To determine the site involved in the effect of TAA salts, we carried out the steady-state and progress curve analysis of BTC hydrolysis by recombinant wild-type human BuChE, by four sel
Trang 1Concentration-dependent reversible activation-inhibition of human butyrylcholinesterase by tetraethylammonium ion
Jure Stojan1, Marko Golicˇnik1, Marie-The´re`se Froment2, Francois Estour2and Patrick Masson2
1
Institute of Biochemistry, Medical Faculty, University of Ljubljana, Slovenia;2Centre de Recherches du Service de Sante´ des Arme´es, Unite´ d’Enzymologie, La Tronche, France
Tetraalkylammonium (TAA) salts are well known reversible
inhibitors of cholinesterases However, at concentrations
around 10 mM, they have been found to activate the
hydrolysis of positively charged substrates, catalyzed by
wild-type human butyrylcholinesterase (EC 3.1.1.8) [Erdoes,
E.G., Foldes, F.F., Zsigmond, E.K., Baart, N & Zwartz,
J.A (1958) Science 128, 92] The present study was
under-taken to determine whether the peripheral anionic site (PAS)
of human BuChE (Y332, D70) and/or the catalytic substrate
binding site (CS) (W82, A328) are involved in this
phenom-enon For this purpose, the kinetics of butyrylthiocholine
(BTC) hydrolysis by wild-type human BuChE, by selected
mutants and by horse BuChE was carried out at 25°C and
pH 7.0 in the presence of tetraethylammonium (TEA) It appears that human enzymes with more intact structure of the PAS show more prominent activation phenomenon The following explanation has been put forward: TEA competes with the substrate at the peripheral site thus inhibiting the substrate hydrolysis at the CS As the inhibition by TEA is less effective than the substrate inhibition itself, it mimics activation At the concentrations around 40 mM, well within the range of TEA competition at both substrate binding sites,
it lowers the activity of all tested enzymes
Keywords: cholinesterases; tetraalkylammonium com-pounds; kinetics; reaction mechanism
Acetylcholinesterase (AChE; EC 3.1.1.7) and
butyrylcholi-nesterase (BuChE; EC 3.1.1.8) are closely related serine
hydrolases [1] No clear physiological function has yet been
assigned to BuChE; it appears to play a role in neurogenesis
and neural disorders [2] and it is of pharmacological and
toxicological importance: it hydrolyses numerous ester
containing drugs [3–5] and, like AChE is inhibited by
similar compounds Thus, an understanding of BuChE
catalysis and inhibition mechanisms is of paramount
importance, especially for the research of new treatments
against organophosphate and carbamate poisoning [6], i.e
for the design of new reactivators of phosphylated
choli-nesterases and of mutated enzymes capable of hydrolyzing
organophosphates or carbamates [7]
BuChE catalysis of charged substrates and inhibition by
charged ligands are complex reactions In particular, they
show homotropic and heterotropic pseudo-cooperative
effects At intermediate substrate concentrations BuChE
hydrolyses its optimal substrate BTC with rates exceeding
those expected by simple Michaelis–Menten dependence
and it is slightly inhibited by excess BTC [8,9] In contrast, AChE shows only negative pseudo-cooperativity at high acetylthiocholine (ATC) concentrations [10,11] Further-more, some cationic ligands, such as TAA salts, choline, and also uncharged trialkylammonium compounds act as acti-vators or inhibitors, depending on both the concentration of the ligand and the substrate [12], the solvent and the presence of cosolvent [13,14] The goal of this work was to locate the site of interaction between BuChE and tetra-alkylammonium (TAA) salts responsible for activation and
to reach a mechanistic explanation of the phenomenon In particular, tetraethylammonium (TEA) at the concentra-tions above 40 mM, reversibly inhibits the wild-type human BuChE, but at the concentrations around 10 mM it accelerates BuChE catalyzed hydrolysis of positively charged substrates
The active site serine, S198 in human BuChE, is located at the bottom of a 20-A˚ deep cleft [15,16] Ligands can bind on two distinct sites: a peripheral anionic site (PAS) located at the mouth of the active site cleft, regarded as the substrate/ ligand recognition site, and the ÔanionicÕ subsite of the CS [1,15] Residues D70 (D72, Torpedo AChE numbering) and Y332 (Y334) are the key elements of the PAS in human BuChE [9,17] For positively charged substrates, the CS is W82 (W84) where the binding occurs through p–cation interactions [7,9,16] Residue A328 (F330), which is also a part of this hydrophobic subsite, was found to be involved
in substrate/inhibitor binding, too [18] To determine the site involved in the effect of TAA salts, we carried out the steady-state and progress curve analysis of BTC hydrolysis
by recombinant wild-type human BuChE, by four selected mutants (Y332A/D70G, Y332D/D70Y, W82A, A328Y) and by commercial horse serum BuChE in the presence of TEA Additionally, we tested the hydrolytic activity toward BTC of the mixture between horse enzyme and W82A
Correspondence to J Stojan, Institute of Biochemistry,
Medical Faculty, Vrazov trg 2, 1000 Ljubljana, Slovenia.
Fax: + 386 1 5437641, Tel.: + 386 1 5437649,
E-mail: stojan@ibmi.mf.uni-lj.si
Abbreviations: AChE, acetylcholinesterase; BuChE,
butyrylcholine-sterase; CS, catalytic site; PAS, peripheral anionic site; TAA,
tetra-alkylamonium; TEA, tetraethylammonium; BTC, butyrylthiocholine;
DTNB, dithiobisnitrobenzoic acid; ATC, acetylthiocholine.
Note: a coordinate file of the homology built model of human
wild-type butyrylcholin-esterase with docked TEA can be downloaded from
http://www2.mf.uni-lj.si/stojan/stojan.html
(Received 27 September 2001, revised 17 December 2001, accepted 19
December 2001)
Trang 2recombinant human enzyme in order to see whether such a
low activity mutant still can tie up substrate by binding it
with high affinity
M A T E R I A L S A N D M E T H O D S
Chemicals and equipment
Butyrylthiocholine and buffer components of biochemical
grade were purchased from Sigma Chemical Co (St Louis,
MO, USA) Tetraethylammonium chloride was obtained
from Fluka (Buchs, Switzerland), chlorpyrifos-oxon (CPO)
was from Dow Chemical Co (Indianapolis, IN, USA) and
diisopropylfluorophosphate (DFP) was from Acrosorganics
France (Noisy-le-Grand, France)
Classical kinetic experiments were performed on a
Beckman DU-7500 diode array spectrophotometer Rapid
kinetic measurements were curried out on a Hi-Tech
(Salisbury, UK) PQ/SF-53 stopped-flow apparatus
connec-ted to a SU-40 spectrophotometer and Apple E-II
micro-computer, equipped with high speed AD converter
Enzyme sources
Recombinant wild-type and mutant human BuChEs Two
amino-acid residues (D70 and Y332) in the PAS and two
(W82 and A328) in the CS, known to play a role in the
binding of positively charged ligands and in inhibition
control of BuChE, were selected The BuChE gene was
mutated to make the single mutants W82A and A328Y and
the double mutants Y332A/D70G and Y332D/D70Y
Wild-type and mutant enzymes were expressed in stably
transfected CHO cells as previously described [9]
Horse Serum BuChE This was purchased from
Worth-ington It was chosen because the major difference between
human and horse BuChEs at the cleft entrance is an
additional negative charge in the loop opposite to the omega
loop As the two enzymes have 90% identical amino-acid
residues [19], we may see the horse enzyme, in terms of
peripheral site differences, as a human A277V/G283D/
P285L triple mutant (W279, D283, I287 homologous, in
Torpedo AChE)
Kinetic experiments and data analysis
Hydrolysis of BTC was measured by Ellman’s method in
0.1M potassium phosphate buffer, pH 7.0 at 25°C [20]
The substrate concentration ranges depended on the human
enzyme mutants: 0.6 lMto 90 mMfor the wild-type, 0.015–
100 mMfor double mutants, 3–100 mMfor W82A mutant
and 0.015–3 mMfor A328Y mutant; the substrate
concen-trations used with the commercial horse serum BuChE were
between 0.05 and 10 mM The concentration of enzyme
active sites E0, was determined by the method of residual
activity using CPO and/or DFP as the titrating reagents
Inhibition experiments were carried out at TEA
concentra-tions from 0 to 100 mM
Initial rate data in the absence of TEA showed, in most
enzymes, deviations from Michaelis–Menten kinetics: at
intermediate substrate concentrations an apparent
activa-tion is seen, while inhibiactiva-tion is detectable at the substrate
concentrations approaching maximum solubility In order
to explain kinetically such observations, we analyzed the data according to the six parameter model (Scheme 1) introduced previously [21]
Sþ SE ÿ!bki
SEAþ P1 ÿ!ak3
SE þ P2
"# K1 "# K2
Sþ E ÿ!ki
EAþ P1 ÿ!k3
E þ P2 Scheme 1
In this scheme, E is the free enzyme, EA the acylated enzyme, while SE and SEA represent the complexes with the substrate molecule bound at the modulation site The products P1 and P2are thiocholine and butyrate, respect-ively K1 and K2 are the equilibrium constants for the substrate binding to the nonproductive site, while ki and
k3 are the rate constants a and b are the partitioning ratios
Mixed equilibrium and steady-state assumptions [22] in the derivation give the following rate equation:
v0 ¼
E0k3½S 1 þ a½SK
2
½S 1 þ ½SK
2
þk3
ÿ
1 þ aK2½Sÿ
1 þ aK1½S
k i
ÿ
1 þ bK1½S
ð1Þ
The corresponding kinetic parameters were evaluated by fitting this equation to the initial rate data obtained in the experiments using recombinant wild-type and the four mutated human enzymes as well as the horse enzyme For the analysis of the experiments in the presence of TEA we made an extension of the model to allow the competition between TEA and BTC at both substrate binding sites and consequently also the occupation of the two sites by two TEA molecules (Scheme 2)
SEI ¢K7 Iþ SE þ S ÿ!bki
SEAþ P1 ÿ!ak3
SEþ P2
"# "# K1 "# K2
EI ¢K5 Iþ E þ S ÿ!ki
EAþ P1 ÿ!k3
Eþ P2
"# "# K3 "# K4 IEI ¢K6 Iþ IE þ S ÿ!dki
IEAþ P1 ÿ!ck3
IEþ P2 Scheme 2
In this scheme, I stands for TEA and c and d are again the corresponding partitioning ratios
An analogous derivation as described for Scheme 1 leads
to the following rate equation:
v0¼
E0k3½S 1 þ aK½S
2þ cK½I
4
½S 1 þ½SK
2þK½I
4
þk3
ÿ 1þaK2½SþcK4½Iÿ
1þK1½SþK3½IþK5½IþK1K7½S½IþK3K6½I2
k i
ÿ 1þbK1½SþdK3½I
ð2Þ Final evaluation of kinetic constants relevant for each individual enzyme was carried out by fitting this equation
Trang 3simultaneously to the data in the absence and presence of
TEA We started with fixed values of parameters obtained
from the analysis without the inhibitor, to determine rough
estimates of TEA binding parameters Eventually, all
parameters were released to achieve the best accordance
between theoretical curves and the data It should be
stressed that some parameters in the reaction Scheme are
closely related to certain parts of data For instance, the
parameter a set to zero, would denote complete blocking of
deacylation Solubility maximum of the substrate, however,
only allows to statistically anticipate the real value unless the
clear plateau is reached [23]
The initial rate data for the W82A mutant differed
substantially from the data for other enzymes It appeared
that the hydrolysis of BTC by this enzyme obeyed
Michaelis–Menten kinetics In order to investigate the
kinetics of this mutant more closely, we measured the
hydrolysis of BTC catalyzed by the W82A mutant, by
the horse enzyme and by the mixture of the two enzymes on
a stopped-flow apparatus Aliquots of two solutions, one
containing the enzyme and the other the substrate and
DTNB were mixed together in the mixing chamber of the
apparatus The absorbance of the reaction mixture was
recorded spectrophotometrically [20] at various
concentra-tions of the substrate in the presence of 0.66 mMDTNB In
order to avoid possible product modulation, we stopped the
measurement when approximately 60 lMconcentration of
the product was formed The stock solutions of the two
enzymes were prepared by dilution of the aliquots with the
same amount of buffer The mixture was prepared by mixing together the aliquots without adding buffer In this way the mixture contained the same concentrations of the two enzymes as the solution of each individual enzyme The activities of the three solutions were now tested at various substrate concentrations in the range from 5 lMto 75 mM The concentration of W82A was 16 lM and that of the horse enzyme was 10 nM The experimental conditions were the same as in classical initial rate measurements (pH 7.0 and 25°C)
We analyzed the data for W82A by fitting a system of stiff differential equations, that described the six-parameter model in Fig 1 under combined steady-state and equilib-rium assumptions (cf [24]) to the data of all experimental progress curves simultaneously Initial rates were obtained
as numerical derivatives at zero time of each progress curve The same procedure was used to evaluate data obtained with commercial horse BuChE The initial rates at various substrate concentrations for the mixture of the two enzymes (W82A mutant and horse serum BuChE) were determined analytically by fitting the equation for single exponential curve to each individual progress curve and than taking derivatives at time zero
Model building Modelling was performed withWHATIF[25], starting with the homology built model of human BuChE (CODE P06276) from Swiss-Model, an automated protein modeling
Fig 1 pS curves for the hydrolysis of butyryl-thiocholine catalyzed by the wild-type, by var-ious human butyrylcholinesterase mutants and
by horse butyrylcholinesterase in 0.1 M phos-phate buffer at pH 7.0 and 25 °C.
Trang 4server [26], on an IBM compatible PC running underLINUX.
Further refinement and the molecular dynamics were
carried out using the macromolecular simulation program
CHARMM[27] on a cluster of four PCs Topology and force
field parameters for TEA fromCHARMMdistribution c27n1
were used Energy minimizations were performed with a
constant dielectric constant (e¼ 1) Electrostatic force was
treated without cutoffs and van der Waals forces were
calculated with the shift method with a cutoff of 10 A˚ All
lysines and arginines were protonated and aspartic and
glutamic acids were deprotonated Histidines were neutral
with a hydrogen on N d1
The corrections of the starting structure were performed
in iterative steps as follows: the protein molecule was put in
the cube of water molecules (9091), subjected to 150
relaxation steps (50 steps of steepest descent optimization,
50 steps of optimization by adopted basis Newton–Raphson
method, 50 steps of steepest descent lattice optimization)
and followed by 10 picoseconds constant pressure and
temperature (CPT) dynamic simulation (300 K, 1 bar, time
step of 1 fs) invoking the Ewald summation for calculating
the electrostatic interactions The last frame was devoided of
all water molecules but those in the coat of 2.9 A˚ around the
protein, relaxed with 100 optimization steps and checked by
the CHECK module in WHATIF The unrealistic protein
portions were exchanged by the DGFIX command or by
using theSCAN LOOPcommand in SPDBVIEWER[26] After
some 20 steps the check score improved substantially, so a
continuous simulation run was performed for 300 ps The
final frame was used in a subsequent simulation involving
TEA Docking was performed by superimposing TEA to
the trimethylamino group of docked acetycholine from
Protein Data Bank entry 2ACE [15] In order to remove
overlapping between existing water molecules and newly
introduced TEA we performed 150 relaxation steps (see
previously) with fixed protein and TEA, followed by further
150 steps without any constrains Finally, the dynamics
simulation as described was run for 180 ps
R E S U L T S
Initial rate data for the hydrolysis of BTC by five (wild-type
and four mutants) recombinant human BuChEs and horse
BuChE are presented in Fig 1 The pS diagrams show that
activation at intermediate substrate concentrations is present
in all selected enzymes except in the W82A mutant, that apparently obeys Michaelis–Menten kinetics Additionally,
to obtain comparable activities, the concentration of this mutant had to be raised almost hundred times in comparison
to the wild-type enzyme and the A328Y mutant and was still
10 times higher than the concentrations of the double mutants Experimental data in all diagrams cannot predict the extent of inhibition at saturating substrate concentrations and in the case of W82A enzyme even the plateau/optimum is not reached On the other hand, the theoretical curves for other enzymes, that were obtained by putting kinetic parameters from Table 1 into the Eqn 1, are in very good agreement with the data and they stipulate complete substrate inhibition In other words, the fitting converged with the parameter a set to zero It should be recalled that the data in the absence and presence of TEA were used for the determination of the kinetic parameters listed in Table 1 Figure 2 shows the dependence of activity on the TEA concentration of all enzymes at different substrate concen-trations From the panels in this figure we can see some important characteristics: (a) activation at low TEA concentrations is clearly visible in the wild-type enzyme, in the A328Y mutant and in the ÔcompensatoryÕ mutant (Y332D/D70Y) It can only be perceived in Y332A/D70G mutant but is absent in the W82A and in horse enzyme (b)
In the wild-type enzyme the activation is the most prom-inent at intermediate substrate concentrations (c) Increas-ing inhibition at higher TEA concentrations is seen in all enzymes and the curves in the presence of the lowest substrate concentration approach to zero This is the most evident in the wild-type and A328Y enzymes The linear decrease in double mutants also indicates such a tendency (d) Interestingly, TEA shows no activation of commercial wild-type horse BuChE at any concentration Moreover, inhibition by TEA is very effective and the rate of hydrolysis clearly approaches zero even at the highest substrate concentration (e) Inhibition by TEA is the most prominent
in the A328Y mutant of human BuChE It occurs at much lower TEA concentrations as in other enzymes, but activation is also present Unlike in the wild-type enzyme, activation in the A328Y mutant appears stronger at higher substrate concentrations (f) Regarding TEA inhibition, the W82A mutant is a special case: the inhibition emerges only
at higher substrate concentrations indicating that either the interaction of TEA with the free enzyme is very weak or an
Table 1 Characteristic constants for the interactions of various human butyrylcholinesterases and horse butyrylcholinesterse with butyrylthiocholine and tetraethylammonium according to Scheme 2 Values in parenthesis are for the Michaelis–Menten mechanism (see Discussion).
Wild-type
(39.5 n M )
Y332D/D70Y (220 n M )
Y332A/D70G (245 n M )
W82A (2.4 l M )
A328Y (39 n M )
Horse BuChE (10 n M )
k i ( M )1 Æs)1) 3.45 ± 0.46 · 10 6
6.88 ± 0.97 · 10 5
7.82 ± 0.57 · 10 5
(1.44) 88.2 ± 1.3 2.08 ± 0.12 · 10 7
8.51 ± 0.2 · 10 6
k 3 (s)1) 467 ± 26 113 ± 2 132 ± 7 (0079) 18.0 ± 0.6 3800 ± 1700 1282 ± 87
K 1 (l M ) 46.9 ± 7.7 292 ± 136 60.9 ± 9.9 17.0 ± 3.4 25.5 ± 2.9 100 ± 9.9
K 2 (m M ) 77.2 ± 12.5 88.6 ± 4.2 85.3 ± 9.3 0.27 ± 0.05 1.03 ± 0.92 38.8 ± 9.3
b 0.028 ± 0.005 0.440 ± 0.056 0.166 ± 0.015 0.0119 ± 0.0022 0.0114 ± 0.0017 0.134 ± 0.032
K 4 (m M ) 177 ± 82.9 129.4 ± 38.6 397 ± 7.5 (340) 5.7 ± 0.4 39.0 ± 8.9 –
Trang 5independent binding of TEA and substrate at low
concen-trations occurs on different sites
In order to find out the reason for the very low activity of
the W82A mutant, we tested the activity of the enzyme
mixture: W82A human BuChE and horse BuChE Figure 3
shows the progress curves obtained in this experiment and
the pS diagram of calculated initial rates It can be clearly
seen that at low substrate concentrations the mixture of the enzymes is less active than the horse enzyme alone Additionally, the theoretical curves for W82A mutant agree very good with the experimental progress curves It should
be stressed, however, that we could only achieve such an agreement with six-parameter model according to Scheme 1 and not with simple Michaelis–Menten reaction mechanism
Fig 2 Dependence of the activity of various human BuChEs and horse BuChE on the con-centration of TEA at various butyrylthiocholine concentrations BTC concentrations for human enzymes are 15 l M , 25 l M , 50 l M ,
100 l M , 1 m M , 2 m M , 3 m M , from the lowest
to the highest curve For horse enzyme BTC concentrations are: 50 l M , 200 l M , 1 m M and
2 m M
Fig 3 Progress curves for the hydrolysis of butyrylthiocholine catalyzed by the horse butyrylcholinesterase, by the W82A mutant of human butyrylcholinesterase and by the enzyme mixture Measurements were performed at substrate concentrations ranging from 5 l M to
75 m M Lower right panel shows the depend-ence of the initial rates in the form of pS curves.
Trang 6Molecular dynamics calculations on the wild-type human
BuChE in water reveal after 180 ps very interesting TEA
positioning From the starting site in the vicinity of W82
indole ring it moved upward the cleft and accommodated
just below Y332 and D70, the major constituents of the PAS
in human BuChE (Fig 4) It seems that the A328 plays a
role in this rapid movement (compare K3values) In A328Y
mutant and in vertebrate AChEs, the homologous F330 or
Y330 would prevent such positioning of TEA
D I S C U S S I O N
The kinetic behavior of ChEs shows deviations from the
Michaelis–Menten model Although it has long been
believed that the only deviation in vertebrate AChEs is
inhibition by excess substrate and that BuChEs are
analo-gically activated, a more detailed investigations on insect
AChEs, nematode enzymes and also BuChEs from various
sources reveal both phenomena [28]
Recent studies on various mutated enzymes showed, that
an appropriate mutation can mask one or the other
deviation, but can also introduce it, if missing (cf
[10,23,29]) Exactly this can be seen from our experiments
in Fig 1 While the pS curve for the wild-type enzyme
shows clearly deviations at intermediate and very high
substrate concentrations none of them is evident in the
W82A mutant However, the progress curves for W82A in
Fig 3, which include the information at very low substrate
concentrations (the plateaus confirm complete hydrolysis),
can only be explained by introducing an additional
deviation from Michaelian kinetics into the reaction
mech-anism (see parameters in Table 1) Moreover, we could also
speculate that unless prevented by the solubility maximum,
inhibition too might occur Consequently, in W82A mutant
a plateau/optimum shift towards higher substrate
concen-trations appeared to take place The explanation for such a
shift may be the very low turnover of this mutant
(kcat¼ 100 min)1, [30]) The probability for the substrate
to encounter the acylation site correctly is so low, that the
possible perturbations at the PAS are kinetically invisible Slow acylation, again, should be the consequence of changed architecture in the catalytic site which firstly, cannot help to accommodate the substrate in forming Michaelis–Menten complex and secondly, enhance the stability of the acylated enzyme
In order to find out whether the extremely poor activity of the W82A mutant is due to low affinity for the substrate and/or to the slow acylation-deacylation, we mixed W82A mutant with horse serum BuChE and tested the hydrolytic activity of the mixture at low BTC concentrations The aim was to perform the experiments where the concentrations of the substrate and the W82A mutant were similar, while the concentration of the horse enzyme was at least thousand times lower Under such conditions it might be incorrectly assumed that the low activity enzyme in such large concentration must tie up substrate by binding it to a number of sites with varying affinity Of course, only a single specific interaction is possible when the enzyme is a reaction partner in stoichiometric amount to the substrate We can conclude therefore that the lower activity of the mixture, compared to horse enzyme alone, is a consequence of good affinity of W82A for BTC (17 lM) and rather ineffective catalysis
Our experiment is the first kinetic evidence that in spite of high substrate affinity the activity of a cholinesterase may be very low It is well known that transition state analogues are extremely good inhibitors As BTC is the substrate, a substantional shift of the pS curve towards high concentra-tions suggests the inability to reach transition state rather to stabilize in it It is well founded therefore to corroborate this finding with apparent activating deviation from Michaelis– Menten kinetics, which has also been reported for several other cholinesterases It was suggested that deviations from Michaelis–Menten kinetics reflect the binding of the substrate molecule to the PAS [9,10,31,32] In enzymes, showing apparent activation, the substrate affinity for the PAS appears to be relatively high, but overall catalytic power of such enzymes is low [23] It seems that inhibition at
Fig 4 Stereo view of important active site residues in superimposed structures of Torpedo acetylcholinesterase (2ACE) and human butyryl-cholinesterase Docked as a tetrahedral adduct is butyrylthiocholine The starting position of tetraethylammonium is position superimposed on the substrate trimethyl group An intermediate position of tetraethylammonium and final position (uppermost) after 180 picoseconds molecular dynamics are also seen Note the overlaping of Torpedo AChE residue F330 and tetraethylammonium in the final position Corresponding A328 in butyrylcholinesterase does not prevent the final orientation of tetraethylammonium Labelling and numbering are according to human butyrylcholinesterase.
Trang 7substrate concentrations in the range of high affinity
binding constant, mimics apparent activation
All this is supported by the inhibitory pattern of TEA on
various enzymes The activation of some enzymes by TEA
in low concentrations might be the consequence of the
competition between the substrate and TEA at the PAS In
comparison to the substrate, TEA inhibits the substrate
hydrolysis less effectively (d > b, see Table 1) This might
be true for all enzymes showing activation by TEA,
especially because in different enzymes it ÔappearsÕ at
different substrate concentrations (compare the wild-type
and the A328Y) At the highest TEA concentrations it
competes with the substrate at both sites, thus, inhibiting the
enzyme The question rises, why some enzymes do not show
activation by TEA, but show substrate affinity at the PAS
Two explanations come to mind The first one would be that
the same substrate orientation at the PAS, in various
enzymes, cannot affect the events at the acylation site As in
the W82A mutant, the missing bulky indole ring at the
bottom of the cleft allows multiple substrate orientations at
the acylation site, thus preventing the influence of the ligand
from the PAS In this enzyme the weak inhibition at the
highest TEA concentrations corroborates the explanation
The second plausible possibility would be different
orien-tation of the substrate at the PAS It might be the case in the
Y332A/D70G double mutant and in the horse enzyme In
addition to an extra negative charge at the mouth of the cleft
in horse enzyme (D283, identical in Torpedo), the substrate
affinity at the PAS of both these enzymes appears lower as
in other tested enzymes
The important role of PAS residues is further supported
by docking and dynamics simulations of TEA in the cleft of
the wild-type human BuChE Similar to the simulations on
human AChE [33], a gradual movement is seen of the TEA
molecule from its starting position at p-electron interactions
with W82 indole ring, upwards to the vicinity of the two
PAS constituting residues, Y332 and D70 Although longer
simulation run might reveal yet another position, such a
movement indicates that at low concentrations TEA might
preferentially occupy PAS, thus, preventing substrate to
bind and to inhibit its own metabolization
Finally, we would like to discuss the significance of
kinetic parameters that we evaluated with our six parameter
model One could argue at this point that the model can
very exactly reproduce the experimental data but does not
reflect the realistic events during the catalytic process and
thus the constants and their values are meaningless Three
points should be emphasized in this connection Firstly, the
kinetic model is one possible reduction of the traditional
reaction scheme generally valid for all cholinesterases It
assumes that Michaelis–Menten complex is not
accumula-ting, but it does not deny it Such an approach is well
justified in the kinetic analysis and the simplification is
introduced according to well known principles [34]
More-over, some parameters can easily be interpreted with the
classical terms of Michalis–Menten kinetics For instance,
k3 in the model represents kcat and ki is in fact kcat/Km
Secondly, the six parameters are sufficient but also
neces-sary to reproduce two deviations from Michaelian kinetics,
for which more and more evidence exists, that they are a
rule rather an exception with cholinesterases It should be
very clear, that many realistic models with more than six
parameters can equally precise reproduce the data, but only
with additional, more or less realistic assumptions Our kinetic model needs no additional assumptions and a unique set of six parameters can be evaluated if the two deviations can be inspected
In conclusion, the major arguing point in the interpret-ation of the results obtained by this model is a great difference between the binding of the substrate to the modifier site of free and acylated enzyme (K1vs K2) We have designed the experiment with the mixture of a normal and a low activity enzyme to confirm at least one high affinity substrate binding site in the enzyme showing two deviations from Michaelian kinetics (see K1for W82A) We agree, that the model does not predict the exact spot and orientation in this binding but clearly explains the observed deviation at that substrate concentration as homotropic inhibition rather as activation The concentration dependent activation-inhibition pattern by TMA and other quaternary and tertiary ammonium compounds [12] strongly supports this interpretation
A C K N O W L E D G E M E N T S
We thank Dr Oksana Lockridge (Eppley Institute, University of Nebraska, Omaha, USA) for generously providing us with human butyrylcholinesterase mutants This work was partially supported by the Ministry of Science and Technology of the Republic of Slovenia, Grant No P3-8720-0381 to J S and by DGA/DSP/STTC, grant no 97/08 and 99 CO 029 to P M.
R E F E R E N C E S
1 Massoulie´, J., Pezzementi, L., Bon, S., Krejci, E & Vallette, F.M (1993) Molecular and cellular biology of cholinesterases Prog Neurobiol 41, 31–91.
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