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-

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

´
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

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

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

)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

)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

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

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

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

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-

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

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

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

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

(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

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

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

(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,

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.

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

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.

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.
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