i.e.
[AChE]/[DEPQ] = 6). If all of the fasciculin-AChE complex could
react with DEPQ at the previous rate, only about 17% of the residual
activity should have been inactivated before DEPQ was completely
depleted; in fact we continued to observe 80% inactivation with about
the same k value (data not shown). Repeating the
inactivation reaction again with a ratio [AChE]/[DEPQ] = 60 finally
did result in depletion of the DEPQ but not before more than 40% of
the residual fasciculin-AChE activity was inactivated (upper
trace, Fig. 4). These data indicated that only a few percent of
the total AChE concentration was involved in the observed inactivation reaction.

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Fig. 4.
Inactivation of the residual activity of the
fasciculin-AChE complex by DEPQ at two ratios of the DEPQ to AChE
concentrations. Erythrocyte AChE at 0.16 nM ( ) and
80 nM ( ) was incubated for 10 min with fasciculin at 50 nM ( ) and 500 nM ( ), and inactivation was
initiated by the addition of DEPQ to a final concentration of 1.4 nM. Aliquots were assayed at the indicated times as
outlined under "Experimental Procedures." Assay points v
were normalized to corresponding control residual activities with
fasciculin but without DEPQ
(vDEPQ = 0) and fitted to Equation 7 (lines) to obtain a value of k for each
curve.
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To quantify this point, we titrated several AChE stocks with DEPQ in
the presence and absence of fasciculin by measuring inactivation. Examples of these titrations are shown in Fig.
5. As expected in the absence of
fasciculin, the stoichiometric amount of DEPQ required for complete
inactivation was within about 15% of the AChE active site
concentration calculated from the initial activity (Fig.
5A). In the presence of saturating fasciculin, however, less
than 100% of the residual activity was rapidly inactivated (Fig. 5,
B and C). We fitted these titration data to a
model with two enzyme populations, one that was relatively rapidly
inactivated by DEPQ and the other that reacted with DEPQ at the very
low rate constants measured by the fluorescence assays in Table I. The rapidly inactivated population corresponded to 5% of the total AChE
concentration in Fig. 5B and 40% in Fig. 5C.
These percentages varied among AChE stocks, with erythrocyte AChE
typically giving about 5% and two preparations of recombinant AChE
exhibiting 2 and 40%, respectively. These data thus are consistent
with the assignment of a small but variable fraction of the AChE as a
population that is largely resistant to fasciculin inhibition.

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Fig. 5.
Titration of AChE with DEPQ in the presence
and absence of fasciculin. Erythrocyte (eryth) or
recombinant (rec) AChE was incubated with or without
fasciculin for 10-30 min and mixed with an equal volume of DEPQ for
90-120 min as outlined under "Experimental Procedures." Each point
represents one mixture with the indicated final concentrations of DEPQ,
AChE, and fasciculin. Aliquots (15-20 µl) were then diluted 50-fold
(panels B and C) or 200-fold (panel A)
into the standard acetylthiocholine solution for assay. Observed
v were normalized to vDEPQ = 0
obtained in the absence of DEPQ, and titration lines fitting the
stoichiometric amount of DEPQ required to give complete rapid
inactivation were calculated with the SCoP program. The calculated
concentrations of rapidly inactivated AChE were 32 nM
(panel A), 13 nM (panel B), and 43 nM (panel C) and correspond closely to the
intersections of the lines in the plots. [The SCoP
simulation program (7) was applied to two populations of AChE which
reacted with DEPQ according to Scheme 1 to fit the data in Fig. 5,
B and C. Rate constant assignments for the
fasciculin-inhibited population were taken from Footnote 3;
kOP/KOP for DEPQ with the
fasciculin-resistant population was assigned as 1 × 108 M 1 min 1, and
the measured nonenzymatic DEPQ hydrolysis rate was 1.4 × 10 4 min 1 (data not shown). The fitted
variables were the ratio of the concentrations of the two populations
and the ratio of their acetylthiocholine hydrolysis rates.]
|
|
We next confirmed that the residual activity remaining after the rapid
inactivation by DEPQ in Fig. 5, B and C, in fact
did correspond to the fasciculin-AChE complex. This involved
demonstrating that this residual activity was slowly inactivated by OPs
at the same low rate constants determined with the fluorescence assays in Table I. AChE was incubated with fasciculin, and activity from the
fasciculin-resistant population was removed by rapid inactivation with
10-60 nM DEPQ (see "Experimental Procedures"). The
activity remaining after this treatment (e.g. the activity remaining after 60 min in the lower trace of Fig. 4) was
then progressively inactivated by further incubation with EMPC, and kOP/KOP was determined as
above. These values of
kOP/KOP from inactivation
were now in good agreement with the values of
kOP/KOP obtained for the
reaction of EMPC with the fasciculin-AChE complexes by
fluorescence assay (e.g. 1.3 × 103
M
1 min
1 for erythrocyte AChE in
Table I).
Detection of More Than One Population of AChE in the Presence of
Fasciculin by Fluorometry--
As our last demonstration of the
consistency between the fluorescence- and inactivation-based assays, we
reexamined the release of fluorescent 7HMQ from the reaction of DEPQ
with AChE when fasciculin was present. Because this method does not
depend on residual enzyme activity, the fasciculin-resistant population
can be monitored separately from the fasciculin-AChE complex simply by
altering the time of measurement and the concentration of DEPQ (Fig.
6). In Fig. 6A, DEPQ was
7-14-fold in excess of the expected fasciculin-resistant population of
AChE. A burst of 7HMQ was released in the initial minute of reaction,
and the amplitude of this burst indicated that approximately 2-3% of
the recombinant AChE concentration had reacted. This percentage agreed
with the percentage of rapidly inactivated AChE obtained by an
inactivation titration like those in Fig. 5 for this recombinant AChE
sample (data not shown). When the DEPQ concentration was increased by a
factor of 25 (Fig. 6B), the initial burst in Fig.
6A became too fast to measure, but the slower reaction of
DEPQ with the fasciculin-AChE complex became apparent. As expected, the
amplitude of this reaction corresponded to the total AChE
concentration, and the k value was consistent with the
kOP/KOP determined by
fluorometry for the reaction of DEPQ with AChE in the presence of
fasciculin (Table I).

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Fig. 6.
Resolution of more than one population of
AChE in the presence of fasciculin by fluorometric assay with
DEPQ. Panel A, recombinant AChE (200 nM)
was preincubated with fasciculin (1.0 µM) before mixing
with an equal volume of 380 nM DEPQ in the stopped-flow
accessory, and generation of 7HMQ was monitored by spectrofluorometry
as in Fig. 2. Panel B, recombinant AChE (200 nM)
was preincubated with fasciculin (1.0 µM) before
conventional mixing with an equal volume of 10 µM DEPQ,
and generation of 7HMQ was monitored as in panel A. A value
of k = 0.01 min 1 was estimated by fitting
the data in panel B with Equation 1. Dashed lines
indicate blank DEPQ hydrolysis rates measured in the absence of
AChE.
|
|
 |
DISCUSSION |
In this paper, we report kinetic parameters for the
phosphorylation of AChE by two fluorogenic OPs, EMPC and DEPQ (Fig. 1). Both human erythrocyte AChE and recombinant human AChE produced from a
high level Drosophila S2 cell expression system were
examined. Because this expression system yields more than 20 mg of
purified AChE from 2 liters of medium after 10 days of continuous
culture, it is attractive for the preparation of wild type and
site-specific mutants of AChE for comparative kinetic analyses and
x-ray crystallography. The agreement of the phosphorylation kinetic
parameters in Table I for the two AChEs provides important confirmation
that the recombinant enzyme retains the catalytic properties of
endogenous AChEs. EMPC and DEPQ were particularly useful
organophosphorylation reagents because their reactions with AChE were
observed both directly by fluorometry and indirectly by enzyme
inactivation, their high phosphorylation rate constants approximated
those for OPs used in chemical warfare applications, and their charges
differed, allowing comparison of neutral EMPC with cationic DEPQ. We
focused specifically on the effects of bound peripheral site ligands on AChE phosphorylation by OPs. Characterization of these effects is of
great interest because it may be possible to design a peripheral site
ligand that will block OP inactivation of AChE specifically while
allowing sufficient acetylcholine hydrolysis activity to maintain
synaptic transmission.
To pursue this goal we first compared the effects on AChE
phosphorylation of two ligands that bind specifically to the peripheral site, the small phenanthridinium derivative propidium and the 61-residue polypeptide fasciculin. Propidium is a potent inhibitor of
substrate hydrolysis by AChE, decreasing the second-order rate constant
kcat/Kapp for
acetylthiocholine and phenyl acetate by factors of 15-50 and the
first-order rate constant kcat by factors of
2-10 (7). To account for this inhibition, we proposed a steric
blockade model in which the primary effect of a small peripheral site
ligand like propidium is to slow the association and dissociation rate
constants for ligand binding to the acylation site without significantly altering their ratio, the equilibrium constant (7, 8).
One objective in proposing this model was to demonstrate that
inhibition by peripheral site ligands could be explained without
invoking a conformational change in the acylation site induced by the
binding of ligand to the peripheral site. Our steric blockade model was
supported by direct measurements with the acylation site ligands
huperzine A and TMTFA: bound propidium decreased the association rate
constants 49- and 380-fold and the dissociation rate constants 10- and
60-fold, respectively, relative to the rate constants for these
acylation site ligands with free AChE (7). The model also was supported
by computer simulations of substrate hydrolysis based on Scheme 1. When
the binding of substrate to the acylation site failed to reach
equilibrium, the observed level of propidium inhibition could be
reproduced (7). On the other hand, the model predicts that propidium
should have little effect on the reaction of a substrate that
essentially equilibrates with the acylation site. Few reports in the
literature include data that allow this prediction to be examined, but
it is supported by a recent investigation of aryl acylamidase activity
in AChE (31). The peripheral site ligands propidium and gallamine
failed to inhibit AChE-catalyzed hydrolysis of aryl acylamides, which are hydrolyzed slowly by AChE and thus should equilibrate with the
acylation site (32), but gave typical inhibition of acetylthiocholine hydrolysis.
The reaction of OPs, including EMPC and DEPQ, with AChE appears to
involve equilibration of the OP with the acylation site (see Footnote
3). Table I indicates that propidium did have modest effects on
kOP and
kOP/KOP for both EMPC and
DEPQ. Do these observations invalidate our steric blockade model and
require that the binding of propidium induce a conformational change in
the acylation site? We argue that they do not, if the model is extended
to allow an unfavorable electrostatic interaction or a steric overlap
between propidium at the peripheral site and an OP at the acylation
site in the AChE ternary complex. The need for such an extension in fact has been recognized in our previous studies because small decreases in the affinity of ligands in ternary AChE complexes relative
to the corresponding binary complex are observed consistently (7). For
example, from the rate constants noted above one can calculate that the
affinities of huperzine A and TMTFA for the acylation site decreased by
factors of 5-6 when propidium was bound to the peripheral site.
Computer modeling revealed no steric overlap between the ligands in
these ternary complexes (7), so the decreased affinity must result from
unfavorable electrostatic interaction between these cationic ligands.
Extending these observations to the OPs, a decrease in affinity for
EMPC and DEPQ also was apparent when propidium was bound to the
peripheral site. Insertion of data from Table I into Equation 6
indicated that this decrease (given by
KSI/KI = KS2/KS) was about an
order of magnitude for both OPs. There was also a clear increase in the
kOP for EMPC (a > 1 in Table I)
and a possible increase in kOP for DEPQ when propidium was bound, consistent with an acceleration of first-order phosphorylation rate constants by bound peripheral site ligands reported recently by Radic (33, 34). It has long been known that
kOP/KOP for the reaction
of neutral OPs with AChE varies smoothly and monotonically with the
pKa of the leaving group (27). This suggests that
cleavage of the leaving group ester bond of the OP is prominent in the
rate-limiting step for phosphorylation of AChE. Computer modeling
revealed a clear unfavorable steric overlap between propidium in the
peripheral site and the leaving group of either EMPC or DEPQ in the
acylation site (data not shown; 35). This steric overlap could
contribute to the decrease in affinity for both neutral EMPC and
cationic DEPQ, and it could induce molecular or electronic strain
caused by the proximity of propidium to the OP in the ternary complex
to increase kOP. This increase would not require
an induced conformational change in the acylation site when propidium
alone is bound but would result from a change in ligand configuration
or overall conformation in the ternary complex.
The consequences of fasciculin binding to the peripheral site of AChE
on phosphorylation kinetic parameters for EMPC and DEPQ were
qualitatively different from those of propidium. Values of kOP/KOP were decreased by
factors of 103 to 105, and
kOP was decreased by factors of 300-4,000
(Table I). Fasciculin has been shown to present a substantial steric
blockade to the entrance and exit of ligands that bind to the acylation
site: association and dissociation rate constants for the binding of N-methylacridinium were decreased 8,000- and 2,000-fold,
respectively, when fasciculin was bound (36). However, steric blockade
of OP association and dissociation rate constants cannot account for
the effects of fasciculin on the phosphorylation rate constants. Because OPs essentially equilibrate with the acylation site, a steric
blockade of OPs by fasciculin that resulted in even a 100,000-fold decrease in association rate constant
(kS2/kS = 10
5) would result in less than a 10% decrease in
kOP/KOP and no change in
kOP (see Footnote 3). The pronounced fasciculin
inhibition of AChE phosphorylation requires an additional interaction
between fasciculin and the acylation site. One possibility might be an unfavorable steric overlap between fasciculin at the peripheral site
and an OP at the acylation site in the AChE ternary complex, but the
three-dimensional structure of the fasciculin-AChE complex shows no
penetration of the acylation site by fasciculin which would lead to
such an interaction. Therefore, the additional interaction must involve
a conformational change in the acylation site induced by bound
fasciculin. Crystal structure analyses of fasciculin-AChE complexes (3,
4) show that fasciculin 2 interacts not only with Trp279 in
the peripheral site but also with residues on the outer surface of an
-loop within 4 Å of Trp84 in the acylation site, well
beyond the region of the peripheral site occupied by propidium (7, 37).
These more extensive surface interactions provide a structural basis
for an inhibitory conformational effect on the acylation site when
fasciculin but not when propidium is bound to the peripheral site.
Second-order phosphorylation rate constants
kOP/KOP obtained for EMPC
or DEPQ alone or in the presence of propidium were in good agreement
when measured either by release of the fluorescent leaving group or by
enzyme inactivation (Table I). In the presence of fasciculin, however,
kOP/KOP values determined
by enzyme inactivation were 100-fold greater for EMPC and
104-fold greater for DEPQ than the corresponding values
measured fluorometrically. Through a series of titrations like those in Fig. 5, this discrepancy was shown to arise from misleading
inactivation measurements caused by a small fraction of the total AChE
(less than 5%, except for one preparation) which remained largely
resistant to inhibition by fasciculin. This fraction thus accounted for most of the enzyme activity in the presence of fasciculin and was
inactivated by both OPs much more rapidly than the fasciculin-AChE complex was phosphorylated. In support of this explanation,
reexamination of the release of fluorescent 7HMQ from DEPQ in the
presence of fasciculin revealed a small initial burst that was in
stoichiometric agreement with the fraction of AChE that was rapidly
inactivated (Fig. 6A). The population of AChE resistant to
fasciculin inhibition may itself be heterogeneous. The titration curves
in Fig. 5, B and C, were not fit precisely by a
model with two forms of AChE, and the deviation suggested a third form
with somewhat less resistance to fasciculin inhibition. In addition,
the initial burst of 7HMQ release in Fig. 6A could not be
fit to a single exponential release of 7HMQ but instead corresponded to
two release reactions, the faster of which corresponded to the
kOP/KOP initially
observed for fasciculin inhibition of inactivation by DEPQ (Table I). Other uncertainties involve the source of the population of
fasciculin-resistant AChE or the process whereby it was produced. Each
AChE preparation we examined, whether erythrocyte or recombinant,
showed this resistant population, but its extent varied among different
affinity chromatography preparations. This population may represent a
portion of the AChE which has undergone an undefined chemical
modification that alters the affinity of fasciculin for the peripheral
site and/or the catalytic efficiency of the fasciculin-AChE complex.
Estimates of kOP/KOP
for this population in the presence of fasciculin from the inactivation
data in Table I were only 2-5-fold lower than kOP/KOP for AChE alone,
and these estimates were relatively insensitive to the saturating
fasciculin concentration (data not shown). Furthermore, calculations
from the titration data in Fig. 5 indicated an acetylthiocholine turnover rate for this population which was about 30% of that for free
AChE (data not shown). These comparisons suggest that catalysis at the
acylation site is only slightly less efficient in the
fasciculin-resistant population than in the predominant conventional
AChE. Fasciculin does appear to interact weakly with this resistant
population, resulting in 3-5-fold decreases in kOP/KOP for EMPC and DEPQ
(Table I). A fasciculin-resistant population also may dominate the
activity of recombinant mouse AChE in the presence of fasciculin: the
addition of fasciculin induced biphasic phosphorylation rates and only
modest decreases in phosphorylation rate constants and TMTFA
association and dissociation rate constants (less than 20-fold; 30). It
is possible that the population of AChE resistant to fasciculin can be
distinguished even in the absence of fasciculin as the fraction of AChE
that underwent a slower reaction with DEPQ in Fig. 2. The amount of
this fraction (about 10% of the total AChE) and its phosphorylation
rate constant (about 10% of the k for the faster phase) are
roughly consistent with the data for the fasciculin-resistant population.
Regardless of the origin of the fasciculin-resistant population, it is
significant because it obscures the kinetic properties of the actual
fasciculin-AChE complex measured by enzyme inactivation, both in our
measurements (Table I) and apparently in those of Radic et
al. (30). We overcame this problem by exploiting the relatively
high sensitivity of the fasciculin-resistant population to DEPQ.
Incubation of small amounts of AChE (3-20 nM) in the presence of fasciculin with 10-60 nM DEPQ was sufficient
to inactivate this population in 60 min, and the kinetic parameters of
the fasciculin-AChE complex then could be measured by inactivation. For
example, measurement of
kOP/KOP for EMPC after
this treatment gave good agreement with the
kOP/KOP values determined
for EMPC with the fasciculin-AChE complex by fluorometry (Table I). In
addition to phosphorylation kinetic parameters, it may be necessary to
employ DEPQ inactivation to reevaluate kinetic parameters for the
reaction of substrates (6) and TMTFA (30) with fasciculin-AChE complexes.
 |
ACKNOWLEDGEMENTS |
We express our gratitude to Dr. Abdul Fauq of
the Mayo Clinic Jacksonville Organic Synthesis Core Facility for
preparation of EMPC and DEPQ used in these studies. We thank Dr. Harvey
Berman of the State University of New York at Buffalo and Dr. Yacov
Ashani of the Israel Institute for Biological Research at Ness-Ziona for initial samples of EMPC and DEPQ, respectively. We also thank Dr.
Avigdor Shafferman of the Israel Institute for Biological Research at
Ness-Ziona for the cDNA for AChE in pACHE10 and Dr. Carlos
Cervenansky for fasciculin 2. We acknowledge the technical assistance
of Dr. John Incardona, Jean Eastman, Dr. Robert Haas, and Pat Thomas in
the initial phases of construction and cloning the recombinant human
AChE gene cassette.
 |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grant NS-16577, United States Army Medical Research Acquisition Activity Grant DAMD 17-98-2-8019, and by grants from the Muscular Dystrophy Association of America.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.
Supported by a Kendall-Mayo postdoctoral fellowship.
§
To whom correspondence should be addressed. Tel.: 904-953-7375;
Fax: 904-953-7370; E-mail: rosenberry{at}mayo.edu.
2
One unit corresponds to 1 µmol of
acetylthiocholine hydrolyzed/min under standard pH-stat assay
conditions (3.67
A412 nm/min in our standard
spectrophotometric assay (17)).
3
To ensure that EMPC and DEPQ were close enough
to equilibrium with AChE in the presence of 30 µM
propidium or 0.5-10 µM fasciculin to justify application
of Equations 4-6, we applied the SCoP simulation program (7) to Scheme
1 with the following rate constant assignments: kS = 1 × 107
M
1 s
1 for EMPC and 2 × 108 M
1 s
1 for DEPQ,
values similar to those assigned previously for neutral and cationic
acetic acid ester substrates (7); kI = kSI = 2 × 108
M
1 s
1 for propidium (7);
kI = kSI = 3 × 107 M
1 s
1 for
fasciculin with free AChE, and kI = kSI = 1 × 107
M
1 s
1 for fasciculin with AChE
in the presence of DTNB and acetylthiocholine (6);
k3 = 5 × 10
6
s
1; k
S,
k
I, k2, and
a from Table I and "Experimental Procedures" (with
k
X = KXkX); k
SI
from KSI from Equation 6; and
k
S2 = kS2
KS
KSI/KI. Simulated values of
kOP and
kOP/KOP were then
compared for complete equilibrium (kS2/kS = 1) and
pronounced steric blockade
(kS2/kS = 0.00001) and
found to differ by less than 10%, justifying the equilibrium assumption.
4
Enzyme activities were standardized to 0.1
A412 nm/min by applying the observed
relationship vstd = 0.1(v/0.1)1/R, where v was
the measured activity, vstd was the standardized activity, and r = 0.95. R was the slope of a
plot of log measured activity versus log enzyme
concentration over a 200-fold range of enzyme dilution.
 |
ABBREVIATIONS |
The abbreviations used are:
AChE, acetylcholinesterase;
DTNB, 5,5'-dithiobis-(2-nitrobenzoic acid);
OP, organophosphate;
EMPC, 7-[(methylethoxyphosphonyl)oxy]-4-methylcoumarin;
7HMC, 7-hydroxy-4-methylcoumarin;
DEPQ, 7-[(diethoxyphosphoryl)oxy]-1-methylquinolinium iodide;
7HMQ, 7-hydroxy-1-methylquinolinium iodide;
TMTFA, m-(N,N,N-trimethylammonio)trifluoroacetophenone.
 |
REFERENCES |
-
Rosenberry, T. L.
(1975)
Adv. Enzymol.
43,
103-218[Medline]
[Order article via Infotrieve]
-
Sussman, J. L.,
Harel, M.,
Frolow, F.,
Oefner, C.,
Goldman, A.,
Toker, L.,
and Silman, I.
(1991)
Science
253,
872-879[Medline]
[Order article via Infotrieve]
-
Bourne, Y.,
Taylor, P.,
and Marchot, P.
(1995)
Cell
83,
503-512[Medline]
[Order article via Infotrieve]
-
Harel, M.,
Kleywegt, G. J.,
Ravelli, R. B. G.,
Silman, I.,
and Sussman, J. L.
(1995)
Structure
3,
1355-1366[Medline]
[Order article via Infotrieve]
-
Taylor, P.,
and Lappi, S.
(1975)
Biochemistry
14,
1989-1997[Medline]
[Order article via Infotrieve]
-
Eastman, J.,
Wilson, E. J.,
Cervenansky, C.,
and Rosenberry, T. L.
(1995)
J. Biol. Chem.
270,
19694-19701[Abstract/Free Full Text]
-
Szegletes, T.,
Mallender, W. D.,
and Rosenberry, T. L.
(1998)
Biochemistry
37,
4206-4216[CrossRef][Medline]
[Order article via Infotrieve]
-
Szegletes, T.,
Mallender, W. D.,
Thomas, P. J.,
and Rosenberry, T. L.
(1999)
Biochemistry
38,
122-133[CrossRef][Medline]
[Order article via Infotrieve]
-
Burgen, A. S. V.
(1949)
Br. J. Pharmacol.
4,
219-228
-
Wilson, I. B.
(1951)
J. Biol. Chem.
190,
111-117[Free Full Text]
-
Aldridge, W. N.,
and Reiner, E.
(1972)
Frontiers of Biology, 26th Ed., p. 328, Elsevier, North Holland, Amsterdam
-
Froede, H. C.,
and Wilson, I. B.
(1971)
in
The Enzymes (Boyer, P. D., ed), 3rd Ed., Vol. 5, pp. 87-114, Academic Press, New York
-
Hart, G. D.,
and O'Brien, R. D.
(1973)
Biochemistry
12,
2940-2945[Medline]
[Order article via Infotrieve]
-
Berman, H. A.,
and Leonard, K.
(1989)
J. Biol. Chem.
264,
3942-3950[Abstract/Free Full Text]
-
Gordon, M. A.,
Carpenter, D. E.,
Barrett, H. W.,
and Wilson, I. B.
(1978)
Anal. Biochem.
85,
519-527[Medline]
[Order article via Infotrieve]
-
Levy, D.,
and Ashani, Y.
(1986)
Biochem. Pharmacol.
35,
1079-1085[CrossRef][Medline]
[Order article via Infotrieve]
-
Rosenberry, T. L.,
and Scoggin, D. M.
(1984)
J. Biol. Chem.
259,
5643-5652[Abstract/Free Full Text]
-
Roberts, W. L.,
Kim, B. H.,
and Rosenberry, T. L.
(1987)
Proc. Natl. Acad. Sci. U. S. A.
84,
7817-7821[Abstract]
-
Ginsburg, S.,
Kitz, R. J.,
and Wilson, I. B.
(1966)
J. Med. Chem.
9,
632-633[Medline]
[Order article via Infotrieve]
-
Kronman, C.,
Velan, B.,
Gozes, Y.,
Leitner, M.,
Flashner, Y.,
Lazar, A.,
Marcus, D.,
Sery, T.,
Papier, Y.,
Grosfeld, H.,
Cohen, S.,
and Shafferman, A.
(1992)
Gene (Amst.)
121,
295-304[Medline]
[Order article via Infotrieve]
-
Incardona, J. P.,
and Rosenberry, T. L.
(1996)
Mol. Biol. Cell
7,
595-611[Abstract]
-
Laemmli, U. K.
(1970)
Nature
227,
680-685[Medline]
[Order article via Infotrieve]
-
Rosenberry, T. L.,
and Bernhard, S. A.
(1971)
Biochemistry
10,
4114-4120[Medline]
[Order article via Infotrieve]
-
Maglothin, J. A.,
Wins, P.,
and Wilson, I. B.
(1975)
Biochim. Biophys. Acta
403,
370-387[Medline]
[Order article via Infotrieve]
-
Ellman, G. L.,
Courtney, K. D.,
Andres, J., V.,
and Featherstone, R. M.
(1961)
Biochem. Pharmacol.
7,
88-95[CrossRef][Medline]
[Order article via Infotrieve]
-
Riddles, P. W.,
Blakeley, R. L.,
and Zerner, B.
(1979)
Anal. Biochem.
94,
75-81[Medline]
[Order article via Infotrieve]
-
Kitz, R. J.,
Ginsburg, S.,
and Wilson, I. B.
(1967)
Mol. Pharmacol.
3,
225-232[Abstract]
-
Hosea, N. A.,
Radic, Z.,
Tsigelny, I.,
Berman, H. A.,
Quinn, D. M.,
and Taylor, P.
(1996)
Biochemistry
35,
10995-11004[CrossRef][Medline]
[Order article via Infotrieve]
-
Berman, H.,
and Leonard, K.
(1990)
Biochemistry
29,
10640-10649[Medline]
[Order article via Infotrieve]
-
Radic, Z.,
Quinn, D. M.,
Vellom, D. C.,
Camp, S.,
and Taylor, P.
(1995)
J. Biol. Chem.
270,
20391-20399[Abstract/Free Full Text]
-
Costagli, C.,
and Galli, A.
(1998)
Biochem. Pharmacol.
55,
1733-1737[CrossRef][Medline]
[Order article via Infotrieve]
-
Barlow, P. N.,
Acheson, S. A.,
Swanson, M. L.,
and Quinn, D. M.
(1987)
J. Am. Chem. Soc.
109,
253-257
-
Radic, Z., and Taylor, P. (1999) Chem.-Biol. Interact., in
press
-
Radic, Z.,
and Taylor, P.
(1998)
in
Structure and Function of Cholinesterases and Related Proteins (Doctor, B. P., Taylor, P., Quinn, D. M., Rotundo, R. L., and Gentry, M. K., eds), pp. 211-214, Plenum Press, New York
-
Albaret, C.,
Lacoutiere, S.,
Ashman, W. P.,
Foment, D.,
and Fortier, P.-L.
(1997)
Proteins
28,
543-555[CrossRef][Medline]
[Order article via Infotrieve]
-
Rosenberry, T. L.,
Rabl, C. R.,
and Neumann, E.
(1996)
Biochemistry
35,
685-690[CrossRef][Medline]
[Order article via Infotrieve]
-
Barak, D.,
Kronman, C.,
Ordentlich, A.,
Ariel, N.,
Bromberg, A.,
Marcus, D.,
Lazar, A.,
Velan, B.,
and Shafferman, A.
(1994)
J. Biol. Chem.
269,
6296-6305[Abstract/Free Full Text]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.