From the Laboratoire de Synthèse et
Physicochimie des Molécules d'Intérêt Biologique UMR
5068, Université Paul Sabatier, 31062 Toulouse, France and the
¶ Institut de Pharmacologie et de Biologie Structurale, CNRS, 205 route de Narbonne, 31077 Toulouse, France
Received for publication, June 25, 2000, and in revised form, February 14, 2001
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ABSTRACT |
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Insect acetylcholinesterase (AChE), an enzyme
whose catalytic site is located at the bottom of a gorge-like
structure, hydrolyzes its substrate over a wide range of concentrations
(from 2 µM to 300 mM). AChE is
activated at low substrate concentrations and inhibited at high
substrate concentrations. Several rival kinetic models have been
developed to try to describe and explain this behavior. One of
these models assumes that activation at low substrate concentrations
partly results from an acceleration of deacetylation of the acetylated
enzyme. To test this hypothesis, we used a monomethylcarbamoylated enzyme, which is considered equivalent to the acylated form of the
enzyme and a non-hydrolyzable substrate analog,
4-oxo-N,N,N-trimethylpentanaminium iodide. It appears that this substrate analog increases the
decarbamoylation rate by a factor of 2.2, suggesting that the substrate
molecule bound at the activation site (Kd = 130 ± 47 µM) accelerates deacetylation. These two
kinetic parameters are consistent with our analysis of the hydrolysis
of the substrate. The location of the active site was investigated by
in vitro mutagenesis. We found that this site is located at
the rim of the active site gorge. Thus, substrate positioning at
the rim of the gorge slows down the entrance of another substrate
molecule into the active site gorge (Marcel, V., Estrada-Mondaca, S.,
Magné, F., Stojan, J., Klaébé, A., and Fournier, D. (2000) J. Biol. Chem. 275, 11603-11609) and also
increases the deacylation step. This results in an acceleration of
enzyme turnover.
The cholinesterases
(ChE),1 including
acetylcholinesterases (AChE) and butyrylcholinesterases (BuChE), are
serine hydrolases that catalyze the hydrolysis of choline esters in two
steps, enzyme acylation followed by deacylation involving a water
molecule (1). This very efficient catalysis shows an acetylcholine
hydrolysis rate close to the diffusion-controlled limit (2). The
process takes place at the acylation site located at the bottom of a
long narrow active site gorge. This site includes a tryptophan (Trp-84 using the numbering of the Torpedo enzyme) that interacts
with the trimethylammonium group of acetylcholine and a serine
(Ser-200) that is acylated and deacylated (hydrolyzed) during substrate turnover (3). Another substrate binding site, located at the mouth of
the active site gorge, has been discovered. This site is non-productive
and is thought to be the initial binding site for substrate on the ChE
catalytic pathway (4, 5). This binding decreases association and
dissociation substrate rate constants at the acylation site and blocks
product release (5, 6).
Analysis of substrate hydrolysis at different concentrations reveals
that insect cholinesterases display activation at low substrate
concentrations similar to vertebrate BuChE and inhibition at high
substrate concentrations similar to vertebrate AChE (7). Analysis of
product-substrate curves and the effects of a competitive inhibitor of
activation led to the hypothesis that substrate binding at the
peripheral site accounts for apparent activation by affecting the
entrance rate constant of a second molecule to the catalytic site (8,
9). Although apparent activation may be explained by steric hindrance
at the mouth of the gorge, it cannot be excluded that another mechanism
participates in the activation. Here, we consider a second hypothesis
proposed by Eriksson and Augustinsson in 1979 (10): the modulation of
the deacylation rate constant follows the binding of an additional
substrate molecule to the acyl-enzyme intermediate.
Enzyme Sources--
Truncated cDNA encoding soluble AChEs
(wild-type and mutated) from Drosophila melanogaster was
expressed with the baculovirus system (11). Secreted AChEs were
purified and stabilized with 1 mg ml Chemicals--
Acetylcholine (ACh), acetylthiocholine (ATCh),
Triton X-100, edrophonium and propidium were purchased from Sigma (St
Louis, MO). Carbaryl, i.e. 1-napthyl methylcarbamate, was
purchased from Cil Cluzeau Info Labo (Sainte-Foi-La-Grande, France).
The substrate analog i.e.
4-oxo-N,N,N-trimethylpentanaminium
iodide (14) was synthesized according to Thanei-Wyss and Waser (15).
7-(methylethoxyphosphinyloxy)-1-methyl-quinolinium iodide was
synthesized as described by Levy and Ashani (Ref. 16; Fig. 1).
Kinetics of Substrate Hydrolysis--
Kinetics were studied at
25 °C in 25 mM phosphate buffer, pH 7, 1 mg
ml Determination of the Decarbamoylation Rate Constant of
AChE--
Enzyme was incubated with carbaryl in 25 mM
phosphate buffer, pH 7, 1 mg ml Determination of Kinetic Constants--
Data were analyzed by
multiple non-linear regression using a non-linear global optimization
method based on simulated
annealing.2
Acceleration of the Decarbamoylation Rate by the Substrate
Analog--
The study of the effect of potential deacetylation
accelerators on acetyl-enzyme was not possible because the
deacetylation rate was too rapid. To slow down this step, we used an
analog of acetyl-enzyme, the carbamoyl-enzyme. The decarbamoylation
rate constant of the wild-type enzyme (0.79 × 10
To test if the substrate molecule accelerates decarbamoylation of the
enzyme, we first used acetylcholine and acetylthiocholine. However,
because some free enzyme always remains in solution, substrate
hydrolysis occurs releasing choline or thiocholine molecules into the
solution. These latter molecules accelerate decarbamoylation (18) by
transcarbamoylation because AChE is able to catalyze transesterification (19, 20). With the Drosophila enzyme, acceleration of decarbamoylation by choline and thiocholine at 1 mM was 4.7 and 140-fold, respectively. Consequently, it was impossible to separate the potential acceleration caused by the substrate molecule from that caused by choline or thiocholine.
Thus to analyze acceleration of decarbamoylation by a substrate
molecule, we synthesized a substrate analog,
4-oxo-N,N,N-trimethylpentanaminium iodide, which cannot be hydrolyzed (Fig.
1). We checked that this molecule is
indeed a competitive inhibitor of the substrate ATCh (data not shown).
The decarbamoylation rate was determined in the presence of various
concentrations of substrate analog from 10-500 µM (Fig.
2). Considering only the decarbamoylation
step and one non-productive binding site, the data were analyzed
according to Scheme 1, where Ec
represents the monomethylcarbamoylated AChE; E, the decarbamoylated
AChE; S, the substrate analog; SEc the substrate analog molecule bound
on the peripheral site; kr, the decarbamoylation
rate constant; a, the coefficient of acceleration or
deceleration; and Kd, the dissociation constant of the substrate analog for the peripheral site.
Effect of Ligands on the Decarbamoylation Rate--
We
investigated the effect of several ligands on the decarbamoylation rate
of monomethylcarbamoylated Drosophila AChE: Triton X-100,
propidium, edrophonium, CHAPS, tetramethylammonium, and decamethonium.
The results are presented in Table I. The
fit of data was achieved with Equation 2, previously used for the substrate analog. All ligands tested showed a significant effect on the
decarbamoylation rate of the enzyme, where a decrease or increase was
observed. This effect has already been reported by Roufogalis and
Thomas (21) who described the acceleration of decarbamoylation by
tetramethylammonium using bovine erythrocyte AChE. However the effect
of ligand binding varies depending on the enzyme species, because a
deceleration by tetramethylammonium using Drosophila AChE
was observed (Table I). Effects were small for monovalent cations; the
strongest activation having a factor of 3 and the greatest inhibition a
factor of 1.6. This suggests that ligand binding to the rim or the
bottom of the gorge has a limited effect on deacylation.
Furthermore, this experiment allowed us to determine the affinity
of each ligand for the carbamoylated enzyme (Table I), which we
compared with its affinity for the free enzyme that we had previously
estimated (9). The affinity of edrophonium for the free enzyme
(Kd = 0.84 µM) was higher than for the carbamoyl form (Kd = 4.7 µM). This
could be explained by the fact that acetylation should change the
edrophonium binding site located at the bottom of the active site
gorge, at the catalytic site (22). By contrast, the affinity of
propidium was not significantly different for the free enzyme
(Kd = 119 nM) and for the carbamoyl form
(Kd = 92 nM). This is in accordance with the observation of Taylor and Lappi (23) who reported that the affinity of propidium is not affected by methanesulfonylation of
Torpedo californica AChE. The affinity of Triton X-100 for the free enzyme and for the carbamoyl form was also not significantly different: Kd = 0.052 g/liter and
Kd = 0.047 g/liter, respectively. Thus, binding of a
ligand at the rim of the active site gorge does not seem to be affected
by acetylation.
Kinetics of Substrate Hydrolysis--
We previously used a simple
model to describe the kinetic pathway of Drosophila AChE
activity as a function of substrate concentrations (8), AChE being
activated at low substrate concentrations and inhibited at high
substrate concentrations. This model indicated that activation of
substrate hydrolysis at low substrate concentrations could be explained
by the binding of a substrate molecule at the peripheral non-productive
site, located at the rim of the active site gorge, which affects the
entrance of another substrate molecule into the active site gorge (9).
However, in this model, an increase of the deacylation rate as a
possible additional explanation of the activation phenomenon was not
considered. Because activation of Drosophila AChE results in
part from an acceleration of the deacetylation (Fig. 2), we had to
change our previous kinetic model. Moreover, a previous model
hypothesized that the affinity of the activation site for the substrate
molecule decreases when the enzyme is acetylated. As carbamoylation did
not affect the affinity of ligands that bind at the rim of the gorge
such as propidium and Triton X-100 (Table I and Ref. 9), we could hypothesize that acetylation does not affect the binding of a substrate
molecule at the rim of the gorge. Consequently, the same parameter
Ks1 (the affinity of the substrate molecule for the peripheral binding site involved in activation) could be applied to
the free enzyme and the acetylated form. Taking into account that
inhibition of substrate hydrolysis occurs for high substrate concentrations (above 1 mM), we propose here a new model,
which considers the existence of two different non-productive sites, where substrate molecules (S1 and S2) bind but are not hydrolyzed (Scheme 2). In this way, both activation of the enzyme activity at low
substrate concentrations and its inhibition at higher substrate concentrations could be explained. Binding of S1 would result in
substrate hydrolysis activation and binding of S2 would result in
substrate hydrolysis inhibition. In Scheme
2, E represents the AChE; EA, the acetyl
enzyme; S1, the substrate molecule responsible for activation; S2, the
substrate molecule responsible for inhibition; a, the
coefficient of acceleration; b, the coefficient of
inhibition of the deacylation rate constant; c, the effect
of peripheral activation site occupation on the entrance of a new
molecule inside the gorge; and d, the effect of peripheral
inhibition site occupation on the entrance of a new molecule inside the
gorge. ki represents the bimolecular rate
constant for acylation and kcat the rate constant for deacylation. Ks1 and
Ks2 represent the dissociation constants of S1
and S2 for the peripheral activation site and the peripheral inhibition
site, respectively. Free substrate molecules and reaction products are
not represented for clarity of the scheme.
The rate of ATCh hydrolysis was measured versus substrate
concentration, and data were fitted using Equation 3, which was derived
from Scheme 2 (Fig. 3, Equation
3). But the fitting yielded a poor estimation of the numerous
kinetic parameters. To be able to fit data accurately, we fixed two
parameters, Ks1 and a, previously estimated by the effect of the substrate analog on the decarbamoylation rate. Parameter d was not necessary to fit data. It
represents the effect of occupation of the inhibition site on the
entrance of a new substrate molecule. To simplify, we can hypothesize
that peripheral inhibition site occupation completely blocks the
entrance of a new molecule inside the gorge (d = 0).
Accordingly, Scheme 2 can be simplified into Scheme
3.
The data of the rate of ATCh hydrolysis versus substrate
concentration were fitted using Equation 4, which was derived from Scheme 3 (Fig. 4, Equation 4),
after having fixed two parameters obtained by studying the effect of
the substrate analog on the decarbamoylation rate of AChE. The
activation coefficient of the deacetylation rate constant
(a) was fixed at 2.2 and the dissociation constant of S1
(Ks1) for the peripheral activation site was
fixed at 130 µM (Table I). The fit was obtained with
0.996 as the global correlation coefficient and 5.2% as the average
relative deviation per point (Fig.
5).
Acceleration of the Decarbamoylation Rate of Mutated
Enzymes--
To locate the amino acids involved in the binding of
substrate analogs and in signal transmission, several enzymes
with a mutation on residues lining the
active site gorge were used (Table II, Fig.
6). The effect of the substrate analog on
the decarbamoylation rate of mutated enzymes was studied from a
concentration of 1 µM to 1 mM substrate
analog. Several mutations induced a decrease in acceleration, which was
in some cases no longer detectable. This could be interpreted either by
a decrease in the affinity of the activation site for the substrate
analog (Kd >1 mM) or by the absence of
the effect of substrate analog binding on the activation site on
decarbamoylation rate (a is not significantly different from
1). For only one mutant, Y374A, the binding of the substrate analog
induced a decrease in the decarbamoylation rate (a <1).
Substrate Binding Sites in Drosophila AChE--
Data from ATCh
hydrolysis versus substrate concentration can be analyzed
considering the existence of three sites: a productive site where
binding of substrate molecule triggers its hydrolysis, and two distinct
non-productive sites, one responsible for the activation and the other
for the inhibition of ATCh hydrolysis by Drosophila AChE.
The productive site, also called the catalytic site, is located at the
bottom of the gorge with Trp-83(84) as a key component interacting with
the quaternary ammonium moiety of choline (24, 25, 26). Some ligands
such as edrophonium and N-methylacridinium are specific for
this site (22, 27). A non-productive site, also called a peripheral
binding site, was first proposed from steady-state kinetic inhibition
with various ligands (28). It has been implicated by equilibrium
dialysis experiments (29), by direct fluorescence (23, 27, 30), and by
NMR (31). Site-directed labeling (24), site-directed mutagenesis (32),
and x-ray crystallography (22, 33) have shown that this peripheral site
is located at the rim of the active site gorge and that Trp-321(279)
was a key component. A multiplicity of subsites, designated as P1
through P4 by Rosenberry (34), has been implicated on the basis of
inhibition kinetics (35, 36). The existence of these subsites were
confirmed by site-directed mutagenesis, which showed that each
inhibitor binds in a distinct manner (32). The presence of several
ligand peripheral sites is consistent with the presence of two
non-productive substrate binding sites, one responsible for the
activation and the other for the inhibition of ATCh hydrolysis by
Drosophila AChE.
Significance of the Model--
The rate of ATCh hydrolysis was
measured versus substrate concentration. Fig. 4 shows the
goodness of the fit (0.996 as global correlation coefficient and 5.2%
as average relative deviation per point), allowing us to consider
Scheme 3 as a good model for the kinetic pathway of
Drosophila AChE activity. Thus we could interpret the
regulation of AChE activity as the binding of substrate molecules at
two non-productive sites, one being responsible for activation and one
for inhibition. However, it remains to be elucidated if these two sites
are independent or overlap, i.e. binding of the substrate
responsible for inhibition depends on the occupation of the activation
site or not. If they are dependent there are two possibilities: either
the substrate molecule responsible for inhibition binds only to
acetylated enzyme and the ternary complex (S1S2E) does not form, or the
substrate binds only to the S1EA complex and the S2EA complex does not
occur. These two possibilities were tested. Product-substrate data did
not fit the equations derived from these two simplified models,
suggesting that the two sites are independent. The deacetylation rate
constant was estimated to be 464 s Residues Affecting Deacylation Rate--
To locate potential sites
involved in the acceleration of the deacetylation rate, we studied the
effect of the binding of different ligands and the effect of a single
mutation on the decarbamoylation rate. Binding of all the ligands
tested affected the decarbamoylation rate. Edrophonium, which binds to
the catalytic site (22), increased the decarbamoylation rate. Propidium
also increased the decarbamoylation rate, binding to the rim of the
gorge on Trp-321(279) in vertebrates (25, 26, 38), similar to
Drosophila, because the dissociation constant of propidium
(8 nM for the wild-type AChE) increases to 12 µM for the W321L
mutant.3 Triton X-100
increased the decarbamoylation rate, binding at the rim of the gorge at
a site distinct from the propidium binding site with Glu-69(70) as the
main residue involved in its binding (9). CHAPS, a non-competitive
inhibitor (39), decreased the decarbamoylation rate. Taking into
account its size, it probably also binds to the rim of the gorge.
Decamethonium accelerated decarbamoylation; this bis-quaternary
ammonium ligand bridges catalytic and peripheral sites (22).
Tetramethylammonium decreased the decarbamoylation rate, and this small
quaternary ligand binds to peripheral and catalytic sites (40). Thus,
the binding of ligands to the catalytic site or to a peripheral site
affects the decarbamoylation rate, where acceleration or deceleration was observed.
Studies using mutated enzymes led to the same result: several mutations
affect the decarbamoylation rate. Mutated residues that affect the
decarbamoylation rate can be located anywhere in the gorge from the rim
to the bottom, i.e. the decarbamoylation rate can be
modified by mutations anywhere in the active site gorge. For example,
F330S, a mutation located in the acyl pocket, E237Q located at the
bottom of the gorge, F371G located at the middle of the gorge, and
V318D located at the rim of the gorge decreased the decarbamoylation
rate constant (kr, Table II).
Localization of the Substrate Activation Site--
The
acceleration in decarbamoylation rate by substrate analog was
consistent with the existence of only one substrate activation site.
This substrate activation site has been recently located at the rim of
the gorge with Glu-69(70) as the main component, using an inhibitor
competitive with activation (Triton X-100; Ref. 9). This location is
consistent with mutational effects because the three mutations located
at position 69 completely abolished activation of the decarbamoylation
rate by the substrate analog. However, other mutations also suppressed
activation by the substrate analog. We can assume that they are
involved in signal transmission because they are located between the
rim and the bottom of the gorge.
This result consolidates two hypotheses that have been proposed to
explain the activation of hydrolysis by substrate molecules in human
BuChE. In the first hypothesis, a substrate molecule binds to the
acyl-enzyme and accelerates deacylation (10, 41). In the second,
binding of a substrate molecule at the rim of the gorge allosterically
regulates the catalytic activity through a conformational change (42,
43). Results obtained for activation in insect AChE show that these two
hypotheses are not exclusive; binding of a substrate molecule at the
rim of the gorge accelerates deacylation (Table I).
Transmission of Information from the Rim to the Bottom of the
Gorge--
Several reports suggest that peripheral sites are
allosteric and affect the reactivity of the esterase site. Specific
inhibitors for some peripheral sites enhance the decarbamoylation rate
of the carbamoylated enzyme or dephosphorylation of the phosphorylated enzyme (18, 44, 45). Here, we confirm these results because Triton
X-100 and propidium that bind to the rim of the gorge increased the
rate of decarbamoylation, which takes place at the bottom of the gorge.
This allostery was also implicated by the effect of mutations at the
rim of the gorge. Mutations at the peripheral site, Asp-70(72) in human
BuChE, have been shown to be involved in activation by the substrate
(4, 46) and in dealkylation of the phosphoryl enzyme (47). Results
obtained with the Drosophila enzyme are in accordance
because mutations located at the entrance of the gorge (at positions
69(70), 71(72), 321(279), or 375(335)) cancelled the activation of
decarbamoylation, showing allostery between the rim of the gorge and
its bottom.
The question arising now is how the information is transmitted from the
rim to the bottom of the gorge. Several non-exclusive hypotheses can be
put forward. (i) The information is transmitted via the backbone; the
binding of the substrate to the rim of the gorge changes or stabilizes
a loop conformation more favorable for deacetylation. (ii) The
information is transmitted by the substrate molecule; the binding at
the rim of the gorge orients the molecule favorably, which then slides
down and increases the decarbamoylation rate at the bottom, and (iii)
the information is transmitted via motions of the side chains of amino
acids lining the gorge and of water molecules.
The first hypothesis involves the movement of a flexible loop
(
The second hypothesis involves a shift of the substrate molecule that
first binds at the rim, slides down the gorge, and then increases
deacetylation. This hypothesis agrees with the normal path of the
substrate molecule. However, inhibitor effects do not support this
because all inhibitors have a significant effect on decarbamoylation
rate. Some of them such as propidium or CHAPS are too cumbersome to
enter the gorge and most probably remain at the rim.
The third hypothesis involves side chains of the amino acids paving the
gorge. Sussman et al. (3) showed that the active site gorge
is lined by connected aromatic residues. Information could be
transmitted by changes in stacking arrangement or by gear effect.
Certain interactions between aromatic residues have been hypothesized
and some demonstrated. Shafferman et al. (25) assumed that
tyrosine (334) interacts with phenylalanine (330), which may reduce the
deacylation efficiency of the acyl-enzyme complex. Tyrosine 374(334)
has been shown to interact with tyrosine 71(72) in the
Torpedo enzyme (3) and in the Drosophila enzyme (48).
The efficiency of deacetylation is dependent on the position of a water
molecule. The location of water molecules inside the site depends on
the arrangement of the amino acid side chains lining the active site
gorge and on the position of the loops structure forming the gorge
wall. When a molecule binds somewhere in the active site gorge, the
arrangement of water molecules is modified (49) resulting in a change
of deacetylation rate. In this way, the binding of a substrate molecule
at the peripheral site would accelerate or decelerate deacylation if
the water molecule involved in the hydrolysis is closer or further from
the acetyl-serine in the new arrangement. The fact that a large number
of mutations affects the acceleration of decarbamoylation by the
substrate supports this last hypothesis and suggests that the
information is transmitted through the motions of the network of side
chains lining the gorge and by water molecules.
Roles of the Activation Site--
The binding of a substrate
molecule at the rim of the gorge seems to have several effects. It
changes the probability of a substrate molecule entering the active
site gorge (9); it may correctly orient positively charged substrates
to slide down to the bottom of the gorge (4); it may affect the
entrance of another substrate molecule inside the site (9); and it may increase deacylation of the serine (this study) and therefore clean up
the active site gorge before the entrance of a new substrate molecule.
Thus, the activation site seems to be a regulator of the traffic
between substrate molecules that enter the gorge and product molecules
that exit the gorge.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 BSA as previously
reported (12). Residue numbering was according to the sequence of the
mature Drosophila AChE (13), and the numbering in
parentheses corresponds to the Torpedo AChE sequence.
1 BSA. Hydrolysis of acetylthiocholine iodide was
followed spectrophotometrically at 412 nm using the method of Ellman
et al. (17) at substrate concentrations from 2 µM to 300 mM. Active site titration was carried out using 7-(methylethoxyphosphinyloxy)-1-methyl-quinolinium iodide.
1 BSA until more than 95%
of the enzyme was inhibited. The mixture was loaded onto a gel
filtration column (P10, Amersham Pharmacia Biotech) and eluted with 25 mM phosphate buffer, pH 7, 1 mg ml
1 BSA.
Enzyme fractions were collected. The decarbamoylation rate was followed
with time at 25 °C for 9 h by sampling aliquots of the reaction
mixture and by estimating free enzyme concentration spectrophotometrically through its activity with 1 mM ATCh.
The reaction can be described by a simple first-order rate equation. The decarbamoylation rate constant observed
(kr obs) was calculated by non-linear
regression analysis using the equation,
where [E]t represents the free enzyme
concentration at time t, [E]0 the initial concentration
of free enzyme, and [Ec]0 the initial concentration of
monomethylcarbamoylated enzyme.
(Eq. 1)
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
4
s
1) is about 106-fold slower than that of deacetylation.
The fit of data using Equation 2, which is derived from Scheme 1,
gave the following results: the decarbamoylation rate constant kr was estimated to be 79 ± 4 × 10
(Eq. 2)
6 s
1; the substrate analog activates
decarbamoylation with a coefficient of acceleration a
estimated at 2.2 ± 0.1; and the dissociation constant
Kd was estimated to be 130 ± 47 µM.
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Fig. 1.
Substrates and inhibitors.
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Fig. 2.
Acceleration of decarbamoylation in the
presence of the substrate analog.
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Scheme 1.
Effect of various ligands on the decarbamoylation rate
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Scheme 2.
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Fig. 3.
The derivation of Equation 3 from Scheme
2.
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Scheme 3.
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Fig. 4.
The derivation of Equation 4 from Scheme
3.
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Fig. 5.
Product-substrate curves for
acetylthiocholine (ATCh) hydrolysis by wild-type
Drosophila AChE. The curve was drawn
by fitting experimental data using Equation 4 (Scheme 3).
Regulation of the decarbamoylation rate of mutated enzyme by the
substrate analog
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Fig. 6.
View of the active site of
Drosophila AChE depicting the residues mutated in this
study.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1. By taking into
account the acceleration of the deacetylation rate constant of 2.2 (Table I), we calculated a turnover number of 1020 s
1, as
illustrated in Fig. 2 and which is similar to another estimation made
for Drosophila AChE turnover, 1783 s
1 (37). In
Scheme 3, the affinity of the substrate molecule for the peripheral
binding site involved in the activation is considered to be the same
for the free enzyme (E) and the acetylated form (EA). This is
consistent with the observation of inhibitors binding at a peripheral
site that is not modified by the carbamoylation of the enzyme. In
contrast, this is contradictory to an hypothesis of our previous
model, where catalytic site occupation lowers the affinity of the
substrate for the peripheral site.
-loop) from Cys-66(65) to Cys-93(94) (47). This hypothesis is
consistent with the observation that mutation of several residues located on the
-loop affects the acceleration of decarbamoylation by
substrate binding at the peripheral site (Table II). However, mutations
of some residues, such as Tyr-370 or Tyr-374, also have an important
effect. As these residues are not located on the loop, according to
this hypothesis, their mutation would affect the conformation of the
-loop.
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FOOTNOTES |
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* This research was supported by grants from DGA and CEE.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.
§ Recipient of a doctoral fellowship from CONACyT, Mexico.
To whom correspondence should be addressed: Laboratoire de
Synthèse et Physicochimie des Molécules
d'Intérêt Biologique, Groupe de Biochimie des
Protéines, Université Paul Sabatier, Bat 4R3, 31062 Toulouse, France. E-mail: fournier@cict.fr.
Published, JBC Papers in Press, February 16, 2001, DOI 10.1074/jbc.M005555200
2 J. Czaplicki, V. Marcel, and D. Fournier, manuscript in preparation.
3 D. Fournier, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: AChE, acetylcholinesterase; ChE, cholinesterase; BuChE, butyrylcholinesterase; ACh, acetylcholine; ATCh, acetylthiocholine; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; BSA, bovine serum albumin.
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REFERENCES |
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1. | Wilson, I. B., and Cabib, E. (1956) J. Am. Chem. Soc. 78, 202-207 |
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