Báo cáo Y học: Concentration-dependent reversible activation-inhibition of human butyrylcholinesterase by tetraethylammonium ion pptx

Concentration-dependent reversible activation-inhibition of human
butyrylcholinesterase by tetraethylammonium ion
Jure Stojan
1
, Marko Golic
ˇ
nik
1
, Marie-The
´
re
`
se Froment
2
, Francois Estour
2
and Patrick Masson
2
1
Institute of Biochemistry, Medical Faculty, University of Ljubljana, Slovenia;
2
Centre 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 m
M
, 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 t o determine whether the peripheral anionic s ite (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 °Cand
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 h as been put forward: TEA competes
with the substrate at the perip heral site thus inhibiting the
substrate h ydrolysis a t t he CS. As the inhibitio n by TEA is
less effective than the substrate inhibition itself, it mimics
activation. At the c oncentrations around 40 m
M
, well within
the range of TEA competition at both substrate binding sites,
it lowers the a ctivity of all tested enzymes.
Keywords: cholinesterases; tetraalkylammonium com-
pounds; k inetics; 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 f unction 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, a n 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 interm ediate substrate concentrations BuChE
hydrolyses its optimal substrate BTC with rates e xceeding
those expected by simple Michaelis–Menten dependence
and it is slightly inhibited by excess BTC [8,9]. In contrast,
AChE shows only n egative p seudo-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 t o
locate the s ite of i nteraction betwee n BuChE and tetra-
alkylammonium (TAA) salts responsible for activation a nd
to reach a mechanistic explanation of the phenomenon. In
particular, tetraethylammonium (TEA) at t he concentra-
tions above 40 m

M
, r eversibly inhibits the wild-type human
BuChE, but at the concentrations around 10 m
M
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 s ites: a peripheral anionic site ( PAS) located at
the mouth of the ac tive 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, w e 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 1 543 7641, Tel.: + 386 1 5437649,
E-mail:
Abbreviations: AChE, acetylcholinesterase; BuChE, butyrylcholine-
sterase; CS, catalytic site; PAS, peripheral anionic site; TAA, tetra-
alkylamonium; TEA, tetraethylammonium; BTC, b utyrylthiocholine;
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
/>(Received 27 September 2001, revised 17 December 2001, accepted 19
December 2001 )
Eur. J. Biochem. 269, 1154–1161 (2002) Ó FEBS 2002
recombinant human enzyme in order to see whether such a
low activity mutant still can tie up substrate by binding it
with high affinity.
MATERIALS AND METHODS
Chemicals and equipment
Butyrylthiocholine and buffer components of biochemical
grade were purchased from Sigma Chemical Co. (St Louis,
MO, USA). Te traethylammonium 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 c urried out on a Hi-Tech
(Salisbury, UK) PQ/SF-53 stopped-flow apparatu s 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 a nd mutant enzymes were expressed in stably
transfected CHO cells as previously described [9].
Horse Serum BuChE. This was purchased from Worth-
ington. It was c hosen because the major difference b etween
human and horse BuChEs at the cleft entrance is an
additional negative charge in t he loop opposite to t he omega
loop. As the two enzymes have 90% identical amino-acid
residues [ 19], we may see t he horse enzyme, in t erms of
peripheral site differences, as a human A277V/G283D/
P285L triple mutant (W279, D283, I287 h omologous, in
Torpedo AChE).
Kinetic experiments and data analysis
Hydrolysis of BTC was measured by Ellman’s method in
0.1
M
potassium phosphate buffer, pH 7.0 at 2 5 °C [20].
The substrate concentration ranges depended on the human
enzyme mutants: 0.6 l
M
to 90 m
M
for the wild-type, 0.015–
100 m

M
for double mutants, 3–100 m
M
for W82A mutant
and 0.015–3 m
M
for A328Y mutant; the substrate concen-
trations used with the commercial horse serum BuChE were
between 0.05 and 10 m
M
. The concentration of enzyme
active sites E
0
, was determined by the method of residual
activity using CPO and/or DFP as the titrating reagents.
Inhibition experiments w ere c arried out at TEA concentra-
tions from 0 to 100 m
M
.
Initial rate data in the absence of TEA showed, in most
enzymes, de viations from Michaelis–Menten k inetics: a t
intermediate substrate concentrations an apparent activa-
tion is seen, while inhibition 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].
S þ SE À!
bk
i

SEA þ P
1
À!
ak
3
SE þ P
2
"# K
1
"# K
2
SS
þþ
SþEÀ!
k
i
EA þ P
1
À!
k
3
E þ P
2
Scheme 1.
In this scheme, E is the f ree enzyme, EA the acylated
enzyme, while SE and SEA represent the complexes with
the substrate molecule bound at the modulation site. The
products P
1
and P

2
are thiocholine and butyrate, respect-
ively. K
1
and K
2
are the equilibrium constants for the
substrate binding to the nonproductive site, while k
i
and
k
3
are t he rate constants. a and b are the partitioning
ratios.
Mixed equilibrium and steady-state assumptions [22] in
the derivation give the following rate equation:
v
0
¼
E
0
k
3
½S 1 þ a
½S
K
2

½S1þ
½S

K
2

þ
k
3
À
1þa
½S
K
2
ÁÀ
1 þa
½S
K
1
Á
k
i
À
1 þb
½S
K
1
Á
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 o f 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 ¢
K
7
I þ SE þ S À!
bk
i
SEA þ P
1
À!
ak
3
SE þ P
2
"# "# K
1
"# K
2
SS S
þþ þ
EI ¢
K
5
I þ E þ S À!
k
i

EA þ P
1
À!
k
3
E þ P
2
þþ þ
II I
"# "# K
3
"# K
4
IEI ¢
K
6
I þ IE þ S À!
dk
i
IEA þ P
1
À!
ck
3
IE þ P
2
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:
v
0
¼
E
0
k
3
½S 1 þ a
½S
K
2
þ c
½I
K
4

½S1þ
½S
K
2
þ
½I
K
4

þ
k
3
À

1þa
½S
K
2
þc
½I
K
4
ÁÀ
1þ
½S
K
1
þ
½I
K
3
þ
½I
K
5
þ
½S½I
K
1
K
7
þ
½I
2

K
3
K
6
Á
k
i
À
1þb
½S
K
1
þd
½I
K
3
Á
ð2Þ
Final evaluation o f kinetic constants relevant for e ach
individual enzyme was carried out by fitting this equation
Ó FEBS 2002 Activation inhibition of human butyrylcholinesterase (Eur. J. Biochem. 269) 1155
simultaneously to the data in the absence and presence of
TEA. We started with fixed values of parameters obtained
from the analysis w ithout the inhibitor, to determine rough
estimates of TEA binding parameters. Eventually, all
parameters were released to achieve t he 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 o f

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 an d by the mixture o f the two enzymes on
a stopped-flow apparatus. Aliquots of two solutions, one
containing the e nzyme and the other the s ubstrate and
DTNB were mixed together in t he mixing chamber of the
apparatus. The absorbance of the r eaction mixture was
recorded spectrophotometrically [20] at various concentra-
tions of the s ubstrate in the presence of 0.66 m
M
DTNB. In
order to avoid possible product modulation, we stopped the
measurement when approximately 60 l
M
concentration of
the product w as formed. T he stock s olutions of the t wo
enzymes were pre pared by d ilution 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 c oncentrations in the range from 5 l

M
to 75 m
M
.
The concentration of W82A was 16 l
M
andthatofthe
horse enzyme was 10 n
M
. 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 w as used to evaluate data o btained
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 f or single exponential
curve to each individual progress curve and than t aking
derivatives at time zero.
Model building
Modelling was performed with
WHATIF
[25], starting with
the homology built model of human BuChE (CODE

P06276) from Swiss-Model, an automated protein modeling
Fig. 1. pS c urves for the hydrolysis of butyryl-
thiocholine c atalyzed by the w ild-type, by v ar-
ious human butyrylcholinesterase mutants and
by horse butyrylcholinesterase in 0.1
M
phos-
phate buffer at p H 7.0 and 25 °C.
1156 J. Stojan et al. (Eur. J. Biochem. 269) Ó FEBS 2002
server [26], on an IBM compatible PC running under
LINUX
.
Further refinement and the molecular dynamics were
carried out using t he macromolecular simulation program
CHARMM
[27] on a cluster of fou r PCs. Topology and force
field parameters for TEA from
CHARMM
distribution c 27n1
were used. Energy minimizations were performed with a
constant dielectric constant (e ¼ 1). Electrostatic force was
treated w ithout cutoffs and van der Waals forces were
calculated with the shift method with a cutoff of 10 A
˚
.All
lysines and arginines were p rotonated and aspartic and
glutamic acids were deprotonate d. Histidines were neutral
withahydrogenonNd1.
The corrections of the starting structure were performed
in iterative steps as follows: the protein molecule w as 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 p icoseconds 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 p rotein
portions were exchanged b y t he
DGFIX
command o r by
using the
SCAN LOOP
command 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 a cetycholine f rom

Protein Data Bank entry 2ACE [15]. In order to remove
overlapping between existing wate r mole cules and newly
introduced TEA we performed 150 relaxation steps (see
previously) with fi xed protein and TEA, followed by further
150 steps without any constrains. Finally, the dynamics
simulation as described was run for 180 ps.
RESULTS
Initial rate data for the hydrolysis of BTC by five ( wild-type
and four mutants) r ecombinant 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 t his
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 p resence of TEA were used for the
determination of the k inetic parameters listed in T able 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 T EA
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 s uch 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 p rominent
in the A 328Y mutant of human BuChE. It occurs at much
lower TEA concentrations as in other enzymes, but
activation is also present. Unlike in th e wild-type enzyme,
activation in the A328Y mutant appears stronger at higher
substrate concentrations. (f) Regarding TEA inhibition, the
W82A mutant i s a special case: the inhibition emerges only
at higher substrate concentrations indicating that either t he
interaction of TEA with the free enzyme is very weak or an
Table 1. Characteristic constants for the interactions of various h uman butyrylcholinesterases and horse butyrylcholinesterse w ith butyrylthio choline
and tetraethylammonium according to S cheme 2 . Values in parenthesis are for the Michaelis–Menten m echanism (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
a 0 0 0 0.0242 ± 0.0032 0.117 ± 0.059 0.376 ± 0.053
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
3
(m
M

) 8.58 ± 3.91 23.8 ± 2.2 13.1 ± 3.4 – 0.325 ± 0.029 7.19 ± 2.8
K
4
(m
M
) 177 ± 82.9 129.4 ± 38.6 397 ± 7.5 (340) 5.7 ± 0.4 39.0 ± 8.9 –
c 0.393 ± 0.096 0.257 ± 0.118 0.407 ± 0.030 – 0 –
d 0.926 ± 0.395 0.915 ± 0.236 0.511 ± 0.049 – 0.093 ± 0.013 –
K
6
(m
M
) 2.97 ± 1.15 59.8 ± 27.0 296 ± 93 – 1.75 ± 0.22 93.7 ± 12.7
Ó FEBS 2002 Activation inhibition of human butyrylcholinesterase (Eur. J. Biochem. 269) 1157
independent 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 W 82A 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 p S diagram of calculated initial rates. It can be clearly
seen that at low s ubstrate 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 t he activity of various
human BuChEs and horse BuChE on the con-

centration of TEA at various butyrylthiocholine
concentrations. BT C concentrations for
human enzymes are 15 l
M
,25l
M
,50l
M
,
100 l
M
,1m
M
,2m
M
,3m
M
, from t he lowest
to the h ighest curve. For horse enzyme BTC
concentratio ns are: 50 l
M
, 200 l
M
,1m
M
and
2m
M
.
Fig. 3. Progress curves for the hydrolysis of

butyrylthiocholine catalyzed by the h orse
butyrylcholinesterase, by t he W82A mutant of
human butyrylcholinesterase and by the enzyme
mixture. Measurements w ere 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.
1158 J. Stojan et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Molecular 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 t he 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 (c ompare K
3
values). In A328Y
mutant and in vertebrate AChEs, the homologous F330 or
Y330 would prevent such positioning of TEA.
DISCUSSION
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 d etailed investigations on insect

AChEs, n ematode 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 i ntrodu ce it, i f 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 th em 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
(k
cat
¼ 100 min
)1
, [30]). The probability for the substrate
to encou nter 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 w ere s imilar, 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 affin ity. Of course, only a s ingle
specific interaction is possible when the enzyme is a reaction
partner i n stoichiometric a mount to the substrate. We can
conclude therefore that the lower activity of the mixture,
compared t o horse enzyme alone, is a consequence of good
affinity of W82A for BTC (17 l
M
) and rather ineffective
catalysis.
Our experiment is t he 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 s hift of the p S c urve to wards 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 c holinesterases. It was suggested that deviations from
Michaelis–Menten kinetics reflect the binding of the
substrate molecule t o the PAS [9,10,31,32]. In enzymes,
showing apparent activation, the substrate affinity for the
PAS appears t o be r elatively high, but overall catalytic
power of such enzymes is low [23]. I t seems that inhibition at
Fig. 4. Stereo view of important a ctive site residues in superimp osed structures of Torpedo acetylcholinesterase (2ACE) and human butyryl-
cholinesterase. Docked as a tetrahedral adduct is butyrylthio choline. The starting position of tetraethylammo nium is position sup erimposed on the
substrate trimethyl grou p. 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 p revent the fi nal orientation of tetraethylammonium. Labelling and numbering are a ccording to human
butyrylcholinesterase.
Ó FEBS 2002 Activation inhibition of human butyrylcholinesterase (Eur. J. Biochem. 269) 1159
substrate concentrations in the range of high affinity
binding constant, mimics apparent activation.
All this i s supported b y the inhibitory pattern of TEA on
various enzymes. The activation of some enzymes by TEA
in low concentrations might b e the conseq uence of t he
competition between the substrate and TEA at the P AS. In
comparison to t he substrate, T EA inhibits the s ubstrate
hydrolysis less effectively (d > b, see Table 1). This might
be true for all enzymes sh owing activation by TEA,
especially because in different enzymes it ÔappearsÕ at
different substrate c oncentrations (compare the wild-type
and the A328Y). At the highest TEA concentration s it
competes with the substrate at both sites, thus, inhibiting the
enzyme. The question rises, why some enzymes do not show
activation by TEA, b ut show substrate affinity at the PAS.
Two explanations come to mind. The first one would be that

thesamesubstrateorientationatthePAS,invarious
enzymes, cannot affect the events a t the acylation site. As in
the W82A mutant, t he missing bulky indole r ing a t t he
bottom of the cleft allows multiple s ubstrate 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 d ouble mutant and in the horse enzyme. In
addition to an extra n egative charge at the mouth of the cleft
in horse enzyme (D283, identical in Torpedo), t he 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 o f TEA in the cleft o f
the w ild-type human BuChE. Similar to the simulations on
human AChE [33], a gradual movement is seen of the TEA
molecule from its startin g 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 y et another position, such a
movement indicates that a t 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 w e evaluated with our six parameter
model. One could argue at this point that the model can
very exactly reproduce the experimental data bu t does not
reflect the realistic events during the catalytic process and

thus th e constants and their values are meaningless. Three
points should be emphasized in this c onnection. 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 simplificatio n 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,
k
3
in the model represents k
cat
and k
i
is in fact k
cat
/K
m
.
Secondly, the six parameters are sufficient but also neces-
sary to reproduce two deviations from Michaelian k inetics,
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 r eproduce the data, but only
with additional, more o r less realistic a ssumptions. Our
kinetic model needs no additional assumptions and a
unique set of six parameters can be e valuated 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 (K
1
vs. K
2
). We
have designed the experiment w ith t he mixture of a normal
and a low activity enzyme t o co nfirm at least one high
affinity substrate binding site in the enzyme showing two
deviations from Michaelian kine tics (see K
1
for W82A). W e
agree, that the model does not predict the exact spot and
orientation i n this binding but clearly explains the observed
deviation at t hat substrate concentration a s 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.
ACKNOWLEDGEMENTS
We thank Dr Oksana Lockrid ge (Eppley Institute, University of
Nebraska, Omaha, USA) for generously providing us with human
butyrylcholinesterase mutants. T his work was p artially supported by
the Ministry of S cience and Tech nology of the Republic of Slovenia,
Grant N o. P3-8720-0381 to J. S. and by DGA/DSP/STTC, grant no.
97/08 and 99 C O 029 to P. M.
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