Kinetic analysis of effector modulation of
butyrylcholinesterase-catalysed hydrolysis of acetanilides
and homologous esters
Patrick Masson
1
, Marie-The
´
re
`
se Froment
1
, Emilie Gillon
1
, Florian Nachon
1
, Oksana Lockridge
2
and
Lawrence M. Schopfer
2
1 Unite
´
d’Enzymologie, De
´
partement de Toxicologie, Centre de Recherches du Service de Sante
´
des Arme
´
es, La Tronche Cedex, France
2 University of Nebraska Medical Center, Eppley Institute, Omaha, NE, USA
Keywords

positively charged substrates. The affinity of D70G for tyramine was lower
than that of the wild-type enzyme. Tyramine activation of hydrolysis for
neutral substrates by D70G was linear. Tyramine was found to be a pure
competitive inhibitor of hydrolysis for positively charged substrates with
both wild-type butyrylcholinesterase and D70G. Serotonin inhibited both
esterase and aryl acylamidase activities for both positively charged and
neutral substrates. Inhibition of wild-type butyrylcholinesterase was hyper-
bolic (i.e. partial) with neutral substrates and linear with positively charged
substrates. Inhibition of D70G was linear with all substrates. A comparison
of the effects of tyramine and serotonin on D70G versus the wild-type
enzyme indicated that: (a) the peripheral anionic site is involved in the non-
linear activation and inhibition of the wild-type enzyme; and (b) in the
presence of charged substrates, the ligand does not bind to the peripheral
anionic site, so that ligand effects are linear, reflecting their sole interaction
with the active site binding locus. Benzalkonium acted as an activator at
low concentrations with neutral substrates. High concentrations of ben-
zalkonium caused parabolic inhibition of the activity with neutral sub-
strates for both wild-type butyrylcholinesterase and D70G, suggesting
multiple binding sites. Benzalkonium caused linear, noncompetitive inhibi-
tion of the positively charged aryl acetanilide m-(acetamido) N,N,N-trime-
thylanilinium for D70G, and an unusual mixed-type inhibition ⁄ activation
(a > b > 1) for wild-type butyrylcholinesterase with this substrate. No
fundamental difference was observed between the effects of ligands on
the butyrylcholinesterase-catalysed hydrolysis of esters and amides. Thus,
Abbreviations
AAA, aryl acylamidase; ASCh, acetylthiocholine; ATMA, m-(acetamido) N,N,N-trimethylanilinium; BuChE, butyrylcholinesterase; DFP,
diisopropylfluorophosphate; NATAc, N-acetylanthranilic acid; Nbs
2
, 5,5¢-dithiobis(2-nitrobenzoic acid); o-NA, o-nitroaniline; o-NAC,
o-nitroacetanilide; o-NP, o-nitrophenol; o-NPA, o-nitrophenylacetate; o-NTFNAC, o-nitrotrifluoroacetanilide; o-NTMNPA, o-N-trimethylnitro-

Human plasma BuChE is of toxicological and phar-
macological importance, because it scavenges and det-
oxifies numerous carboxyl ester drugs and prodrugs
[16–18], and carbamyl and phosphoryl esters, including
nerve agents [19]. Numerous widely used chemicals are
aryl acylamides (drugs: acetaminophen, phenacetin,
flutamide, isocarboxazid, lidocaine, butanilicaine; pesti-
cide: acephate; herbicides and fungicides: acetochlor,
propanil and butachlor). The AAA activity of BuChE
in plasma and tissues could participate in the metabo-
lism of these aryl acylamide drugs and xenobiotics.
However, the potential detoxification role of the AAA
activity of BuChE needs to be addressed.
Known AAAs are serine hydrolases that catalyse the
deacylation of N-acyl arylamines [20,21]. Several
AAAs have been identified in mammalian tissues
[22,23]. Certain acryl acylamidases are identical to
carboxylesterases [24]. Albumin also displays an AAA
activity [25,26]. A correspondence between certain
molecular forms of AAAs and cholinesterases has been
demonstrated in different organs [22]. Deacetylation of
retinal melatonin into 5-methoxytryptamine is cataly-
sed by an eye AAA [27]. However, no clear physiologi-
cal function has yet been ascribed to most mammalian
AAAs. At a minimum, AAAs are toxicologically rele-
vant because they deacylate arylamide xenobiotics
[20,21,28].
The crystal structures of acetylcholinesterase and
BuChE reveal that these enzymes have a common
architecture, with only one catalytic triad located at

effectors, depending on the integrity of the peripheral anionic site, reflect
the allosteric ‘cross-talk’ between the peripheral anionic site and the cata-
lytic centre.
Modulation of butyrylcholinesterase catalytic activitiy P. Masson et al.
2618 FEBS Journal 275 (2008) 2617–2631 ª 2008 The Authors Journal compilation ª 2008 FEBS
the ester site. Moreover, certain of these studies were
performed using partially purified enzymes from sera
[22,36,42], biological fluids [37,43] or commercial prep-
arations [32,42] that very probably contained serum
albumin as a contaminant. Human serum albumin has
been found to display intrinsic AAA activity [25,26].
Thus, in order to provide a complete analysis and to
clarify debated issues, we investigated the effects of
tyramine, serotonin and benzalkonium on the BuChE-
catalysed hydrolysis of neutral and charged aryl acetyl
amides [o-NAC, o-nitrotrifluoroacetanilide (o-NTF-
NAC) and m-(acetamido) N,N,N-trimethylanilinium
(ATMA)] and acetyl esters [o-nitrophenylacetate
(o-NPA) and acetylthiocholine (ASCh)] under steady-
state conditions. All of these substrates give the same
acyl enzyme intermediate. Effects on wild-type human
BuChE and its PAS mutant D70G were compared.
Because the presence of contaminating proteins dis-
playing AAA activity, e.g. albumin, in the BuChE
preparation could have biased the results, experiments
were carried out on highly purified recombinant
enzymes free of albumin and any other AAAs. It was
found that there was no fundamental difference in the
mechanisms of inhibition and activation for either the
AAA or esterase activities by these ligands. In

¼
K
s
k
3
ðk
2
þ k
3
Þ
¼
K
s
½1 þðk
2
=k
3
Þ
ð2Þ
k
cat
¼
k
2
k
3
ðk
2
þ k
3

1 þ K
m
=½S

1 þ b½S=K
ss
1 þ½S=K
ss

ð5Þ
where K
ss
is the dissociation constant of co mplexes S
p
E
and S
p
ES (K
ss
> K
m
). The parameter b reflects t he
efficiency with which S
p
ES forms p roducts. When b >1,
there is s ubstrate a ctivation; when b < 1, there is substrate
inhibition; when b = 1, the enzyme kinetics obey the
simple Michaelis–Menten model (Eqn 1). BuChE shows
substrate activation w ith ATMA (b = 1.53, K
ss

of o-NAC by wild-type BuChE [22,36,37,39]. The acti-
vating effect of tyramine yields the expected hyperbolic
Dixon and Cornish–Bowden plots (Fig. 1A,B) for
hydrolysis of both o-NPA and o-NAC. This nonessen-
tial activation can be mathematically treated in a man-
ner similar to partial mixed-type inhibition (see
Experimental procedures, Scheme 3). Similar activating
effects on the BuChE-catalysed hydrolysis of o-NPA
have been reported for the positively charged ligands
dibucaine [45], amiloride [46] and tetraalkylammonium
compounds [47]. This activation was interpreted in
Table 1. Effect of ligands (tyramine, serotonin and benzalkonium) on esterase and AAA activities of BuChE with the neutral substrates o-
NPA versus o-NAC. Values are means ± standard error from three to five independent determinations. H, hyperbolic; L, linear; P, parabolic.
These terms refer to the appearance of the Dixon plots. Hyperbolic curves appear when there is partial inhibition or when there is activation.
Parabolic curves indicate multiple ligand binding. A, activation; C, competitive; I, inhibition. ND, not determined.
Substrate
Tyramine Serotonin Benzalkonium
o-NPA o-NAC o-NPA o-NAC o-NPA o-NAC
Wild-type: I or A type HA HA HCI LCI PCI HA + PCI
K
a
(mM) 1 ± 0.3 0.8 ± 0.2 – – –
b
0.03 ± 0.01
K
i
(mM) – – 1.7 ± 0.5 7.7 ± 0.2 0.18 ± 0.02 0.37 ± 0.03
a ND (< 1) 0.4 1.9 ± 1.2 – – –
b 2.8 5.5 0.6 ± 0.3 ) ––
b ⁄ a ND (> 3) 14 ± 6 0.3 ± 0.3 – – –

ASCh ATMA ASCh ATMA ASCh
a
ATMA
Wild-type: I or A type LCI LCI LCI LUI LMI
b
LMI
c
K
a
(mM) –––––ND
d
K
i
(mM) 0.78 ± 0.07 2.4 ± 0.9 0.53 ± 0.19 0.09 ± 0.03 1.03 lM
b
0.05 lM
d
a 0 0 0 0 4.52
b
5.7
d
b 00000
b
3.1
D70G I or A type LCI LCI LCI LNI ND
a
LNI
K
a
(mM) ––––––

that linearly accelerates catalysis (bk
cat
with b > 1).
The degree of activation in the presence of tyramine
was higher for the hydrolysis of o-NAC than for the
hydrolysis of o-NPA. This was determined from the
nonactivated and asymptotic limits in the nonlinear
hyperbolic acceleration plots (Fig. 1A,B), which pro-
vided estimates of b. For o-NPA hydrolysis b = 2.8,
and for o-NAC hydrolysis b = 5.5. For o-NAC,
because the hydrolysis kinetics were performed under
first-order conditions, the b ⁄ a ratio was determined
using Eqn (14) (see Experimental procedures): b ⁄ a =
14 ± 6 and a = 0.4 (Table 1). For o-NPA, experi-
ments were performed at [S] close to K
m
, so that
Eqn (13) (see Experimental procedures), which
describes velocity, gives inaccurate values for a
(a < 1) and therefore b ⁄ a >3.
The difference in the extent of activation can be
explained by differences in the rate-limiting steps.
Because the rate-limiting step for the hydrolysis of
o-NAC is acylation [35], it follows that the activating
effects of tyramine take place at the level of acylation.
For the hydrolysis of o-NPA, both acylation and deac-
ylation are partly rate limiting [35]. If, by analogy with
its effect on o-NAC, the activating effects of tyramine
reflect the acceleration of acylation, the activation of
hydrolysis of o-NPA should become limited by the

i
= K
a
This strongly suggests that
[Tyramine] m
M
1n(1/ΔA
λ 410nm
),min
0
10
20
30
40
50
60
A

B
[Tyramine] m
M
0 1 2 3 4 5 6 0 1 2 3 4 5 6
[S]/n
0
2
4
6
8
10
12

Cornish–Bowden plots of [S] ⁄ v versus [tyra-
mine]. Nonlinear Dixon plots are expected
for activation. (B) o-NAC (d,1m
M; s,
2m
M; , 3.5 mM; h,5mM); Dixon plots of
v
)1
versus [tyramine].
P. Masson et al. Modulation of butyrylcholinesterase catalytic activitiy
FEBS Journal 275 (2008) 2617–2631 ª 2008 The Authors Journal compilation ª 2008 FEBS 2621
both the competitive inhibition of hydrolysis of posi-
tively charged substrates and activation of hydrolysis
of neutral substrates result from tyramine binding to
PAS. Such qualitatively opposite effects can be tenta-
tively interpreted in terms of allosteric inhibition ⁄ acti-
vation: the binding of tyramine to PAS induces a
conformational change that affects the formation of
the productive enzyme–substrate complex. It should be
remembered that PAS and the binding locus (W82) of
the active site are connected through an W loop
[29,44]. For positively charged substrates, the confor-
mational change prevents the productive binding of
substrate, probably by disrupting the W82–p-cation
interaction; in contrast, for neutral substrates, the con-
formational change optimizes the enzyme–substrate
orientation in the active site pocket for acylation.
Effects of serotonin
It was found that serotonin inhibited both esterase and
AAA activities of BuChE (Tables 1 and 2), in contrast

benzalkonium has been reported previously [42]. With
o-NPA and o-NAC, benzalkonium shows parabolic
competitive inhibition. Parabolic inhibition suggests
that the binding of more than one benzalkonium con-
tributes to the inhibition (Fig. 4). The multiplicity of
cation binding sites was revealed with phenox-
azine ⁄ phenothiazine dyes for wild-type BuChE [48],
[Serotonin] mM
0
10
20
30
40
50
A

B
[Serotonin] mM
0 2 4 6 8 10 12 14 16
0 2 4 6 8 10 12 14 16
[S]/n
[S]/n
0
1
2
3
4
5
6
7

Fig. 2. Inhibitory effect of serotonin on wild-
type BuChE-catalysed hydrolysis of o-NPA
and o-NAC in 0.1
M phosphate buffer at
25 °C. (A) o-NPA (d, 0.1 m
M; s, 0.2 mM; ,
0.4 m
M; h, 0.6 mM; , 0.8 mM): left panel,
Dixon plots of v
)1
versus [serotonin]; right
panel, Cornish–Bowden plots of [S] ⁄ v
versus [serotonin]. Nonlinear plots indicate
partial inhibition. (B) o-NAC (d,2m
M; s,
4m
M; ,10mM): left panel, Dixon plots of
v
)1
versus [serotonin]; right panel, Cornish–
Bowden plots of [S] ⁄ v versus [serotonin].
Converging Dixon plots and parallel
Cornish–Bowden plots indicate competitive
inhibition.
Modulation of butyrylcholinesterase catalytic activitiy P. Masson et al.
2622 FEBS Journal 275 (2008) 2617–2631 ª 2008 The Authors Journal compilation ª 2008 FEBS
and with propidium for a mutant (A277W ⁄ G283D)
having PAS similar to that of acetylcholinesterase [49].
The fact that benzalkonium acts as an apparent
activator at low concentrations with o-NAC and not

). The fact that D70G
is slightly activated at low benzalkonium concentra-
tions, whereas the wild-type enzyme is not, indicates
that b ⁄ a ‡ 1 for D70G, whereas b ⁄ a < 1 for the wild-
type enzyme. This subtle difference in behaviour
between the two enzyme forms reflects the higher
conformational plasticity of the active site gorge of
D70G compared with that of the wild-type enzyme for
acylation with neutral ester.
Effects of benzalkonium on ATMA hydrolysis
Hydrolysis of ATMA by wild-type BuChE in the pres-
ence of increasing concentrations of benzalkonium
gave unusual Lineweaver–Burk plots (Fig. 5A) in
which the lines intersected in the upper right quadrant
at 1 ⁄ [S]
cross
$ 2 ± 0.5 mm
)1
. This is consistent with
benzalkonium being an inhibitor at low substrate con-
centration and an activator as the substrate concentra-
tion is increased. The highest ATMA concentration
(0.5 mm) was below K
ss
= 0.70 mm [35], so that acti-
vation by excess substrate did not take place. This pat-
tern of inhibition has been reported previously for
decamethonium inhibition of the hydrolysis of 7-acet-
oxy-4-methylcoumarin by acetylcholinesterase [50].
The inhibition of wild-type BuChE hydrolysis of

ð6Þ
with the coordinates of the intersecting point:
1=½S
cross
¼
b À 1
K
m
ða À bÞ
ð7Þ
1=V
max;cross
¼
a À 1
V
max
ða À bÞ
ð8Þ
This very rare situation in which ligand L is an
inhibitor at low [S] and an activator at high [S],
[benzalkonium] m
M
ΔA
430nm
/min
0.0
0.1
0.2 0.3
0.4
0.5

plot shows only benzalkonium concentrations greater than 0.1 m
M.
The substrate concentration was 1 m
M o-NAC. Nonlinearity
indicates multiple binding.
P. Masson et al. Modulation of butyrylcholinesterase catalytic activitiy
FEBS Journal 275 (2008) 2617–2631 ª 2008 The Authors Journal compilation ª 2008 FEBS 2623
beyond [S]
cross
, is symmetrical to system C5 of partial
and mixed inhibition as described by Segel [51].
The values for a and b can be determined from the
re-plots of 1 ⁄ Dslope versus 1 ⁄ [L] and 1 ⁄ Dintercept ver-
sus 1 ⁄ [L] [51]; Dslope of the Lineweaver–Burk plot is
the difference between the slope at ligand concentra-
tion [L] and the slope without ligand (Eqn 9):
Dslope ¼
aK
m
ð½LþK
i
Þ
V
max
ðb½LþaK
i
Þ
À
K
m

m
(a – b), the intercept on the
[L]
)1
axis is – b ⁄ aK
i
and the slope is aK
i
V
max
⁄ K
m
(a ) b).
Dintercept is the difference between the intercept of
the Lineweaver–Burk plot at ligand concentration [L]
and the intercept without ligand (Eqn 11):
Dintercept ¼
ð½LþaK
i
Þ
V
max
ðb½LþaK
i
Þ
À
1
V
max
ð11Þ

, and K
i
was determined from the intercept on the
[L]
)1
axis of the Dslope
)1
re-plot (Fig. 5A, inset). This
gave a = 5.7 and K
i
= 0.05 lm .
The high value of a reflects the decreased affinity of
benzalkonium for the enzyme–substrate complex. This
result is consistent with the proposal that the binding
site for benzalkonium is either at PAS or in the active
site gorge close to PAS. The binding of benzalkonium
would then have to induce a conformational change at
the active site that is responsible for the increase in k
cat
at high [S] beyond [S]
cross
. Thus, the activating effect
of benzalkonium produces an effect similar to the acti-
vation by excess substrate that has been found to be
dependent on the integrity of PAS [44,49,52,53].
Because the rate-limiting step for the hydrolysis of
ATMA is acylation (k
2
> k
3

A
B
1/[ATMA] mM
1/v (1/ΔA
λ290 nm
), min
0
1000
2000
3000
4000
5000
0 2 4 6 8 10
0.0
0.2 0.4
0.6
0.8 1.0
1/[benzalkonium] µM
–10
0
10 20
0.001
0.00 3
0.004
0.005
0.00 2
1/
Δ
sl op e
wi ld t yp e

to be purely noncompetitive, i.e. the lines in the
Lineweaver–Burk plot cross on the y-axis (Fig. 5B) with
K
i
=13±2lm. Thus, the affinity of D70G for ben-
zalkonium is at least 260-fold weaker than that of the
wild-type enzyme. This suggests that PAS is the binding
site for benzalkonium, and supports the proposal that
the complexity encountered with the wild-type enzyme
reflects the binding of benzalkonium to PAS.
Effects of benzalkonium on ASCh hydrolysis
Under our experimental conditions, a study of the
inhibition of ASCh hydrolysis was not possible
because 5,5¢ -dithiobis(2-nitrobenzoic acid) (Nbs
2
) pre-
cipitated with benzalkonium. However, the inhibition
of BuChE-catalysed hydrolysis of ASCh by benzalko-
nium has been reported [42]. It is unclear how these
authors avoided the precipitation problem. In that
study, the inhibition of human BuChE was found to
be of the partial mixed type. Unfortunately, the experi-
ments were performed at high substrate concentration,
in the concentration range corresponding to substrate
activation (cf. Experimental procedures, Scheme 2,
Eqn 10). Thus, the reported K
i
value (1.03 lm) [42]
probably reflects the inhibition of substrate activation.
That is, benzalkonium is probably competing with the

Interaction of propanil with BuChE
Propanil (3¢,4¢-dichloroacetanilide) was not hydrolysed
by wild-type BuChE under our experimental condi-
tions, i.e. [E] > [S]. Yet, propanil binds to BuChE and
linearly inhibits the hydrolysis of o-NTFNAC and
ASCh over a large substrate concentration range. Inhi-
bition constants were determined from Dixon plots
and Cornish–Bowden plots (data not shown). Propanil
is a pure competitive inhibitor (a = 0) of the BuChE-
catalysed hydrolysis of both substrates: K
i
= 0.49 ±
0.05 mm with ASCh, and K
i
= 0.74 ± 0.58 mm with
o-NTFNAC. Thus, propanil interferes with the forma-
tion of ES, but not with S
p
EorS
p
ES.
In the BuChE–ASCh complex, the choline head
group strongly interacts with W82 [29]. The fact that
propanil is a competitive inhibitor suggests that it also
binds to the p-cation binding site W82. These results
imply that other acetyl anilide substrates (i.e. o-NAC,
o-NTFNAC, ATMA) may bind to W82 in the active
centre. This would place the substrate in the BuChE–
acetanilide substrate complexes into a favourable posi-
tion for the use of the catalytic triad Ser198 ⁄

single nucleophilic serine, Ser198, for both activities
[35]. Second, studies on mutant forms, e.g. silent allo-
zyme and S198C ⁄ D mutants of BuChE, support the
kinetic findings with wild-type BuChE and rule out the
P. Masson et al. Modulation of butyrylcholinesterase catalytic activitiy
FEBS Journal 275 (2008) 2617–2631 ª 2008 The Authors Journal compilation ª 2008 FEBS 2625
hypothesis that a nucleophile other than Ser198 is
responsible for the AAA activity [35]. Third, inspection
of the three-dimensional structure of human BuChE
shows that Ser224 is deeply buried inside the protein,
with Oc pointing away from the surface, about 6–7 A
˚
from the bulk solvent [29] (Fig. 6A). Therefore, no
access for substrate to Ser224 is possible.
The latter problem was acknowledged by the
authors of the Ser224 proposal. However, it was
argued that the binding of ligands such as benzalkoni-
um may induce a conformational change that activates
a Ser224 ⁄ His438 ⁄ E197 triad [33]. Our present results
show that the effect of benzalkonium on the AAA
activity of BuChE can be interpreted without postulat-
ing the unmasking of an alternative nucleophile. A
conformational change of the enzyme that would give
accessibility to Ser224 is unlikely, because it would
require a large movement of the main chain and subse-
quent disorganization of the central b-sheet. Moreover,
catalysis relies on optimal angles and distances
between the nucleophile and the base in order to allow
the formation of short, strong hydrogen bonds. The
observed spatial position of Ser224 and His438 does

compounds on the AAA and esterase activities of
human BuChE.
The concentrations of tyramine and serotonin that
activate or inhibit the AAA activity of BuChE (and
also its esterase activity) are several orders of magni-
tude higher than the concentrations of these com-
pounds that can be encountered in vivo under
physiological conditions or even during pathological
processes. The concentration of serotonin in human
plasma of normal subjects is 9 nm [62]; it is increased
several fold as a consequence of migraine headache,
schizophrenia, hypertension or carcinoid syndrome.
The concentration of tyramine in the plasma of nor-
mal subjects is about 7 nm [63]; it is increased as a
Ser198
Ser224
A
B
Ser224
Ser198
His438
Glu325
wat
wat
2.7
2.8
2.5
2.9
2.7
Fig. 6. (A) Overall view of the three-dimensional structure of

Exogenous aryl acylamide compounds, drugs and
xenobiotics are mostly metabolized in the liver and
other organs by amidase-carboxylesterases [20,21].
There is no carboxylesterase in human plasma
[60,65,66]. The only AAA activities present in human
plasma are those of albumin and BuChE. Both activi-
ties are very slow with acetanilides (o-NAC, o-NTF-
NAC) [26,35]. In addition, BuChE does not hydrolyse
m-nitroacetanilide [35], the fungicide propanil (m,p-di-
chlorocacetanilide) (present study) or drugs such as
acetaminophen (p-hydroxyacetanilide) and phenacetin
(p-ethoxyacetanilide) (P. Masson & M. T. Froment,
unpublished results). Although BuChE may interact
with high concentrations of these compounds, e.g. the
inhibition of AAA activity by propanil occurs at a K
i
value of about 0.6 mm, such concentrations are far
higher than would be expected to occur in blood dur-
ing treatments or intoxications, even in the most severe
cases. Therefore, it is unlikely that BuChE plays a
significant role in the metabolism of endogenous or
exogenous aryl acylamides.
Experimental procedures
Chemicals
o-NAC was obtained from Merck (Limonest, France) and
benzalkonium chloride was obtained from Interchim
(Montluc¸ on, France). Tyramine, serotonin (5-hydroxytryp-
tamine), o-NPA, o-nitrophenol (o-NP) and o-nitroaniline
(o-NA) were purchased from Sigma Chemical France (Saint
Quentin Fallavier, France). NATAc and diisopropylfluoro-

charged acetanilide (NATAc) and neutral acetanilides
(o-NAC and o-NTFNAC). The stock solution of ATMA
was in phosphate buffer, that of o-NAC was in methanol,
and those of o-NTFNAC and NATAc were in 50% acetoni-
trile. Assays with ATMA were carried out according to John-
son et al. [69]. The substrate concentration ranged from
0.025 to 5 mm. The hydrolysis of ATMA was recorded for
30 min at 290 nm (e
o)NTMNPA
= 1850 m
)1
Æcm
)1
), and rate
measurements were performed on the steady-state phase [26].
Assays with NATAc were carried out according to Kolken-
brock et al. [70]. The hydrolysis of NATAc was recorded for
30 min at 293 and 325 nm. Assays with o-NAC were carried
out according to the method of Hoagland and Graf [71]. The
final methanol concentration in the assays was 5%. The sub-
strate concentration in the assays was in the range 0.05–
4 mm. Assays with o-NTFNAC were carried out according to
Darvesh et al. [34]. The final concentration of acetonitrile in
the assays was 3.5%. The hydrolysis of o-NAC was recorded
for 45 min at 430 nm for o-NA (e
o-NA
= 4000 m
)1
Æcm
)1

hydrolysis of o-NAP can be investigated up to saturation.
However, because of the lower solubility of o-NAC and
o-NTFNAC, studies with these substrates were carried out
at concentrations much lower than K
m
(cf. [35]). The
steady-state catalytic parameters K
m
, k
cat
, K
ss
and b were
determined by nonlinear computed fitting of Eqns (1,5)
using the sigma plot 4 program (Jandel Science, San
Raphael, CA, USA). The active site concentration [E] of
highly purified enzyme preparations was determined
according to [73] with DFP as the titrant. The active site
concentration of wild-type BuChE was 0.16 ± 0.01 lm,
and that of the D70G mutant was 0.196 ± 0.02 lm.
Effects of ligands on steady-state kinetics
The effects of the ligands (L: tyramine, serotonin and ben-
zalkonium) on the AAA and esterase activities of wild-type
BuChE and its D70G mutant were investigated. Assays
were performed in 0.1 m phosphate buffer pH 7.0 at 25 °C.
The effect of each ligand on related pairs of substrates
(neutral substrates o-NAC and o-NPA, and positively
charged substrates ATMA and ASCh) was compared. To
avoid complications caused by substrate activation with
ATMA and ASCh, assays were performed at low and inter-

½E½S
aK
m
fð½LþKÞ=ðb½LþaK Þgþ½Sfð½LþaKÞ=ðb½LþaK Þg
ð13Þ
At low substrate concentrations, [S] > K
m
:
v ¼
k
cat
f1 þðb½LÞ= ðaK Þg
K
m
f1 þð½L=KÞg
½E½Sð14Þ
If a = 0, inhibition is competitive; if a = 1, inhibition is
noncompetitive; if a > 1, inhibition is mixed. If the ternary
complex ESL is nonproductive (b = 0), there is linear
mixed inhibition. If ESL is productive with b > 1, there is
activation. Activation and inhibition of mixed type are
symmetric. If ESL makes product with 0 < b < 1 and
b ⁄ a < 1, there is nonlinear hyperbolic inhibition. In hyper-
bolic activation, 0 < a < 1 and b ⁄ a > 1. More complex
situations that can be encountered, e.g. multiple binding,
are examined in the Results and Discussion section. It
should be noted that only the b ⁄ a ratio can be determined
from Eqn (14) (cf. Table 1; activation of BuChE-catalysed
hydrolysis of o-NAC by tyramine).
Kinetic constants were determined by: (a) linearizing the

interest in this study. This work was supported by De
´
le
´
-
gation Ge
´
ne
´
rale pour l’Armement/Programme d’Etudes
Amont (DGA/PEA) No. 01 08 07 ⁄ 03 CO 010-05 to
PM.
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