Tài liệu Báo cáo khoa học: High activity of human butyrylcholinesterase at low pH in the presence of excess butyrylthiocholine pptx

High activity of human butyrylcholinesterase at low pH
in the presence of excess butyrylthiocholine
Patrick Masson
1
, Florian Nachon
1,2
, Cynthia F. Bartels
2
, Marie-Therese Froment
1
, Fabien Ribes
1
,
Cedric Matthews
1
and Oksana Lockridge
2
1
Centre de Recherches du Service de Sante
´
des Arme
´
es, Unite
´
d’Enzymologie, La Tronche, France;
2
Eppley Institute,
University of Nebraska Medical Center, Omaha, Nebraska, USA
Butyrylcholinesterase is a serine esterase, closely related to
acetylcholinesterase. Both enzymes employ a catalytic triad
mechanism for catalysis, similar to that used by serine pro-

teases such as a-chymotrypsin. Enzymes of this type are
generally considered to be inactive at pH values below 5,
because the histidine member of the catalytic triad becomes
protonated. We have found that butyrylcholinesterase
retains activity at pH £ 5, under conditions of excess
substrate activation. This low-pH activity appears with wild-
type butyrylcholinesterase as well as with all mutants we
examined: A328G, A328I, A328F, A328Y, A328W, E197Q,
L286W, V288W and Y332A (residue A328 is at the bottom
of the active-site gorge, near the p-cation-binding site; E197
is next to the active-site serine S198; L286 and V288 form the
acyl-binding pocket; and Y332 is a component of the
peripheral anionic site). For example, the k
cat
value at
pH 5.0 for activity in the presence of excess substrate was
32 900 ± 4400 min
)1
for wild-type, 55 200 ± 1600 min
)1
for A328F, and 28 700 ± 700 min
)1
for A328W. This
activity is titratable, with pK
a
values of 6.0–6.6, suggesting
that the catalytic histidine is protonated at pH 5. The
existence of activity when the catalytic histidine is protonated
indicates that the catalytic-triad mechanism of butyrylcho-
linesterase does not operate for catalysis at low pH. The

mechanism explaining the catalytic behaviour of butyryl-
cholinesterase at low pH in the presence of excess substrate
remains to be elucidated.
Keywords: butyrylcholinesterase; excess substrate activation;
mutant enzyme; pH dependence; steady-state kinetics.
Human butyrylcholinesterase (EC 3.1.1.8; BuChE) is a
serine esterase, which is present in vertebrates. It is routinely
isolated from plasma [1] where it is considered to be of
pharmacological and toxicological importance because it
hydrolyzes numerous ester-containing drugs [2] and scav-
enges toxic esters, such as organophosphates [3]. Its primary
amino-acid sequence is 54% identical with that of Torpedo
californica acetylcholinesterase (EC 3.1.1.7; AChE) [4]. A
3D model for human BuChE has been built [5] from the
known co-ordinates for the 3D structure of T. californica
AChE [6]. This model agrees with the general features of the
recently determined X-ray structure of human BuChE [7,8].
In particular, most of the essential features of the catalytic
site (i.e. a catalytic triad of Ser-His-Glu, an oxyanion hole, a
p-cation-binding site, and an acyl-binding pocket) are the
same in AChE and BuChE (Fig. 1). The acyl-binding
pocket, which is responsible for the difference in substrate
specificity between the two enzymes, is larger in BuChE
[5,8–10]. The active site for both enzymes is located at the
bottom of a 20-A
˚
deep gorge. An aspartate residue
[D70(72)] is located at the mouth of the gorge. [Italicized
numbers in parentheses (N ) after amino-acid numbers refer
to residue numbering in T. californica AChE. In human

BuChE, the corresponding residue is N)2.] This aspartate,
part of the peripheral anionic site, contributes to the affinity
of positively charged substrates for the active site, and is a
major factor in the binding of excess substrate to these
enzymes [11,12]. Neither AChE nor BuChE follows Micha-
elis–Menten kinetics with positively charged substrates.
Under standard conditions, i.e. at neutral pH and 25 °C,
AChE has been shown to be inhibited by excess substrate,
whereas BuChE is activated [13]. However, we recently
reported that AChE may display substrate activation at low
pH [14]. The complete mechanism by which activation or
inhibition of cholinesterases by excess substrate occurs is
still controversial, but it is now accepted that binding of a
second molecule of substrate on the peripheral anionic site
(PAS) induces a conformational change that triggers the
process.
For some time, we have been interested in the molecular
basis of substrate activation in wild-type and mutants of
human BuChE [11,15,16], as well as substrate activation in
wild-type and mutants of human and Bungarus fasciatus
AChE at low pH [14]. The term substrate activation
describes the situation in which excess substrate causes an
increase in the turnover number (k
cat
)ofanenzyme.For
wild-type BuChE reacting with butyrylthiocholine (BTC),
Correspondence to P. Masson, Centre de Recherches du Service de
Sante
´
des Arme

´
es, Unite
´
d’Enzymologie, B.P. 87, 38702 La Tronche
Cedex, France. Fax: + 33 4 76 63 69 63, Tel.: + 33 4 76 63 69 59;
E-mail:
Abbreviations: AChE, acetylcholinesterase; BuChE, butyrylcholine-
sterase; BTC, butyrylthiocholine; DTNB, 5,5¢-dithiobis-
2-nitrobenzoic acid; PAS, peripheral anionic site.
Enzymes: butyrylcholinesterase (EC 3.1.1.8; BuChE); acetylcholin-
esterase (EC 3.1.1.7; AChE).
(Received 5 August 2002, revised 29 October 2002,
accepted 25 November 2002)
Eur. J. Biochem. 270, 315–324 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03388.x
the turnover number is determined by BTC concentrations
in the 10–100 micromolar range. Millimolar levels of BTC
cause the activity to rise above this turnover number,
eventually reaching a new, excess-substrate-defined turn-
over number, or bk
cat
(with b > 1). With wild-type human
BuChE, the turnover number for BTC increases 2.5–3-fold
(b ¼ 2.5–3) in the presence of excess BTC (at pH 7.0).
Mutations at the 328(330) position (A328 is at the bottom of
theactivesitegorge,nearthep-cation site) have been
reported to cause a marked decrease in this substrate
activation, i.e. A328F is activated only 20% (b ¼ 1.2), at
pH 7.0 [17], whereas A328Y is inhibited by 20% (b ¼ 0.8),
at pH 8.0 [9].
To clarify the cause of these differences in substrate

activation between the wild-type and A328 mutants, we
examined the pH dependence of BTC turnover. Although
we found the pK
a
values for k
cat
and bk
cat
of the mutants to
be slightly decreased relative to wild-type, these decreases
could not explain the observed differences in substrate
activation. However, in the course of our studies, we
observed that the bk
cat
values did not fall to zero as the pH
was lowered from 8.5 to 5.0, for either wild-type or mutant
BuChE. Rather they approached a substantial, nonzero
limit. For example, the limiting value for bk
cat
at low pH
was 32 900 ± 4400 min
)1
for wild-type, 55 200 ±
1600 min
)1
for A328F, and 28 700 ± 700 min
)1
for
A328W. Similar observations were made for wild-type
human and B. fasciatus AChEs and their mutants modified

on the equivalent residues [14]. Consistent with previous
reports, we found that k
cat
did approach zero as the pH
decreased from 8.5 to 5.0. The pK
a
values that we found for
both k
cat
and bk
cat
are consistent with titration of the
catalytic histidine, H438(440). The persistence of activity in
the presence of the protonated form of the catalytic histidine
is inconsistent with the generally accepted mechanism for
hydrolysis by cholinesterases [18,19]. This mechanism
utilizes the catalytic histidine as an acceptor for a proton
from the catalytic serine, therefore protonation of the
histidine would be expected to block catalysis. Rather,
the appearance of activity under conditions in which the
catalytic histidine is protonated indicates a change in the
mechanism of BuChE and AChE. Work is in progress to
probe the mechanism that could explain these observations.
Materials and Methods
Chemicals
Butyrylthiocholine iodide (BTC) and 5,5¢-dithiobis-2-nitro-
benzoic acid (DTNB) were purchased from Sigma Chemical
Co., St Louis, MO, USA. Chlorpyrifos-oxon was from
Chem Services Inc., West Chester, PA, USA (catalog
number MET-674B). All other chemicals, including buffer

components, were of biochemical grade.
Mutagenesis and expression of recombinant BuChE
Mutagenesis and expression were performed as described
previously [17]. Briefly, mutations in human BuChE were
created, then amplified by PCR using Pfu polymerase.
Fragments containing the mutation were cloned into the
plasmid pGS. The plasmid was transfected into CHO-KI
cells by calcium phosphate coprecipitation. Stable cell lines
were selected in methionine sulfoximine. Expressed BuChE
was secreted from these cells and collected into serum-free
medium.
Purification of BuChE
Mutant forms of BuChE were purified from culture
medium as previously described [1,17]. Briefly, the culture
medium was passed over a procainamide–Sepharose affinity
column which retained the BuChE, which was then
selectively eluted with 0.2
M
procainamide hydrochloride.
Further purification was obtained by ion-exchange chro-
matography on DE52 (Whatman, Clifton, NJ, USA) using
an NaCl gradient for elution. The resulting enzyme was
typically 70–95% pure. Wild-type BuChE used in this work
was purified from human plasma using the same combina-
tion of affinity and DE52 chromatography.
The concentration of each BuChE mutant was deter-
mined by titration with chlorpyrifos-oxon as proposed by
Amitai et al. [20]. Chlorpyrifos-oxon concentration was
standardized against wild-type BuChE of known concen-
tration.

Enzyme assay
Initial rate of turnover of BTC was measured by the method
of Ellman et al.[21]in0.1
M
sodium phosphate buffer, pH
variable from 5.0 to 8.5 and, in 0.1
M
sodium acetate buffer,
pH ranging from 4.0 to 5.25. The ionic strength of
phosphate buffers varied from 0.1 to 0.29, and that of
acetate buffers varied from 0.014 to 0.075. Such changes in
ionic strength are known to have no effect on k
cat
of
BuChE-catalyzed hydrolysis of cationic substrates
[11,13,22]. Buffers contained 0.33 m
M
DTNB and 0.01–
50 m
M
BTC, at 25 °C. Product formation was followed by
Fig. 1. Side view of the active-site gorge of acylated (butyrylated) human
BuChE. The arrow indicates the entrance of the gorge. D70 and Y332
are the peripheral anionic site residues. The active site is at the bottom
of the gorge: the substrate binding subsite is W84 and A328; the acyl-
binding pocket is formed from L286 and V288; the catalytic triad is
S198, H438 and E325. Residue 197 next to the catalytic serine is
involved in stabilization of transition states.
316 P. Masson et al.(Eur. J. Biochem. 270) Ó FEBS 2003
the change in A

420
. Observed rates were corrected for
spontaneous hydrolysis of BTC and for spontaneous
reduction of DTNB from blank samples. The observed
rate, in terms of mol productÆmin
)1
ÆL
)1
, was obtained by
dividing the DA
420
Æmin
)1
by the absorption coefficient for
the 5-thio-2-nitrobenzoic acid (TNB), which is the product
of the reaction of thiocholine with DTNB. This calculation
was complicated by the fact that TNB has a pK
a
of 4.53 [23],
which gives rise to a decrease in absorption coefficient at pH
values below 7.0. Consequently, we measured the absorp-
tion coefficients at 420 nm for TNB, at pH values between
5.0 and 8.5. Representative absorption coefficients are:
8590
M
)1
Æcm
)1
at pH 5.0; 11 100
M

)1
Æcm
)1
at pH 5.5;
12 500
M
)1
Æcm
)1
at pH 6.0; 13 200
M
)1
Æcm
)1
at pH 7.0;
and 13 300
M
)1
Æcm
)1
at pH 8.0.
Data analysis
Steady-state turnover of BTC with wild-type BuChE
exhibits the phenomenon of excess substrate activation.
This is illustrated in Scheme 1. This scheme is also suitable
for excess substrate inhibition.
This scheme is described by Eqn (1):
k
app
¼

k
cat
þ
bÃk
cat
þ½S
K
ss
1 þ
K
m
½S

1 þ
½S
K
ss

ð1Þ
where k
app
is the apparent rate, in terms of mol
productÆ(mol BuChE)
)1
Æmin
)1
, [S] is the concentration of
BTC. k
cat
is the turnover number (min

)1
)when
[S] << K
ss
, K
m
is the Michaelis–Menten constant, bk
cat
is the turnover number (min
)1
) when [S] >> K
ss
,andK
ss
is the dissociation constant for excess BTC [10,24]. The
parameter b reflects the efficiency of product formation
from the ternary complex (SES). When b >1, there is
substrate activation. When b <1, there is substrate
inhibition. When b ¼ 1, the enzyme follows Michaelis–
Menten kinetics. The k
cat
, K
m
, K
ss
and b values were
obtained by nonlinear fitting of the apparent rate vs. BTC
concentration data to Eqn (1), using SigmaPlot v4.16
(Jandel Scientific, San Rafael, CA, USA). The value for
bk

cat
was obtained by multiplying k
cat
by b.
Results
pH dependence of turnover
The values of k
cat
, bk
cat
, K
m
and K
ss
were obtained by fitting
the apparent rate (k
app
) vs. BTC concentration data to eqn
(1), at each pH value (data not shown). To within the limits
of experimental error, K
m
was independent of pH for all
enzymes. This is consistent with earlier reports on the pH
dependence of K
m
for human BuChE [25,26].
The value of K
ss
increased as the pH was lowered from
8.5 to 5.2, for all enzymes. This indicates that the binding of

BTC to the excess-substrate activation site is becoming
weaker as the pH is lowered. The change in K
ss
varied from
fivefold to 20-fold, depending on the enzyme. At pH 8.5, K
ss
had essentially stopped changing with pH, having reached a
limiting value for high pH. As the pH was lowered, the value
of K
ss
became progressively larger; however, by pH 5.2 a
clear inflection point had not yet developed. Therefore, pK
a
values for K
ss
could not be determined; only an upper limit
of 5.0 could be estimated. Such a low pK
a
is consistent with
the involvement of an acidic amino acid in the binding of
excess BTC. Excess-substrate activation for human BuChE
has been attributed to binding of positively charged
substrates, such as BTC, to D70 in the peripheral anionic
site [11,27]. The pH dependence of K
ss
is consistent with
protonation of D70. The pH dependence data for the D70G
mutant supports this statement. Indeed, although the D70G
mutant shows a slight activation by excess substrate
(b ¼ 1.2 ± 0.2) at BTC concentrations higher than 2 m

M
[11,15,27], its high K
ss
value (> 1 m
M
[27]) does not
significantly change with pH (not shown). However, D70 is
not the only residue involved in substrate activation: the
D70N mutant was shown to be strongly activated by excess
substrate [27] and even the D70G mutant shows substrate
activation similar to that of wild-type enzyme in the
presence of high concentrations of sugars or polyols [28].
No further discussion on the pH dependence of K
m
or K
ss
will be presented, so that we can focus attention on the pH
dependence of k
cat
and bk
cat
.
Figure 2 shows the pH dependence of k
cat
and bk
cat
for
wild-type BuChE and the mutants A328G, A328I, A328F,
A328Y and A328W, using BTC as substrate. Simple
inspection of Fig. 2 reveals that both k

cat
and bk
cat
exhibit
well-defined pH titration profiles, with the minimum rates
occurring at low pH. The values for k
cat
approach zero by
pH 5.0. However, the bk
cat
values clearly approach a
nonzero limiting activity at low pH. Limiting activity is the
plateau value in a titration curve. All of the titrations extend
over a range of at least 3 pH units, indicating that they are
more than 90% complete by pH 5. Therefore, the nonzero
limiting rates for bk
cat
at low pH cannot be attributed to
incomplete titration.
The data in Fig. 2 were in turn fitted to an expression for
asinglepK
a
(see the legend to Table 1 for details). The
fitting results are tabulated in Table 1.
Wild-type BuChE and all of the A328 mutants show
limiting rates for k
cat
at low pH (k
H
)thatare10%orlessof

their limiting rates at high pH (k
A
). Therefore, the limiting
values of k
cat
at low pH may be considered to be effectively
zero. This is despite the fact that most of these k
H
values are
statistically greater than zero. The pK
a
values of 6.5–7.0 for
these enzymes are all consistent with the titration of a
histidine. These results for k
cat
are consistent with the what
is commonly found for wild-type BuChE and wild-type
AChE (see [32] for a review). In these reports, the titrating
histidine has consistently been taken to be the catalytic
histidine, i.e. H438(440) in BuChE.
The bk
cat
values also gave well-behaved titrations for
wild-type BuChE and all of the A328 mutants, except
A328W. The pK
a
values of 6.0–6.5 are consistent with
titration of a histidine. By analogy with k
cat
,thetitrating

Scheme 1. Steady state turnover of BTC.
Ó FEBS 2003 High activity of BuChE at low pH (Eur. J. Biochem. 270) 317
histidine is probably the catalytic histidine, H438. It is
noteworthy that the limiting values of bk
cat
at low pH are
decidedly greater than zero (e.g. k
H
¼ 32 900 ± 4400 min
)1
for wild-type BuChE or k
H
¼ 28 700 ± 700 min
)1
for
A328W). Thus, in the presence of excess BTC, BuChE is
active even though H438 is protonated. The existence of
substantial activity for BuChE when the catalytic histidine is
protonated is an unprecedented observation, which has
significant implications for the mechanism.
We would like to emphasize that the activity that we
measure for bk
cat
does not approach zero at low pH. For a
titration that ends at zero activity for the fully protonated
histidine, theory predicts that at 1 pH unit below the
pK
a
, only 10% of the histidine is unprotonated and that
therefore only 10% of the activity will remain. Our results

in Fig. 2 show that for A328F 71% remains, for A328I
32% remains, for wild-type 40% remains, and for A328G
42% remains. In all four cases, the remaining activity at
1 pH unit below the pK
a
is much higher than the theoretical
prediction.
As the major point of this paper rests on the observation
that there is significant turnover of BTC by BuChE at low
pH, it is important to control for artifactual sources of
turnover. The first point to be made is that, to our
knowledge, BTC is not hydrolyzed, to any significant extent,
by any enzyme other than BuChE. AChE will hydrolyze
BTC slowly, but our recombinant enzymes were collected
into serum-free medium, which contains no AChE. As we
are seeing high bk
cat
values for BTC turnover at low pH, this
activity is probably not due to a contaminating enzyme.
Secondly, all of the rate data have been corrected for
spontaneous hydrolysis of BTC and chemical reduction of
DTNB. Thirdly, to control for unexpected contaminations
in the expressed enzymes, which might have arisen from the
cell culture, we examined wild-type BuChE that had been
purified to homogeneity (tetrameric G
4
form) from human
plasma. As can be seen in Figs 2 and 3, wild-type BuChE
also had substantial activity at low pH, in the presence of
excess substrate. It is unlikely that both naturally occurring

and cultured BuChE would show the same contaminations;
therefore, artifactual hydrolysis from contamination is
unlikely.
Further titration of wild-type BuChE
The titration of wild-type BuChE was extended from
pH 8.5 to 4.0. At pH 4.0, the k
cat
activity was effectively
zero, and bk
cat
activity was approaching zero (Fig. 3). The
titration of k
cat
was monophasic, with a pK
a
of 6.7 ± 0.09.
However, the titration of bk
cat
was very broad, extending
for more than 4.5 pH units. The profile was clearly biphasic,
with pK
a
values of 4.63 ± 0.24 and 6.68 ± 0.20 and a rate
for the singly protonated species of 41 400 ± 4500 min
)1
.
Complete elimination of the bk
cat
activity required proto-
nation of two amino acids. This biphasic titration accen-

tuates the fact that the intermediate, singly protonated
species is active. That is to say, complete protonation of
an amino acid with a pK
a
of 6.68 results in an enzyme that
still hydrolyzes BTC at a rate of 41 400 min
)1
.Aswehave
suggested, the most likely candidate for this group is
the catalytic histidine, H438. The pK
a
of 4.63 suggests
the involvement of an acidic residue in the activity at
low pH.
Low-pH activity and residue E197
Selwood et al. [34] reported two pK
a
values for the pH
dependence of the reaction of Electrophorus electricus and
T. californica AChEs with BTC. The higher pK
a
(6.3 and
6.1, respectively) was attributed to the titration of the
catalytic histidine, H440 (corresponding to H438 in human
BuChE). Protonation of this residue in electric eel AChE left
a preparation that retained % 30% of the activity found at
high pH. The residual activity could be abolished by further
titration, yielding a pK
a
of 4.7. Similarly, the pH profile for

T. californica AChE yielded a pK
a
of 5.0. This pK
a
was
attributed to residue E199, an active-site residue corres-
ponding to E197 in human BuChE. They proposed that
titration of H440 resulted in a change in mechanism Ôfrom
triad catalysis to one that likely involves general base
catalysis by E199 of direct water attack on the scissyl
carbonylÕ. The similarity between their results and ours is
evident and suggests that the low-pH activity of BuChE, in
the presence of excess BTC, may be due to general base
catalysis by E197.
Owing to this similarity, it became necessary to test the
involvement of residue E197(199) in the activity of BuChE
at low pH. To accomplish this, we determined the pH
Fig. 2. pH dependence for the turnover number (k
cat
) and the excess-
substrate-activated turnover number (bk
cat
) of wild-type human BuChE
and various 328-position mutants. Each panel represents a different
mutant form of BuChE, as indicated. In each panel, the solid circles
indicate the measured k
cat
values, the solid squares indicate the
measured bk
cat

values, and the lines are the result of fitting the meas-
ured rates to an expression for a single pK
a
(see the legend to Table 1
for details). The values for k
cat
and bk
cat
,ateachpH,weretakenfrom
fitting of k
app
vs. BTC concentration data for each mutant (data not
shown).
318 P. Masson et al.(Eur. J. Biochem. 270) Ó FEBS 2003
dependence of the activity of the BuChE mutant E197Q.
We reasoned that glutamine at position 197 was equiv-
alent to protonation of E197. If general base catalysis by
E197 was responsible for the residual activity seen with
wild-type BuChE, in the presence of excess BTC at low
pH, then E197Q would not be able to support that
activity. The excess-substrate activity of E197Q (bk
cat
)
should then titrate to zero with a pK
a
% 6.5 (correspond-
ing to the catalytic histidine, H438). Figure 4 shows that
this expectation was not realized. The bk
cat
rates of E197Q

approach a substantial, limiting rate at low pH. The
change in bk
cat
between high pH and low pH is not large,
but the trend is clear, and it yields a pK
a
of 6.17 ± 0.56.
Thus, the suggestion that E197 is responsible for the low-
pH activity of wild-type BuChE in the presence of excess
BTC is not supported.
Moreover, it should be noted that the pK
a
of mutant
E1997Q for k
cat
is shifted by % 1 pH unit below that of k
cat
of wild-type. Such a shift supports the assumption that the
observed pK
a
is related to His438 because it is consistent
with the fact that the electrostatic stabilizing effect of the
E197 side chain on the protonated form of H438 is
abolished in the E197Q mutant.
Role of position 328 in the excess-substrate effect
Mutations at position 328 seem to modulate the behaviour
of BuChE, rather than to introduce qualitatively new
behaviour. The most obvious indication of this modulation
appears at high pH where the limiting value of bk
cat

tends to
approach the limiting value of k
cat
as the size of the residue
at position 328 increases (Fig. 2). For example, the differ-
ence between bk
cat
and k
cat
at high pH is 68 400 min
)1
for
wild-type BuChE (A328), 19 700 min
)1
for A328F, and
13 000 min
)1
for A328W. Thus, the larger residues seem to
interfere with the ability of excess substrate to increase the
activity at high pH. However, the aliphatic or aromatic
character of side chains has to be considered too. At low
pH, the size of the residue in position 328 has no consistent
effect on bk
cat
.Thevaluesforbk
cat
are generally between
20 000 and 40 000 min
)1
(Fig. 2). The net effect is that the

value of bk
cat
at high pH becomes closer to its value at low
pH as the size of the residue at position 328 gets larger
(Fig. 2). For example, the difference between bk
cat
at high
pH and bk
cat
at low pH is 65 600 min
)1
for wild-type
(A238), 22 700 min
)1
for A328F, and 0 for A328W.
From the effect that the size of the 328 residue has on
bk
cat
, it is tempting to suggest that the 328 position (which is
part of the substrate binding site) plays a special role in the
excess substrate effect. However, mutations at other loca-
tionsintheactivesitealsoperturbthepHdependenceof
bk
cat
. E197Q (part of the esteratic site) shows a pH
dependence for bk
cat
that is similar to that for A328F
(Fig. 4). V288W (in the acyl-binding pocket) and Y332A (in
the PAS) show pH dependencies more like A328I, i.e. the

difference between bk
cat
and k
cat
at high pH is smaller than
for wild-type, and the difference between bk
cat
at high pH
and bk
cat
at low pH is relatively small (Fig. 5). On the other
hand, L286W (also in the acyl-binding pocket) is similar to
wild-type BuChE, i.e. the difference between bk
cat
and k
cat
at high pH is large relative to the difference at low pH, and
the difference between bk
cat
at high pH and bk
cat
at low pH
is relatively large (Fig. 5). In all of these enzymes, there is a
substantial activity for bk
cat
at low pH, strengthening the
Table 1. pH dependence of k
cat
and bk
cat

for BuChE mutants. Values for the parameters were determined by fitting the data from Figs 1, 3 and 4 to the
expression:
k ¼
k
H
þ k
A
à 10
ðpHÀpK
a
Þ
1 þ 10
pHÀpK
a
which is an algebraic rearrangement of the more common expression for the dependence of rate on pH involving a single pK
a
[29]:
pH ¼ pK
a
À log
k À k
A
k
H
À k

The term k stands for the observed rate, k
A
stands for the limiting rate at high pH, and k
H

stands for the limiting rate at low pH. This is a general expression
for a pH titration, which does not exclude the possibility of a nonzero limiting rate at either pH extreme. The rearrangement was required in order to obtain
the dependent variable (k) in terms of the independent variable (pH). Fitting was performed using
SIGMAPLOT
v.4.16. k
cat
is the turnover number in
the absence of excess substrate. bk
cat
is the turnover number in the presence of excess substrate. Residue volumes were taken from Zamyatnin [30].
Hydrophobicity ratings were taken from Karplus’ ÔpureÕ hydrophobicity scale [31]. NA, not applicable. There is no change in bk
cat
with pH for A328W.
BuChE
k
cat
bk
cat
Residue volume
(A
˚
3
) Hydrophobicity
k
H
(min
)1
)
k
A

(min
)1
)pK
a
k
H
(min
)1
)
k
H
(min
)1
)pK
a
A328G 2000 ± 1100 26 100 ± 1000 6.78 ± 0.11 26 800 ± 1700 70 400 ± 800 6.23 ± 0.07 60.1 1.18
Wild-type 2800 ± 1100 30 100 ± 1200 6.83 ± 0.10 32 900 ± 4400 98 500 ± 3600 6.56 ± 0.15 88.6 2.15
A328I 1230 ± 1100 27 900 ± 1340 6.79 ± 0.11 13 600 ± 3640 51 100 ± 1480 6.02 ± 0.15 166.7 3.88
A328F 6600 ± 1300 58 200 ± 1800 6.58 ± 0.06 55 200 ± 1600 77 900 ± 2200 6.57 ± 0.18 189.9 3.46
A328Y 8200 ± 3000 73 300 ± 3100 7.03 ± 0.11 33 400 ± 9400 71 900 ± 2600 6.12 ± 0.32 193.6 2.81
A328W 3200 ± 1900 41 000 ± 1700 6.59 ± 0.13 28 700 ± 700 28 700 ± 700 NA 227.8 4.11
E197Q 3200 ± 740 12 000 ± 280 5.85 ± 0.12 12 800 ± 1200 16 700 ± 730 6.17 ± 0.56 – –
L286W 4300 ± 2700 16 200 ± 2100 6.26 ± 0.45 17 400 ± 4100 93 000 ± 2700 6.17 ± 0.10 – –
V288W 6300 ± 1100 52 200 ± 1800 6.70 ± 0.07 62 900 ± 4200 89 300 ± 4200 6.08 ± 0.29 – –
Y332A 2900 ± 1400 48 400 ± 1400 6.51 ± 0.07 27 500 ± 3400 64 700 ± 2600 6.29 ± 0.19 – –
Ó FEBS 2003 High activity of BuChE at low pH (Eur. J. Biochem. 270) 319
argument that this high activity at low pH in the presence of
excess substrate is a common feature of BTC hydrolysis by
BuChE.
Mutations in the acyl-binding pocket of mouse AChE

(F297A and F297I) or in the hydrogen-bonding network
(E450Q) have also been shown to alter the excess-substrate
effect. In these cases, excess-substrate inhibition was
switched into excess-substrate activation [10,35,36]. More-
over, mutations in the p-cation-binding site of human and
snake AChE have been found to cause activation by excess
acetylthiocholine at low pH [14]. Taken together, these
observations suggest that any change to the structure of the
active site may alter the excess-substrate effect. Thus the
response of cholinesterases to excess-substrate binding
appears to involve the intricate interplay of a variety of
residues in the active site.
pH dependence of A328W
The A328W mutant has a remarkable pH vs. activity profile
(Fig. 2, bottom right panel). At low pH, bk
cat
is larger than
k
cat
, but at high pH bk
cat
is smaller than k
cat
.Thatistosay,
A328W goes from substrate activation, at low pH, to
substrate inhibition, at high pH. A similar observation was
made by Kalow [25] using benzoylcholine as substrate for
wild-type human BuChE and by us with benzoylthiocholine
as substrate on the same enzyme. In particular, the pH-
dependence study of benzoylthiocholine hydrolysis by wild-

type BuChE showed a progressive shift from activation by
excess substrate (b > 1) at low pH to inhibition by excess
substrate (b < 1) at pH > 7.1 (unpublished).
There is no reason to believe that excess substrate binds to
a different site at high pH than it does at low pH. Therefore
the switch from substrate activation to substrate inhibition
most probably reflects a pH-dependent difference in the
response of the protein to excess-substrate binding. That is
to say, the structure of the BuChE active site changes in
response to excess-substrate binding, and this change is
different at high pH than it is at low pH.
Fig. 4. pH dependence for the turnover number (k
cat
) in the absence of
excess substrate and for the turnover number (bk
cat
) in the presence of
excess substrate, of the human BuChE mutant E197Q. The solid circles
indicate the measured k
cat
values, the solid squares indicate the
measured bk
cat
values, and the lines are the result of fitting the meas-
ured rates to an expression for a single pK
a
(see the legend to Table 1
for details). The values for k
cat
and bk

cat
,ateachpH,weretakenfrom
fittings of k
app
vs. BTC concentration data (data not shown).
Fig. 5. pH dependence for the turnover number (k
cat
) in the absence of
excess substrate and for the turnover number (bk
cat
) in the presence of
excess substrate, of various BuChE mutants. Each panel represents a
different mutant form of BuChE, as indicated. In each panel, the solid
circles indicate the measured k
cat
values, the solid squares indicate the
measured bk
cat
values, and the lines are the result of fitting the meas-
ured rates to an expression for a single pK
a
(see the legend to Table 1
for details). The values for k
cat
and bk
cat
,ateachpH,weretakenfrom
fittings of k
app
vs. BTC concentration data (see Materials and Meth-

ods) for each mutant (data not shown).
Fig. 3. pH dependence for the turnover number (k
cat
) in the absence of
excess substrate and for the turnover number (bk
cat
) in the presence of
excess substrate, of wild-type human BuChE over the pH range 4–8.5.
From pH 5 to 8.5 the assays were performed in 0.1
M
sodium phos-
phate buffers. From pH 4 to 4.75. the assays were performed in 0.1
M
sodium acetate buffers. The solid circles indicate the measured k
cat
values, the solid squares indicate the measured bk
cat
values, and the
lines are the result of fittings. The k
cat
rates were fitted to an expression
for a single pK
a
(see the legend to Table 1 for details). The bk
cat
rates
werefittedtoanexpressionfortwopK
a
values [33].
k ¼

k
A
à K
2
à K
4
þ k
H
ýHÃK
4
þ k
H2
ýH
2
K
2
à K
4
þ½HÃK
4
þ½H
2
The term k is the observed rate, k
A
is the limiting rate at high pH, k
H
is
the rate for singly protonated form, k
H2
is the limiting rate at low pH

(zerointhiscase),K
2
is the dissociation constant for the first proto-
nation, K
4
is the dissociation constant for the second protonation, and
[H] is the hydrogen ion concentration. The values for k
cat
and bk
cat
,at
each pH, were taken from fittings of k
app
vs. BTC concentration data
(data not shown).
320 P. Masson et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Dependence of p
K
a
on the size/hydrophobicity
or aromaticity of residue 328
The original motivation for these studies was the hypo-
thesis that the residue in position 328 significantly
perturbed the pK
a
values for k
cat
and bk
cat
. This, however,

is not the case. There is only a slight dependence of pK
a
on the size of the residue at position 328 (Fig. 6). It is,
however, noteworthy, that with any given mutant the pK
a
for bk
cat
is generally lower than the pK
a
for k
cat
(Table 1).
This difference indicates that excess-substrate binding to
the PAS site of BuChE affects the environment of H438.
It could be due to either the presumed conformational
change induced on binding [11,12,15,37,38] or simply the
presence of an additional positive charge close to the
active site (in the form of BTC or any positively charged
substrate) making protonation of the catalytic histidine
more difficult.
Plots of pK
a
values against the nonpolar surface area of
the residue [31] or against any of a variety of residue
hydrophobicity scales, e.g. Chothia’s residue accessible
surface area scale [39] or Nozaki and Tanford’s water/
organic solvent partition scale [40], were similar to those in
Fig. 6 (data not shown). It is not surprising that the
correlation of pK
a

values with residue size is similar to the
correlation of pK
a
values with residue hydrophobicity, as
Chothia [41] has pointed out that hydrophobicity is
directly related to the accessible surface area of the
residue, i.e. size. In view of this, we believe that it is not
possible to conclude whether the variations in pK
a
of bk
cat
of the A328 mutants are due to a steric or a hydropho-
bic effect. Moreover, results with the bulkiest residue
(mutant A328W) do not fit the pattern, suggesting
that the tryptophan ring may affect the H438 pK
a
through
p-cation interactions.
Discussion
The central observation in this paper is that BuChE retains
significant hydrolytic activity after protonation of what
appears to be the catalytic histidine, H438. This occurs
under the influence of binding of excess substrate to the
PAS, i.e. for bk
cat
, but not at lower substrate concentrations,
i.e. for k
cat
in Scheme 1. This creates a major mechanistic
puzzle.

How can BuChE manage to turnover at a rate of
% 20 000–50 000 min
)1
when the catalytic histidine
appears to be protonated?
Let us review the accepted general mechanism of serine
hydrolase catalysis. The mechanism of serine esterases is
generally considered to be analogous to that of the serine
proteases [18]. It goes through four steps [18,42,43]
represented in Scheme 2 (human BuChE amino-acid
numbering). First, the carbonyl carbon of the substrate
undergoes a nucleophile attack by the Oc of the catalytic
serine, while the proton is shuttled to the catalytic histidine.
This results in the formation of a tetrahedral transition-
state intermediate, the negative charge on the former
carbonyl oxygen being stabilized by interactions with the
dipoles of the oxyanion hole. Secondly, the alcohol product
is released, picking up a proton from the catalytic histidine.
This results in the formation of a transient acyl-enzyme
adduct. The alcohol product is exchanged with a molecule
of water. Thirdly, the acyl-enzyme undergoes a nucleophile
Scheme 2. Proton shuttle mechanism.
Fig. 6. Dependence of pK
a
for k
cat
(filled circles) and bk
cat
(open
squares) on the volume of the residue at position 328. The pK

a
values
were taken from fitting the data of Fig. 1 to an expression for a single
pK
a
(see Table 1). The residue volume was taken from Zamyatnin [30]
(see Table 1). The letters are the single letter codes for the amino acids
at the 328 position. They are provided to help the reader to associate
the data with the mutant. The lines are presented to emphasize the
trend in these data. They have no analytical significance.
Ó FEBS 2003 High activity of BuChE at low pH (Eur. J. Biochem. 270) 321
attack by this molecule and the dissociated proton is
transferred to the catalytic histidine. This results in the
formation of a second tetrahedral transition-state interme-
diate, the negative charge on the former carbonyl oxygen
being again stabilized by the oxyanion hole. Fourthly, the
catalytic serine is released, picking up a proton from
the catalytic histidine. This results in regeneration of the
starting enzyme.
There are currently two proposals for the driving force
behind catalysis: the low-barrier hydrogen-bond model
[44,45] and the electrostatic stabilization of the transition
state model [46,47].
According to the low-barrier energy model, substrate
binding drives a conformational change to form a
Michaelis complex in which steric compression is intro-
duced between the histidine and carboxylate (aspartate in
chymotrypsin, glutamate in cholinesterases) of the cata-
lytic triad. Compression of the His-Asp/Glu diad causes
the basicity of the histidine to increase, so that it is able to

accept/abstract the Oc proton from the catalytic serine.
Transferring a proton from the catalytic serine to the
catalytic histidine relieves the steric compression by
forming a short, strong hydrogen bond between the
protonated histidine and the carboxylate. In this way, the
ability of the histidine to accept the proton from the serine
Oc is greatly increased. The presence of a short, strong
hydrogen bond was shown by NMR studies for both
human AChE and human BuChE complexes with com-
pounds mimicking the transition state [48,49].
In the electrostatic transition-state stabilization model,
the dipoles of the oxyanion hole are considered to be
optimally prealigned to strongly polarize the carbonyl of the
substrate. The carbonyl carbon becomes very electrophilic
and as the bond between the serine c-oxygen and the
carbonyl carbon forms, the pK
a
of the Oc proton falls to a
point where it can be released to the catalytic histidine,
which is positioned to accept it. Thus, no free energy is spent
on re-orienting the dipoles of the protein in the transition
state. This leads to a large decrease in transition-state energy
for the enzyme reaction compared with the chemical
hydrolysis reaction in water, and thereby, a large increase
in the rate for catalysis. It is noteworthy that the ability of
the oxyanion hole to stabilize the tetrahedral transition state
is well illustrated in the recently solved X-ray structure of
human BuChE [8]. In this structure, the enzyme is not free,
but the acidic product of substrate hydrolysis (butyrate) is
still loosely bound to the catalytic serine (bond length is

2.16 A
˚
). The carbonyl carbon of the butyrate adopts a
partial tetrahedral character. Such a distortion results from
the strong polarization of the C–O bond by the dipoles of
the oxyanion hole in conjunction with the influence of a
close nucleophile like the Oc of the catalytic serine. The
same type of adduct was observed previously for Strepto-
myces griseus protease A [50]. Thus, the butyrate–BuChE,
quasi-tetrahedral complex is more stable than the free or the
nonhydrated acyl-enzyme.
We have found that the protonated form of BuChE, in
the presence of excess substrate, i.e. bk
cat
, is active at low pH
(see Fig. 2). It is assumed that the change in bk
cat
as a
function of pH reflects protonation of the catalytic histidine.
This observation generates a problem for any mechanism in
which the catalytic histidine is the proton acceptor for the
catalytic serine, because the likelihood of a protonated
histidine accepting an additional proton is very low. Thus,
at low pH and in the presence of excess substrate, any model
for catalysis by human BuChE based on the histidine being
the proton acceptor becomes untenable. The protonated
form of the catalytic histidine may accept a proton in the
transition state, therefore serving as a general base catalyst,
only if a concerted proton transfer to the leaving group of
the substrate occurs.

Proton transfer
The requirement of an acceptor for the Oc proton from the
catalytic serine is a critical component of the mechanism for
the serine proteases/esterases. Based on the 3D structures of
a-chymotrypsin [44], AChE [6], and human BuChE [8], the
most logical recipient for the serine proton is the catalytic
histidine. Most models use the histidine to shuttle protons:
first, between the serine and the leaving group; and then
between the attacking water and the serine. In its role as a
proton shuttle, the catalytic histidine formally accepts a
proton from a donor and then delivers it to the acceptor
(Scheme 3).
On the other hand, our data at low pH indicate that
catalysis by BuChE and AChE [14], in the presence of excess
substrate, can proceed readily even when the catalytic
histidine is protonated. Formal transfer of an additional
proton to the cationic form of the histidine is not
reasonable. This is the dilemma. One solution would be a
concerted proton transfer, in the transition state, via the
catalytic histidine. The concerted proton transfer would be
between the serine and the leaving group and later between
the water and the serine.
Cyclic transition-state structures have been proposed for
nonenzymatic acid catalysis of ester hydrolysis ([51] and
references therein). Such a concerted transition state would
not conflict with electrostatic stabilization of the transition
state. Finally, a concerted proton transfer mechanism could
also explain catalysis by AChE at low pH [14]. How-
ever, further studies are needed to test this hypothetical
mechanism.

Thus, we suggest that BuChE and AChE may use two
different mechanisms for transferring protons. At high pH,
where the catalytic histidine is unprotonated, both choli-
nesterases use the traditional proton shuttle mechanism
(Scheme 3) both for k
cat
and bk
cat
. At low pH (where the
catalytic histidine is protonated), and in the presence of
excess substrate, the binding of which induces a conform-
ational change, cholinesterases use another mechanism
which remains to be elucidated.
Scheme 3. Proton shuttle transition state.
322 P. Masson et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Conclusion
We have found that, as for AChE [14,34], the turnover of
human BuChE reaches a substantial, nonzero limiting rate
at low pH, in the presence of excess positively charged
substrate. This observation suggests that catalysis at low
pH, in the presence of excess substrate, does not involve the
classical acid-base triad-mediated mechanism. However,
involvement of general base catalysis by a carboxylate, i.e.
E197(199), was disproved.
The observations of high activity from BuChE and
AChE at low pH is a new and important finding which
requires further investigation to dissect the molecular
mechanisms of hydrolysis of substrates by cholinesterases
under extremes of pH and substrate concentration.
Acknowledgements

We are grateful to Lawrence M. Schopfer for fruitful discussions and
constant support during this work. This work was supported by US
Medical Research and Materiel Command grants DAMD 35-1905-
2010-00 (to O.L.), DGA/DSP/STTC 99CO-029/PEA and DGA/
ODCA Washington 00-2-032-0-00 (to P.M. and O.L.) and by a
National Cancer Institute grant, P30CA36727, to the Eppley
Institute. The opinions and assertions contained herein should not
be construed as the official views of the US Army or the Department
of Defense.
References
1. Lockridge, O. (1990) Genetic variants of human serum choli-
nesterase influence metabolism of the muscle relaxant succinyl-
choline. Pharmacol. Ther. 47, 35–60.
2. Lockridge, O. (1992) Genetic variants of human serum butyryl-
cholinesterase influence the metabolism of the muscle relaxant
succinylcholine. In Pharmacogenetics of Drug Metabolism (Kalow,
W., ed.), pp. 15–50. Pergamon Press, New York.
3. Raveh, L., Grunwald, J., Marcus, D., Papier, Y., Choen, E. &
Ashani, Y. (1993) Human butyrylcholinesterase as a general
prophylactic antidote for nerve agent toxicity. In vitro and
in vivo quantitative characterization. Biochem. Pharmacol. 45,
2465–2474.
4. Lockridge, O., Bartels, C.F., Vaughan, T.A., Wong, C.K., Nor-
ton, S.E. & Johnson, L.L. (1987) Complete amino acid sequence
of human serum cholinesterase. J. Biol. Chem. 262, 549–557.
5. Harel, M., Sussman, J.L., Krejci, E., Bon, S., Chanal, P.,
Massoulie, J. & Silman. I. (1992) Conversion of acetylcholinester-
ase to butyrylcholinesterase: modeling and mutagenesis. Proc.
Natl. Acad. Sci. USA 89, 10827–10831.
6. Sussman, J.L., Harel, M., Frolow, F., Oefner, C., Goldman, A.,

Toker, L. & Silman, I. (1991) Atomic structure of acetyl-
cholinesterase from Torpedo californica: a prototypic acetylcho-
line-binding protein. Science 253, 872–879.
7. Nachon, F., Nicolet, Y., Viguie
´
, N., Masson, P., Fontecilla-
Camps, J.C. & Lockridge, O. (2002) Engineering of a monomeric
and low-glycosylated form of human butyrylcholinesterase. Eur.
J. Biochem. 269, 630–637.
8. Nicolet,Y.,Nachon,F.,Masson,P.,Lockridge,O.&Fontecilla-
Camps, J.C. (2003) Crystal structure of recombinant human
butyrylcholinesterase: new insights into the catalytic mechanisms
of cholinesterases. Proceedings of the XIth Cholinergic Mechanisms
Symposium (Fisher, A. & Soreq, H., eds). Martin Dunitz Ltd,
London (in press).
9. Saxena, A., Redman, A.M.G., Jiang, X., Lockridge, O. & Doctor,
B.P. (1997) Differences in active site gorge dimensions of choli-
nesterases revealed by binding of inhibitors to human butyryl-
cholinesterase. Biochemistry 36, 14642–14651.
10. Radic, Z., Pickering, N.A., Vellom, D.C., Camp, S. & Taylor, P.
(1993) Three distinct domains in the cholinesterase molecule
confer selectivity for acetyl- and butyrylcholinesterase inhibitors.
Biochemistry 32, 12074–12084.
11. Masson, P., Froment, M T., Bartels, C.F. & Lockridge, O. (1996)
Asp70 in the peripheral anionic site of human butyrycholinester-
ase. Eur. J. Biochem. 235, 36–48.
12. Mallender, W.D., Szegletes, T. & Rosenberry, T.L. (2000) Acetyl-
thiocholine binds to Asp74 at the peripheral site of human
acetylcholinesterase as the first step in the catalytic pathway.
Biochemistry 39, 7753–7763.

13. Tougu, V. (2001) Acetylcholinesterase: mechanism of catalysis and
inhibition. Curr.Med.Chem.1, 155–170.
14. Masson, P., Schopfer, L.M., Bartels, C.F., Froment, M T., Ribes,
F., Nachon, F. & Lockridge, O. (2002) Substrate activation
in acetylcholinesterase induced by low pH or mutation in the
p-cation subsite. Biochim. Biophys. Acta 1594, 313–324.
15. Masson, P., Xie, W., Froment, M T., Levitsky, V., Fortier, P L.,
Albaret, C. & Lockridge, O. (1999) Interaction between the
peripheral site residues of human butyrylcholinesterase, D70 and
Y332, in binding and hydrolysis of substrates. Biochim. Biophys.
Acta 1433, 281–293.
16. Masson, P., Xie, W., Froment, M T. & Lockridge, O. (2001)
Effects of mutations of active site residues and amino acids
interacting with the W loop on substrate activation of butyryl-
cholinesterase. Biochim. Biophys. Acta 1544, 166–176.
17. Xie, W., Varkey-Altamirano, C., Bartels, C.F., Speirs, R.J.,
Cashman, J.R. & Lockridge, O. (1999) An improved cocaine
hydrolase: the A328Y mutant of human butyrylcholinesterase is
4-fold more efficient. Mol. Pharmacol. 55, 83–91.
18. Quinn, D.M. (1987) Acetylcholinesterase: enzyme structure, reac-
tion dynamics, and virtual transition state. Chem. Rev. 87, 955–979.
19. Harel,M.,Quinn,D.M.,Nair,H.K.,Silman,I.&Sussman,J.L.
(1996) The X-ray structure of a transition state analog complex
reveals the molecular origins of the catalytic power and substrate
specificity of acetylcholinesterase. J. Am. Chem. Soc. 118, 2340–
2346.
20. Amitai, G., Moorad, D., Adani, R. & Doctor, B.P. (1998)
Inhibition of acetylcholinesterase and butyrylcholinesterase by
chlorpyrifos-oxon. Biochem. Pharmacol. 56, 293–299.
21. Ellman, G.L., Courtney, K.D., Andres, V. & Featherstone, R.M.

(1961) A new and rapid colorimetric determination of acetyl-
cholinesterase activity. Biochem. Pharmacol. 7, 88–95.
22. Tougu, V. & Kesvatera, T. (1996) Role of ionic interactions in
cholinesterase catalysis. Biochim. Biophys. Acta 1298, 12–30.
23. Riddles, P.W., Blakeley, R.L. & Zerner, B. (1979) Ellman’s
reagent: 5,5¢-dithiobis (2-nitrobenzoic acid), a reexamination.
Anal. Biochem. 94, 75–81.
24. Webb, J.L. (1963) Enzyme and Metabolic Inhibitors,Vol.1,p.36.
Academic Press, New York.
25. Kalow, W. (1964) The influence of pH on the hydrolysis of
benzoylcholine by pseudocholinesterase of human plasma. Can.
J. Physiol. 42, 161–168.
26. Lockridge, O., Blong, R.M., Masson, P., Froment, M T.,
Millard, C.B. & Broomfield, C.A. (1997) A single amino acid
substitution, Gly117His, confers phosphotriesterase (organopho-
sphorus acid anhydride hydrolase) activity on human butyryl-
cholinesterase. Biochemistry 36, 786–795.
27. Masson, P., Legrand, P., Bartels, C.F., Froment, M T., Schopfer,
L.M. & Lockridge, O. (1997) Role of aspartate 70 and tryptophan
82 in binding of succinyldithiocholine to human butyryl-
cholinesterase. Biochemistry 36, 2266–2277.
28. Levitsky, V., Xie, W., Froment, M T., Lockridge, O. &
Masson, P. (1999) Polyol-induced activation by excess substrate of
Ó FEBS 2003 High activity of BuChE at low pH (Eur. J. Biochem. 270) 323
the D70G butyrylcholinesterase mutant. Biochim. Biophys. Acta
1429, 422–430.
29. Irvin, J.L. & Irvin, E.M. (1947) Spectrophotometric and
potentiometric evaluation of apparent acid dissociation
exponents of various 4-aminoquinolines. J. Am. Chem. Soc. 69,
1091–1099.

30. Zamyatnin, A.A. (1972) Protein volume in solution. Prog.
Biophys. Mol. Biol. 24, 109–123.
31. Karplus, P.A. (1997) Hydrophobicity regained. Protein Sci. 6,
1302–1307.
32. Main, A.R. (1979) Mode of action of anticholinesterases. Phar-
macol. Ther. 6, 579–628.
33. Fersht, A. (1999) Structure and Mechanism in Protein Science,
pp. 172–173. W.H. Freeman, San Francisco.
34. Selwood,T.,Feaster,S.R.,States,M.J.,Pryor,A.N.&Quinn,
D.M. (1993) Parallel mechanisms in acetylcholinesterase-
catalyzed hydrolysis of choline esters. J. Am. Chem. Soc. 115,
10477–10482.
35. Hosea, N.A., Berman, H.A. & Taylor, P. (1995) Specificity
and orientation of trigonal carboxyl esters and tetrahedral alkyl-
phosphonyl esters in cholinesterase. Biochemistry 34, 11528–
11536.
36. Hosea, N.A., Radic, Z., Tsigelny, I., Berman, H.A., Quinn, D.M.
& Taylor, P. (1996) Aspartate 74 as a primary determinant in
acetylcholinesterase governing specificity to cationic organophos-
phates. Biochemistry 35, 10995–11004.
37. Radic, Z., Reiner, E. & Taylor, P. (1991) Role of the peripheral
anionic site on acetylcholinesterase: inhibition by substrates and
coumarin derivatives. Mol. Pharmacol. 39, 98–104.
38. Shafferman, A., Belan, B., Ordentlich, A., Kronman, C., Grosfeld,
H.,Leitner,M.,Flashner,Y.,Cohen,S.,Barak,D.&Ariel,N.
(1992) Substrate inhibition of acetylcholinesterase: residues
affecting signal transduction from the surface to the catalytic
center. EMBO J. 11, 3561–3568.
39. Chothia, C. (1976) The nature of the accessible and buried surfaces
in proteins. J. Mol. Biol. 105, 1–14.

40. Nozaki, Y. & Tanford, C. (1971) The solubility of amino acids and
two glycine peptides in aqueous ethanol and dioxane solutions.
J. Biol. Chem. 246, 2211–2217.
41. Chothia, C. (1974) Hydrophobic bonding and accessible surface
area in proteins. Nature (London) 248, 338–339.
42. Kraut, J. (1977) Serine proteases: structure and mechanism of
catalysis. Annu. Rev. Biochem. 46, 331–358.
43. Blow, D.M., Birktoft, J.J. & Hartley, B.S. (1969) Role of a buried
acid group in the mechanism of action of chymotrypsin. Nature
(London) 221, 337–340.
44. Cleland, W.W., Frey, P.A. & Gerlt, J.A. (1998) The low barrier
hydrogen bond in enzymatic catalysis. J. Biol. Chem. 273, 25529–
25532.
45. Cassidy, C.S., Lin, J. & Frey, P.A. (1997) A new concept for the
mechanism of action of chymotrypsin: the role of the low barrier
hydrogen bond. Biochemistry 36, 4576–4584.
46. Warshel, A. (1998) Electrostatic origin of the catalytic power of
enzymes and the role of preorganized active sites. J. Biol. Chem.
273, 27035–27038.
47. Fuxreiter, M. & Warshel, A. (1998) Origin of the catalytic power
of acetylcholinesterase: computer simulation studies. J. Am. Chem.
Soc. 120, 183–194.
48. Viragh, C., Harris, T.K., Reddy, P.M., Massiah, M.A., Mildvan,
A.S. & Kovach, I.M. (2000) NMR evidence for a short, strong,
hydrogen bond at the active site of a cholinesterase. Biochemistry
39, 16200–16205.
49. Massiah, M.A., Viragh, C., Reddy, P.M., Kovach, I.M., Johnson,
J., Rosenberry, T.L. & Mildvan, A.S. (2001) Short, strong
hydrogen bonds at the active site of human acetylcholinesterase:
proton NMR studies. Biochemistry 40, 5682–5690.

50. James, M.N., Sielecki, A.R., Brayer, G.D., Delbaere, L.T. &
Bauer, C.A. (1980) Structures of product and inhibitor complexes
of Streptomyces griseus protease A at 1.8 A
˚
resolution. A model
for serine protease catalysis. J. Mol. Biol. 144, 43–88.
51. Bender, M.L. (1960) Mechanisms of catalysis of nucleophilic
reactions of carboxylic acid derivatives. Quart. Rev. 60, 53–113.
324 P. Masson et al.(Eur. J. Biochem. 270) Ó FEBS 2003