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Kinetics of inhibition of acetylcholinesterase in the

presence of acetonitrile
Markus Pietsch, Leonie Christian, Therese Inhester, Susanne Petzold and Michael Gütschow
Pharmaceutical Chemistry I, Pharmaceutical Institute, University of Bonn, Germany

Keywords The hydrolysis of acetylthiocholine by acetylcholinesterase from Electro-


acetylcholinesterase; enzyme kinetics; phorus electricus was investigated in the presence of the inhibitors tacrine,
gallamine triethiodide; hyperbolic mixed-type
gallamine and compound 1. The interaction of the enzyme with the sub-
inhibition; tacrine hydrochloride
strate and the inhibitors was characterized by the parameters KI, a¢, b or b,
Correspondence Km and Vmax, which were determined directly and simultaneously from
M. Pietsch, School of Chemistry & Physics, nonlinear Michaelis–Menten plots. Tacrine was shown to act as a mixed-
The University of Adelaide, Adelaide, SA type inhibitor with a strong noncompetitive component (a¢  1) and to
5005, Australia completely block deacylation of the acyl-enzyme. In contrast, acetylcholin-
Fax: +61 8 8303 4358 esterase inhibition by gallamine followed the ‘steric blockade hypothesis’,
Tel: +61 8 8303 5360
i.e. only substrate association to as well as substrate ⁄ product dissociation
E-mail: markus.pietsch@adelaide.edu.au
from the active site were reduced in the presence of the inhibitor. The rela-
(Received 15 August 2008, revised 10 tive efficiency of the acetylcholinesterase–gallamine complex for the cataly-
January 2009, accepted 11 February 2009) sis of substrate conversion was determined to be 1.7–25% of that of the
free enzyme. Substrate hydrolysis and the inhibition of acetylcholinesterase
doi:10.1111/j.1742-4658.2009.06957.x were also investigated in the presence of 6% acetonitrile, and a competitive
pseudo-inhibition was observed for acetonitrile (KI = 0.25 m). The interac-
tion of acetylcholinesterase with acetonitrile and tacrine or gallamine
resulted in a seven- to 10-fold increase in the KI values, whereas the princi-
pal mode of inhibition was not affected by the organic solvent. The deter-
mination of the inhibitory parameters of compound 1 in the presence of
acetonitrile revealed that the substance acts as a hyperbolic mixed-type
inhibitor of acetylcholinesterase. The complex formed by the enzyme and
the inhibitor still catalysed product formation with 8.7–9.6% relative
efficiency.

Acetylcholinesterase (AChE, EC 3.1.1.7) is a serine active site at the bottom of a 20 Å deep gorge. Sub-
hydrolase [1], which belongs to the a ⁄ b hydrolase strate binding is facilitated by another component of
family [2,3]. The enzyme hydrolyses a broad range of the active site, the anionic site, which is characterized
ester and amide substrates, showing the highest speci- by several conserved aromatic residues, such as Trp84
ficity for acetylselenocholine, acetylthiocholine (ATCh) and Phe330. These residues have been shown to inter-
and acetylcholine (ACh) [4]. Substrate cleavage act with the quaternary ammonium groups of ACh or
proceeds via a two-step mechanism: acylation of the ATCh via cation–p interactions [7–12]. Further
enzyme, followed by deacylation involving a water stabilization of the quaternary moiety arises from an
molecule [5–7]. This process is mediated by the cata- electrostatic interaction with the acidic side-chain of
lytic triad Ser200–His440–Glu327 (Torpedo californica Glu199 [7,12]. A second substrate-binding site, the
AChE, TcAChE, numbering [8]) located within the peripheral anionic site (PAS), lies essentially on the

Abbreviations
ACh, acetylcholine; AChE, acetylcholinesterase; AD, Alzheimer’s disease; AP2238, 3-(4-{[benzyl(methyl)amino]methyl}phenyl)-6,7-dimethoxy-
2H-2-chromenone; ATCh, acetylthiocholine; Ab, amyloid-b; MeCN, acetonitrile; Nbs2, 5,5¢-dithiobis(2-nitrobenzoic acid); PAS, peripheral
anionic site; Tc, Torpedo californica.

2292 FEBS Journal 276 (2009) 2292–2307 ª 2009 The Authors Journal compilation ª 2009 FEBS
M. Pietsch et al. Inhibition kinetics of acetylcholinesterase

surface of AChE [8] and consists of five residues, in an improvement in cholinergic neurotransmission
Tyr70, Asp72, Tyr121, Trp279 and Tyr334, clustered and cognitive function [38–40].
around the entrance of the active site gorge [13–17]. With regard to the involvement of PAS in the pro-
PAS binds ACh transiently as the first step in the cata- cesses of AD, the use of PAS inhibitors and dual-site
lytic pathway, enhancing the catalytic efficiency by inhibitors of AChE allows for the inhibition of the cat-
trapping the substrate on its way to the active site, and alytic activity of the enzyme and also lowers the inci-
allosterically modulates catalysis [7,12,18–22]. dence of Ab fibril assembly [33,41,42]. Prototypes of
The principal physiological function of AChE, AChE inhibitors known to bind at the active site, PAS
mediated by the active site of the enzyme, is the rapid or both sites simultaneously are tacrine, gallamine and
hydrolysis of the neurotransmitter ACh at cholinergic donepezil (Fig. 1), respectively. The crystal structures
synapses and neuromuscular junctions, resulting in of each complexed with AChE have been published
the termination of the nerve impulse. In addition to [10,41,43]. In a previous study, we described 7-benzyl-
this ‘classical’ function, several ‘nonclassical’ activities 5,6,7,8-tetrahydro-2-isopropylamino-4H-pyrido[4¢,3¢:4,5]-
of AChE have been reported, which are associated thieno[2,3-d][1,3]thiazin-4-one (compound 1) (Fig. 1),
with PAS [9,21,23,24]. AChE is involved in neurite which inhibits AChE in the submicromolar range.
growth [25], haematopoiesis and osteogenesis [26], Kinetic analysis and structural similarities between
and acts as an adhesion protein in synaptic develop- donepezil, AP2238 (Fig. 1) and compound 1 suggest
ment and maintenance [9]. AChE has also been that these substances act as dual-site inhibitors of
shown to promote the pathophysiological assembly of AChE and bind along the active site gorge [42–44].
the amyloid-b (Ab) peptide into amyloid fibrils On the basis of these results, we performed a detailed
in vitro [27,28] and in vivo [29,30], with complexes of kinetic study with the prototype inhibitors tacrine and
AChE and Ab displaying an enhanced neurotoxicity gallamine, as well as compound 1. The interaction of
in comparison with fibrils formed by Ab alone the inhibitors with AChE from Electrophorus electricus
[31–33]. AChE has been found to be associated with was characterized using the kinetic models of AChE
amyloid plaques and neurofibrillary tangles, two inhibition, shown in Scheme 1 [45,46] and Scheme 3
hallmarks of Alzheimer’s disease (AD), and may con- [47], as well as the simplified model for hyperbolic
tribute to their development [34–36]. A third charac- mixed-type inhibitors (Scheme 2), i.e. general modifiers
teristic symptom of AD is the decrease in cholinergic [48–50]. The analysis presented herein allowed for the
neurons, which causes a loss of cholinergic neuro- simultaneous determination of the kinetic parameters
transmission and may be responsible for the common KI, a¢, b or b, Km and Vmax directly from nonlinear
signs of memory failure [37,38]. This ‘cholinergic Michaelis–Menten plots. Recently, the interaction of
hypothesis’ provided the rationale for the current gallamine and tacrine with AChE was found to be
major therapeutic approach to AD: the inhibition of dependent on the presence of acetonitrile (MeCN) [51],
the catalytic function of AChE, thereby increasing the a cosolvent frequently used in AChE inhibition assays
bioavailability of ACh at the synaptic cleft, resulting [44,51–54]. In our ongoing investigations, this finding

N O
N
NH2 O 3I O
N
O O
x HCl N
N O

Tacrine hydrochloride Gallamine triethiodide Donepezil

H
N S N N
N
O
S
O O O O

AP2238 1

Fig. 1. Inhibitors of AChE.

FEBS Journal 276 (2009) 2292–2307 ª 2009 The Authors Journal compilation ª 2009 FEBS 2293
Inhibition kinetics of acetylcholinesterase M. Pietsch et al.

kS k2 k3 (IC50 = 0.047 ± 0.001 lm, no MeCN; IC50 = 0.34 ±


S + E ES P1 + EA E + P2
k–S H2O 0.02 lm, 6% MeCN) and gallamine (IC50 = 1100 ±
+ + +
60 lm, no MeCN; IC50 = 2930 ± 140 lm, 6%
I I I MeCN) were in good agreement with results from a
previous study [51]. In the case of compound 1, AChE
kI k–I kSI k–SI kAI k–AI
inhibition was only determined in the presence of 6%
kS2
MeCN because of a lack of solubility in the absence of
ak2 bk3
S + EI ESI P1 + EAI EI + P2 an organic cosolvent. As the enzyme was not
k–S2 H2O
completely inhibited at high concentrations of
Scheme 1. Kinetic model of AChE inhibition. compound 1, residual activity at infinite concentration
of the inhibitor (v[I] fi ¥) had to be considered. Using
KS
Eqn (15) (see Experimental procedures), values of
kP
S + E ES' E + P IC50 = 0.58 ± 0.02 lm and v[I] fi ¥ = 0.094 ± 0.004
+ + (relative to the activity without inhibitor v0) were deter-
I I mined, which confirmed previously reported data [44].
To characterize the inhibition of AChE, a kinetic
KI α'K I model was considered (Scheme 1), which is analogous
to that introduced by Barnett and Rosenberry [45] and
α'K s β'kP
S + EI ESI' EI + P
Szegletes et al. [46]. In this model, the substrate S
binds to the enzyme E to form an initial enzyme–sub-
Scheme 2. Simplified kinetic model of the general modifier strate complex, also called Michaelis complex ES [55].
mechanism. This complex proceeds to an acylated enzyme interme-
diate EA, with the acylation rate constant k2, under
kS k2 k3 simultaneous formation of the first product P1. The
S + E ES P1 + EA E + P2
k–S H2O acyl-enzyme is then hydrolysed with the deacylation
+ +
rate constant k3 to give the second product P2 and the
I I
free enzyme, which enters a new catalytic cycle. If
ATCh is used as substrate, thiocholine and acetate are
kI k–I k AI k–AI
formed as P1 and P2, respectively. The Michaelis con-
bk 3
stant Km and the maximal velocity Vmax, which can be
EI EAI EI + P2 experimentally determined, are expressed by Eqns (1)
H2O
and (2), respectively [56,57]:
Scheme 3. Kinetic model of AChE inhibition excluding the for-
mation of ESI. kS þ k2 K
Km ¼   ¼  S  ð1Þ
kS 1 þ k2 1 þ k2
was analysed in detail by determining the effect of k3 k3
MeCN on the kinetic parameters of AChE inhibition.
k2 ½E0
Vmax ¼   ð2Þ
Results and Discussion 1 þ k2
k3

Characterization of AChE inhibition and


where [E]0 is the total enzyme concentration and KS is
estimation of the inhibitory parameters
the dissociation constant of ES. The parameter kcat is
The inhibition of AChE from E. electricus by tacrine, equal to the quotient Vmax ⁄ [E]0 [46]. For the hydrolysis
gallamine and compound 1 was determined spectro- of ATCh by AChE from E. electricus, values of the
photometrically in a coupled assay with the substrate rate constants k2 (1.23 · 106 min)1) and k3
ATCh and 5,5¢-dithiobis(2-nitrobenzoic acid) (Nbs2). (9.3 · 105 min)1) have been obtained previously by
Inhibition studies were performed in the absence and direct measurements of the acetyl-enzyme. As k2 is
presence of 6% v ⁄ v MeCN at various concentrations of only about 1.3 times larger than k3, both constants are
both the substrate [S] and the inhibitor [I]. For compar- rate influencing [58].
ison, IC50 values were initially determined at a sub- An inhibitor I can bind to each of the three enzyme
strate concentration of 500 lm by plotting the rates species to form a binary enzyme–inhibitor complex EI,
versus [I]. The inhibitory constants obtained for tacrine or the ternary complexes ESI and EAI with the

2294 FEBS Journal 276 (2009) 2292–2307 ª 2009 The Authors Journal compilation ª 2009 FEBS
M. Pietsch et al. Inhibition kinetics of acetylcholinesterase

enzyme–substrate complex and the acyl-enzyme, hyperbolic expression for v is, in general, more advan-
respectively [46]. The EI complex is capable of bind- tageous, as this method includes no transformation of
ing S, and catalyses product formation via ESI and the primary data, lower standard deviations and less
EAI, with the parameters a and b describing the fac- bias in parameter estimates compared with algorithms
tors by which k2 and k3 are altered. As a general using linearized plots [60]. To apply such a nonlinear
steady-state solution for the reaction rate v in optimization, we simplified the kinetic model outlined
Scheme 1 (based on extension of the Michaelis–Men- in Scheme 1 to that of the general modifier mechanism
ten expression) is too complex for useful comparison (Scheme 2) [48]. In this model, ES and EA are not
with experimental data [45,46,48,59], a virtual equilib- considered separately, but summed in a complex ES¢
rium has been assumed for all reversible reactions in that includes all enzyme–substrate intermediates. In an
Scheme 1 (i.e. k)S  k2, k)S2  ak2, k)SI  k2, analogous manner, ESI¢ represents the complexes of I
k)SI  ak2, k)AI  k3, k)AI  bk3) [45,46,59]. The with the substrate-bound enzyme and the acyl-enzyme
resulting expression for v is given by Eqn (3) with the [14]. The dissociation constants of S from these binary
dissociation constants KX expressed by the quotients and ternary complexes are KS = k)S ⁄ kS and
k)X ⁄ kX. a¢KS = k)S2 ⁄ kS2, respectively [49]. Both ES¢ and ESI¢

Vmax ½S
v ¼ 0  1 0 0  1 0   11 ð3Þ
I I I
B1þ K C B k B 1þK C  k B 1 þ K CC
Km B
@
 IC þ ½S B 3 B  C þ
SI 2 B AICC
a I A @ k2 þ k3 @ a I A k2 þ k3 @ b I AA
1þ 1þ 1þ
KSI KSI KAI

A direct analysis of AChE inhibition using Eqn (3) are capable of product formation (with P being both
is not possible, as contributions from the inhibition of thiocholine and acetate [14]), governed by the catalytic
both acylation and deacylation complicate the interpre- constants kP and b¢kP, respectively. The parameter b¢
tation of the data. In addition, estimates for a and b reflects the efficiency of hydrolysis of ESI¢ compared
are not separately available. Therefore, the parameters with that of ES¢. This type of inhibition (b¢ > 0) is
a and b were introduced to facilitate calculation. At referred to as hyperbolic inhibition, as the shape of a
saturating concentrations of I, these parameters are reciprocal velocity 1 ⁄ v versus [I] plot is hyperbolic. In
expressed by Eqns (4) and (5), respectively [46]: contrast, a value of b¢ = 0 causes a linear dependence
of 1 ⁄ v on [I], and thus the inhibition is called linear
aKI aKS
a ¼ ¼ ð4Þ [49,50]. The dissociation constant of EI is KI = k)I ⁄ kI
KSI KS2
[49] and thus defined as in Scheme 1, whereas a¢KI
ab ðk2 þ k3 Þ reflects a composite of constants for inhibitor binding
b ¼ ð5Þ to the enzyme–substrate complex and the acyl-enzyme
ak2 þ bk3
[14]. The factor a¢ corresponds to the ratio of a¢KI and
Under these conditions, i.e. [I] fi ¥, S exclusively KI. As the overall equilibrium constant for the forma-
binds to the EI complex and is converted to the pro- tion of ESI¢ must be the same regardless of a path via
ducts via ESI and EAI. The reaction rate, v[I] fi ¥, at a ES¢ or EI, the same factor a¢ must be included in the
given substrate concentration is defined by a combina- model [49,61]. Derivation of the general velocity equa-
tion of Eqns (3–5): tion for the system in Scheme 2 can be performed
bVmax ½S assuming a rapid equilibrium or steady-state condition.
v½I!1 ¼ ð6Þ The first method gives a relatively simple expression,
b
Km þ ½S whereas the steady-state approach results in a very
a
complex expression containing squared [S] and [I]
The kinetic parameters KI, a and b are usually deter- terms. However, the steady-state velocity equation
mined by linearization of the Michaelis–Menten equa- simplifies to the same form as the rapid equilibrium
tion (Eqn 3) on the basis of the Lineweaver–Burk plot velocity equation when pseudo-equilibrium conditions
and replotting of the slopes and intercepts obtained prevail (i.e. k)S  kP), as, in this case, the Michaelis
(after normalization) [46,59]. However, it was shown constant Km = (k)S + kP) ⁄ kS substitutes for KS =
that an iterative nonlinear optimization based on the k)S ⁄ kS in the velocity equation [49,61,62]. In addition,

FEBS Journal 276 (2009) 2292–2307 ª 2009 The Authors Journal compilation ª 2009 FEBS 2295
Inhibition kinetics of acetylcholinesterase M. Pietsch et al.

Km has been found to be similar to KS for several sub- ATCh, as well as the nonhydrolysable substrate ana-
strates of AChE [63]. For the purposes of the present logue 4-oxo-N,N,N-trimethylpentanaminium [7,12],
study, a rapid equilibrium and an irreversible character revealed that these compounds also interact with Trp84
of the catalytic step were assumed. Under these condi- and Phe330 (TcAChE numbering [8]). Thus, it is unli-
tions, the Michaelis–Menten equation for the general kely that tacrine binds to the Michaelis complex, i.e. no
type of inhibition shown in Scheme 2 is as follows: ternary complex ESI is formed. However, in the crystal
structure of AChE with tacrine, the immediate vicinity
V ½S
v ¼ 0   1max 0 1 ð7Þ of the catalytic serine is not occupied by the inhibitor
I I [10], and thus tacrine is probably able to bind to the EA
B 1þ K C B 1 þ a0 K C
Km B
@
 C þ ½S B
I  IC complex [67]. Under these conditions, the kinetic model
b0 I A @ b0 I A in Scheme 1 can be simplified to that shown in
1þ 0 1þ 0
a KI a KI Scheme 3. As I does not interact with ES to form ESI,
At saturating concentrations of I, the products are the value of the dissociation constant KSI in Scheme 1
exclusively formed from ESI¢ with a rate constant b¢kP. becomes very large and the quotient KI ⁄ KSI is virtually
Under these conditions, the rate v[I] fi ¥ can be zero. Equation (4) reveals that the ratios KI ⁄ KSI and
expressed by Eqn (8) [44,49]: KS ⁄ KS2 are equal, and thus the dissociation constant KS2
must also become very large (i.e. the formation of ESI
b0 Vmax ½S does not occur via binding of S to EI). An identical
v½I!1 ¼ ð8Þ
a0 Km þ ½S model as depicted in Scheme 3 has been used by Krupka
and Laidler [47] to explain AChE inhibition caused by
As the rate v[I] fi ¥ must be the same, regardless of the interaction of I with E and EA. The Michaelis–Men-
whether the model in Scheme 1 or 2 is applied, Eqns (6) ten equation for this type of inhibition is obtained by
and (8) can be set as equal. Therefore, the parameters a simplification of Eqn (3) with KSI fi ¥:
and b in Eqn (6) can be expressed by means of a¢ and b¢
Vmax ½S
as follows: b = b¢ and a = b¢ ⁄ a¢ = b ⁄ a¢. In addition, v¼ 0 0   11
the value KI is equally defined in both kinetic models    1þ I
I 
B k    B K CC
(Schemes 1 and 2); thus, all three parameters character- Km 1þ K þ ½S B 3 k2 B AI CC
I @ k2 þk3 þ k2 þk3 @1þb I AA
izing the inhibition according to the kinetic model in
Scheme 1, i.e. KI, a and b, can be determined on the KAI
basis of the simplified model depicted in Scheme 2. This ð9Þ
methodology was applied to the inhibition of AChE by
gallamine (in the absence of MeCN) and compound 1 At saturating concentrations of I, the rate v[I] fi ¥ is
(with 6% MeCN). The PAS inhibitor gallamine (with- equal to zero when calculated using Eqn (9). This means
out MeCN) has already been reported to follow the that the kinetic model in Scheme 3 and Eqn (9) are only
kinetic model in Scheme 1 [46], whereas inhibition by applicable if complete inhibition occurs.
compound 1 was found not to be complete at saturating In analogy with the kinetic model in Scheme 2, the
concentrations of I, i.e. v[I] fi ¥ and therefore b are dissociation constant KAI, obtained from Scheme 3,
greater than zero. was termed a¢KI with a¢ corresponding to the ratio of
For inhibitors attacking the active site of AChE and a¢KI and KI. Additional rearrangement of Eqn (9)
containing a positively charged quaternary nitrogen results in the following expression for v:
atom, it has been reported that they act not only by
binding to the free enzyme at the same site as the sub- Vmax ½S
v ¼  
0  1 ð10Þ
strate, but also by adding to the acyl-enzyme. However, I 1 þ bk3
these compounds do not inhibit through attachment to B1 þ
B  k 2 C C
   B a KI 1 þ k3 C
0
the Michaelis complex [47,55,64–66]. An example of I B k C
Km 1 þ K þ ½S B  2
C
such an inhibitor is tacrine, which has been shown to I B 1þb I C
B 0 C
occupy the anionic binding site of TcAChE by being @ a KI A
sandwiched between the aromatic rings of Trp84 and
Phe330, mainly through p–p interactions and cation–p
interactions. In addition, a direct hydrogen bond is Equation (10) was used to analyse AChE inhibition by
formed between the acridinic protonated nitrogen of the tacrine in the absence and presence of 6% MeCN.
inhibitor and the carbonyl oxygen of His440 [10]. Crys- In the present study, we applied an iterative nonlinear
tallographic studies on AChE complexed with ACh and optimization based on the hyperbolic Michaelis–Menten

2296 FEBS Journal 276 (2009) 2292–2307 ª 2009 The Authors Journal compilation ª 2009 FEBS
M. Pietsch et al. Inhibition kinetics of acetylcholinesterase

Eqn (7) (with b¢ = b) and Eqn (10) to calculate the Eqn (13), which is similar to the specific velocity plot
parameters KI, a¢, b (Eqn 7) or b (Eqn 10), Km and Vmax (Doc. S1, Fig. S2A,B, see Supporting information):
simultaneously from plots of rate versus [S] in the  
0  bk3 1
presence of various inhibitor concentrations. However, I 1þ
provisional estimates of the kinetic parameters were B  k2  C
B1 þ C
necessary prior to the computer-assisted iterative deter- B 0 1 þ k3  C  
B a KI  C 
B I C
mination [60]. To obtain such estimates for Km and v0
¼B  
k2
 1 þ C r þ 1þ I
B KI C
Vmax, we determined these parameters separately for vI B 1þ 0
b I C 1þr KI
B a KI C
each set of data (tacrine without MeCN, tacrine with B C
@ A
MeCN, gallamine without MeCN, gallamine with
MeCN, and compound 1 with MeCN) in the absence of
ð13Þ
inhibitor. The data were analysed by a nonlinear regres-
sion according to Eqn (11), which represents a simplifi- Investigations of AChE inhibition by tacrine, in the
cation of Eqn (3) with [I] = 0: absence and presence of 6% MeCN, on the basis of
the modified specific velocity plot (Eqn 13, data not
Vmax ½S
v ¼ ð11Þ shown) indicated a mixed-type inhibition that tended
Km þ ½S to noncompetitive inhibition in both cases with KI =
0.038 lm, a¢ = 0.91 and KI = 0.25 lm, a¢ = 1.0,
Using this method, the following values of Km and respectively. The value b was determined to be equal
Vmax were calculated for the five sets of data: tacrine, to –0.004 for the enzyme–inhibitor interaction, both
no MeCN: Km = 101 ± 14 lm, Vmax = 110 ± 3%; with and without MeCN. As b < 0 cannot be defined
tacrine, 6% MeCN: Km = 684 ± 41 lm, Vmax = by the mechanism in Scheme 3, b was set to zero for
229 ± 5%; gallamine, no MeCN: Km = 135 ± the calculation of the kinetic parameters by Eqn (10).
19 lm, Vmax = 116 ± 3%; gallamine, 6% MeCN: AChE inhibition by gallamine without MeCN and
Km = 671 ± 20 lm, Vmax = 235 ± 3%; compound compound 1 in the presence of 6% MeCN, analysed
1, 6% MeCN: Km = 606 ± 32 lm, Vmax = 229 ± according to Eqn (12) (Fig. S1A, see Supporting infor-
4%. (The rate of the AChE-catalysed hydrolysis of mation), was found to follow a hyperbolic mixed-type
500 lm ATCh, corrected by the value of the nonenzy- inhibition with a¢ > 1 and b > 0. This is shown for
matic hydrolysis, was set to 100% in all experiments.) gallamine by the common intersection point of the lines
Provisional estimates for KI, a¢ and b in Eqn (7) in the specific velocity plot at r ⁄ (1 + r) > 1; v0 ⁄ vI = 1
(b¢ = b) were obtained by analysing the data of the (Fig. S1A, see Supporting information), as well as by
AChE inhibition studies with the specific velocity plot discrete intercepts of the replots (Fig. S1B, see Support-
developed by Baici [61]. This method is advantageous ing information) [61]. On the basis of these replots, the
over the commonly used Lineweaver–Burk plot or the parameters KI = 330 lm, a¢ = 5.7 and b = 0.31 were
similar Hanes–Woolf plot [49], as it always gives linear determined. Using this method, KI = 0.52 lm, a¢ = 1.3
plots, independent of whether the inhibition is linear and b = 0.077 were calculated for the inhibition of
or hyperbolic. The type of inhibition can be obtained AChE by compound 1 (data not shown).
by simple inspection of the specific velocity plot In contrast with the study without MeCN, a plot of
(Eqn 12), and linear replots permit the calculation of v0 ⁄ vI versus r ⁄ (1 + r) for AChE inhibition by gallamine
KI, a¢ and b [61]. On the basis of Eqn (12), the quo- in the presence of 6% MeCN showed an array of curves
tient of the rate without inhibitor and the rate in the with a common intersection point close to r ⁄ (1 + r) =
presence of inhibitor, v0 ⁄ vI, was plotted against 1; v0 ⁄ vI = 1 (Fig. S2A, see Supporting information). A
r ⁄ (1 + r), with r being equal to [S] ⁄ Km (Doc. S1, plot of int0 ⁄ (int0)1) versus 1 ⁄ [I], where int0 is the inter-
Fig. S1A,B, see Supporting information): cept on the ordinate axis [r ⁄ (1 + r) = 0], and an initial
  analysis (Fig. S2B, see Supporting information) gave an
1 1  
 I I intercept equal to unity. Such a behaviour indicates
v0 a0 KI KI r 1þ K competitive inhibition [61], which is described by
¼  þ  I ð12Þ
vI b I 1þr b I Eqn (14):
1þ 0 1þ 0
a KI a KI
Vmax ½S
v ¼    ð14Þ
I
Km 1 þ K þ ½S
To obtain provisional estimates for KI, a¢ and b in I
Eqn (10), we developed a graphical method based on

FEBS Journal 276 (2009) 2292–2307 ª 2009 The Authors Journal compilation ª 2009 FEBS 2297
Inhibition kinetics of acetylcholinesterase M. Pietsch et al.

As an approximation, Eqn (14), a simplified form of A 125


the Michaelis–Menten Eqns (3) and (10) valid for com-
petitive inhibitors, and an estimated value KI = 1130 100
lm, obtained on the basis of the modified specific

Rate (%)
velocity plot (Eqn 13; Doc. S1, Fig. S2B, see Support- 75
ing information), were used to quantify the interaction
of AChE with gallamine in the presence of 6% MeCN. 50

25
Determination of the parameters of inhibition
using the Michaelis–Menten equation 0
The final kinetic analysis of the inhibition by tacrine 0 500 1000 1500 2000 2500
(Fig. 2A,B), gallamine (Fig. 3A,B) and compound 1 [ATCh] (µM)
(Fig. 4) in the absence and presence of 6% MeCN was
accomplished using Eqn (7) (b¢ = b), Eqn (10) or B 200
Eqn (14), as outlined above. The rates of enzyme-
160
catalysed substrate cleavage were analysed as a func-
tion of both [S] and [I], and the parameters KI, a¢, b or

Rate (%)
120
b, Km and Vmax were calculated simultaneously
(Table 1). Parameter estimates were taken from (modi-
80
fied) specific velocity plots and Michaelis–Menten plots
in the absence of inhibitor (see above). All the values
40
of Km and Vmax calculated independently by the non-
linear optimization of Eqns (7) and (10) (Table 1) were
0
in good agreement with the parameter estimates 0 500 1000 1500 2000 2500
obtained in the absence of the inhibitors (see above). [ATCh] (µM)
In the case of gallamine investigated in the presence of
MeCN, the calculation of the kinetic parameters was Fig. 2. Inhibition of AChE by tacrine in the absence (A) and pres-
based on Eqn (14), as a competitive mode of inhibition ence (B) of 6% MeCN. Michaelis–Menten plots using mean values
was assumed from the modified specific velocity plot and standard deviations of rates from four separate experiments in
100 mM sodium phosphate, 100 mM NaCl, pH 7.3 with 350 lM
(Fig. S2A,B, see Supporting information). The Km
Nbs2 and 0.033 UÆmL)1 AChE. (A) Concentrations of tacrine were
value of 778 lm obtained differed considerably from as follows: open circles, [I] = 0; filled circles, [I] = 0.025 lM; open
the parameter estimate of 671 lm. In contrast, the cal- squares, [I] = 0.05 lM; filled squares, [I] = 0.1 lM; open triangles,
culated Vmax value of 245% was very similar to the [I] = 0.15 lM; filled triangles, [I] = 0.2 lM; open reversed triangles,
parameter estimate of 235%. This behaviour can be [I] = 0.25 lM. Nonlinear regression according to Eqn (10) gave KI =
explained by the competitive mode of inhibition. The 0.027 ± 0.003 lM, a¢ = 1.4 ± 0.2, Km = 101 ± 5 lM and Vmax =
substrate affinity of the enzyme will be reduced, i.e. 110 ± 1%. (B) Concentrations of tacrine were as follows: open
circles, [I] = 0; filled circles, [I] = 0.125 lM; open squares, [I] =
the apparent Km value will be increased, whereas the
0.25 lM; filled squares, [I] = 0.5 lM; open triangles, [I] = 0.75 lM;
maximum velocity of product formation Vmax is not filled triangles, [I] = 1.0 lM; open reversed triangles, [I] = 1.25 lM.
affected [49,50]. With a given set of data for rates as a Nonlinear regression according to Eqn (10) gave KI = 0.26 ±
function of [S], the determination of Vmax and thus Km 0.02 lM, a¢ = 1.1 ± 0.2, Km = 691 ± 35 lM and Vmax = 229 ± 4%.
becomes less accurate when [S] is low relative to the In (A) and (B), the b values were set to zero, as the starting values
apparent Km value [49]. This occurred in our study for obtained from the modified specific velocity plots (data not shown)
high concentrations of gallamine in the presence of 6% were b < 0.

MeCN.
One possibility to avoid this accuracy problem use higher substrate concentrations to analyse AChE
would be to investigate the enzyme–inhibitor interac- inhibition by gallamine in the presence of 6% MeCN,
tion in the presence of higher substrate concentrations. which might also have revealed a deviation from the
However, in the case of AChE, it is known that sub- apparent competitive inhibition. Instead, we investi-
strate inhibition arises under such conditions, with gated the interaction of the inhibitor with AChE over
PAS being involved in the mechanism [12,19,22,68–70]. a range of [S], where substrate inhibition was not
As gallamine binds to PAS [41], a more complex mode observed [7,14]. The maximum [S] value of 2250 lm
of inhibition might result [71]. Therefore, we did not corresponded to  16–22Km for experiments without

2298 FEBS Journal 276 (2009) 2292–2307 ª 2009 The Authors Journal compilation ª 2009 FEBS
M. Pietsch et al. Inhibition kinetics of acetylcholinesterase

A 125 200

100 160
Rate (%)

Rate (%)
75 120

50 80

25 40

0 0
0 500 1000 1500 2000 2500 0 500 1000 1500 2000 2500
[ATCh] (µM) [ATCh] (µM)

B 200 Fig. 4. Inhibition of AChE by compound 1 in the presence of 6%


MeCN. Michaelis–Menten plot using mean values and standard
160 deviations of rates from four separate experiments in 100 mM
sodium phosphate, 100 mM NaCl, pH 7.3 with 350 lM Nbs2 and
0.033 UÆmL)1 AChE. Concentrations of compound 1 were as fol-
Rate (%)

120
lows: open circles, [I] = 0; filled circles, [I] = 1.5 lM; open squares,
[I] = 3.0 lM; filled squares, [I] = 4.5 lM; open triangles,
80 [I] = 6.0 lM; filled triangles, [I] = 7.5 lM. Nonlinear regression
according to Eqn (7) gave KI = 0.59 ± 0.05 lM, a¢ = 1.1 ± 0.1,
40 b¢ = b = 0.096 ± 0.007, Km = 607 ± 25 lM and Vmax = 230 ± 3%.
A value of a = 0.087 was calculated as the quotient of b and a¢.
0
0 500 1000 1500 2000 2500 inhibition of AChE by tacrine and compound 1 in the
[ATCh] (µM) presence of MeCN (Table 1). The KI value obtained
using the predefined Vmax value in Eqn (14) was
Fig. 3. Inhibition of AChE by gallamine in the absence (A) and pres-
ence (B) of 6% MeCN. Michaelis–Menten plots using mean values
2020 lm (Table 1), whereas an only slightly larger
and standard deviations of rates from four separate experiments in value of 2150 lm resulted when Vmax was determined
100 mM sodium phosphate, 100 mM NaCl, pH 7.3 with 350 lM independently.
Nbs2 and 0.033 UÆmL)1 AChE. (A) Concentrations of gallamine The determination of the factors a¢, b and b
were as follows: open circles, [I] = 0; filled circles, [I] = 500 lM; (Table 1) was performed to obtain an insight into the
open squares, [I] = 1000 lM; filled squares, [I] = 2000 lM; open tri- mode of inhibition, and the KI values (Table 1) pro-
angles, [I] = 3000 lM; filled triangles, [I] = 4000 lM; open reversed
vided information on the inhibitory potency of the
triangles, [I] = 5000 lM. Nonlinear regression according to Eqn (7)
gave KI = 270 ± 20 lM, a¢ = 15 ± 2, b¢ = b = 0.25 ± 0.03,
compounds. As depicted in Schemes 2 and 3, the fac-
Km = 135 ± 8 lM and Vmax = 116 ± 1%. A value of a = 0.017 was tor a¢ defines whether the inhibitor binds to an
calculated as the quotient of b and a¢. (B) Concentrations of gall- enzyme–substrate species (ES and EA combined
amine were as follows: open circles, [I] = 0; filled circles, together as ES¢ in Scheme 2 or EA in Scheme 3) with
[I] = 750 lM; open squares, [I] = 1500 lM; filled squares, a greater affinity than to the free enzyme, or vice
[I] = 3000 lM; open triangles, [I] = 4500 lM; filled triangles, versa. The preference of the inhibitor for binding to an
[I] = 6000 lM; open reversed triangles, [I] = 7500 lM. Nonlinear
enzyme–substrate species is reflected in values where
regression according to Eqn (14) with Vmax being set to 235% gave
KI = 2020 ± 50 lM and Km = 699 ± 10 lM.
a¢ < 1, which indicate mixed-type inhibition with a
pronounced uncompetitive component. A higher affin-
ity of the inhibitor to the free enzyme, seen where
MeCN and 3.2–3.7Km for experiments with MeCN a¢ > 1, corresponds to mixed-type inhibition with a
(Table 1). To incorporate a more accurate Vmax value more competitive character. A pure noncompetitive
in the fitting process, this parameter was set to a value mode of inhibition is characterized by a¢ = 1, i.e. an
of 235%, obtained in the absence of gallamine (see equal affinity of the inhibitor to any form of the
above). An analysis of the data according to Eqn (14) enzyme.
using this set Vmax value gave Km = 699 lm (Table 1). An investigation of AChE inhibition by tacrine in
This value is closer to the parameter estimate of the absence of MeCN, according to the kinetic model
671 lm, as well as the Km values calculated for the in Scheme 3, gave values of KI = 0.027 lm and

FEBS Journal 276 (2009) 2292–2307 ª 2009 The Authors Journal compilation ª 2009 FEBS 2299
Inhibition kinetics of acetylcholinesterase M. Pietsch et al.

Table 1. Inhibition of AChE from Electrophorus electricus. Values with standard error were calculated using data (mean values) from four
separate experiments, at five or six inhibitor concentrations and 10 or 12 substrate concentrations. Analysis of the data was performed using
Eqn (7) (gallamine triethiodide, no MeCN; compound 1, 6% v ⁄ v MeCN; b¢ = b), Eqn (10) (tacrine · HCl, no MeCN; tacrine · HCl, 6% v ⁄ v
MeCN) or Eqn (14) (gallamine triethiodide, 6% v ⁄ v MeCN).

Inhibitor MeCN (% v ⁄ v) KI (lM) a¢a b b Km (lM) Vmax (%)

Tacrine · HCl 0 0.027 ± 0.003 1.4 ± 0.2 0b NDc 101 ± 5 110 ± 1


Tacrine · HCl 6 0.26 ± 0.02 1.1 ± 0.2 0b NDc 691 ± 35 229 ± 4
Gallamine triethiodide 0 270 ± 20 15 ± 2d NDc 0.25 ± 0.03 135 ± 8 116 ± 1
Gallamine triethiodide 6 2020 ± 50 NDe 1 NDc 699 ± 10 235f
Compound 1 6 0.59 ± 0.05 1.1 ± 0.1g NDc 0.096 ± 0.007 607 ± 25 230 ± 3
a
A value of 1.3 for k2 ⁄ k3 has been taken from the literature [58] to calculate a¢ for experiments with tacrine · HCl, no MeCN, and
tacrine · HCl, 6% v ⁄ v MeCN. b Starting value b < 0, thus b was set to zero. c ND, nondeterminable. d a = b ⁄ a¢ = 0.017. e b = 1 in
Scheme 3, thus a¢ is nondeterminable. f Vmax was set to 235%, determined during provisional estimate investigations in the absence of gall-
amine triethiodide. g a = b ⁄ a¢ = 0.087.

a¢ = 1.4 (Table 1). The parameter b was necessarily The parameter b represents the substrate conversion
set to zero for the nonlinear analysis (Table 1) and a catalysed by an inhibitor-bound enzyme species com-
catalytically inactive EAI (Scheme 3) could therefore pared with that by the free enzyme (both Schemes 1
be concluded. Thus, the deacylation of the acyl-enzyme and 2). Our study found a value of b = 0.25 for the
was completely blocked by the inhibitor. A mixed-type interaction of AChE with gallamine (Table 1), which
inhibition, tending more to noncompetitive inhibition, confirmed the parameter estimate based on the specific
was found with tacrine. This was characterized by an velocity plot (see above). Both b and the calculated
a¢ value close to unity, and indicated that the affinity value of a were in agreement with the data obtained
of tacrine towards AChE was only minimally affected by Szegletes et al. [46] for the inhibition of AChE by
by acylation of the active site serine. Such a kinetic gallamine with ATCh as substrate, who reported
behaviour has been reported to occur only if b = 0 b = 0.44 and a = 0.019. Under the assumption of
and the acylation rate constant of substrate conversion equilibrium conditions, a is represented by Eqn (4).
k2 is equal to or larger than the deacylation rate con- Therefore, low values of this parameter require either
stant k3 [47]. Both requirements are fulfilled, as shown that ESI (Scheme 1) is not formed (KS ⁄ KS2 and
by the present study and as reported by Froede and KI ⁄ KSI  0) or that a  0 (Scheme 1). As shown in
Wilson [58], respectively. our study, a depends on both b and a¢, with the rela-
These findings were in agreement with the results of tively large value of the latter parameter indicating a
Nochi et al. [72] (obtained with ATCh and AChE from comparably low affinity of both the substrate and the
E. electricus), who reported KI = 20.4 nm and KI* = inhibitor to form an enzyme–substrate–inhibitor spe-
a¢KI(1 + k3 ⁄ k2) = 38.3 nm for tacrine on the basis of cies (Scheme 2). Thus, we hypothesize that the low a
the kinetic model in Scheme 3 (with b = 0) [72,73]. For value results from a diminished formation of ESI
comparison, we calculated a¢ = 1.1 using these values of rather than from inhibition of acylation (Scheme 1).
KI and KI*. This result also indicates a mixed-type inhibi- Recently, nonequilibrium analysis of AChE inhibition
tion with a pronounced noncompetitive component. by the PAS ligands propidium and gallamine resulted
Inhibition of AChE by gallamine (in the absence of in the construction of the ‘steric blockade hypothesis’
MeCN), analysed according to the kinetic model in (based on the model in Scheme 1). This hypothesis
Scheme 2, was characterized by values of demonstrates that PAS ligands inhibit substrate hydro-
KI = 270 lm, a¢ = 15 and b¢ = b = 0.25. A value of lysis without inducing conformational changes in the
a = 0.017 was calculated as the quotient of b and a¢ active site [46]. Nonequilibrium conditions are charac-
(Table 1). The a¢ value obtained demonstrated the terized by k)S < k2 and k)S2 < ak2 [4,46], and thus
preference of gallamine to bind to the free enzyme a  kS2 ⁄ kS was concluded according to Szegletes et al.
rather than to the enzyme–substrate species ES and [46]. The ‘steric blockade hypothesis’ implies that a
EA (combined in ESI¢, Scheme 2), which indicates a ligand bound to PAS slows down ligand entry into
mixed-type inhibition with a pronounced competitive and exit from the active site of AChE (kS2 < kS and
component. Such a kinetic behaviour has recently been k)S2 < k)S) without affecting the thermodynamics of
reported by Mooser and Sigman [74], who found pure the binding of active site-directed ligands (KS2 = KS).
competitive inhibition (KI = 140–320 lm) for the It also stipulates that the PAS ligand has no effect on
interaction of gallamine with AChE from E. electricus. the rate constants of substrate acylation and deacylation

2300 FEBS Journal 276 (2009) 2292–2307 ª 2009 The Authors Journal compilation ª 2009 FEBS
M. Pietsch et al. Inhibition kinetics of acetylcholinesterase

(a = b = 1), and that bound substrate does not alter study by Ronzani [76], where Km values of 85 and
the interaction of the PAS ligand with the enzyme 750 lm were determined for the AChE-catalysed con-
(kI = kSI = kAI and k)I = k)SI = k)AI). Thus, only version of ATCh in the absence and presence of 6.5%
the ratio k)I ⁄ kI, i.e. the KI value, is relevant [46]. As MeCN, respectively. The latter Km value was calcu-
an extension of the steric blockade model, it was pro- lated on the basis of a competitive mode of inhibition
posed that bound PAS ligands also reduce the dis- suggested for MeCN and KI = 0.16 m [75,76]. For
sociation rate constants for product release from the competitive inhibitors, KI can be calculated according
active site, which becomes rate limiting at high [S] to the equation KI = [I] ⁄ [(Km¢ ⁄ Km))1], where Km¢ is
[19,46]. the Michaelis constant in the presence of a certain
On the basis of the ‘steric blockade hypothesis’, we amount of inhibitor [75,76]. Applying this equation to
concluded that the inhibition of AChE by gallamine our experiments and using mean values of the data
(in the absence of MeCN) decreased the rate constants shown in Table 1 (Km¢ = 666 lm at [MeCN] =
kS2 and k)S2 (Scheme 1) to values  1.7% of kS and 1.15 m, Km = 118 lm without MeCN), we calculated
k)S in our experiments. This conclusion agrees with an equivalent KI value of 0.25 m for MeCN. Consider-
the result of a simulated gallamine inhibition of ATCh ing the cosolvent MeCN as a competitive inhibitor, it
hydrolysis under nonequilibrium conditions [46], where might be included as a ‘second’ inhibitor in the fitting
kS2 and k)S2 were set to 1.5% of kS and k)S, respec- equations to analyse the influence of the ‘first’ inhibi-
tively, to obtain optimal correlation between the calcu- tor (i.e. tacrine, gallamine or compound 1). Such
lated and experimentally determined parameters KI, a attempts have, however, not been made in this study.
and b. The last two parameters can also be used to The inhibition experiments performed in the pres-
characterize the relative efficiency of EI to catalyse ence of 6% MeCN and tacrine or 6% MeCN and gall-
substrate conversion: a is defined as the ratio of the amine were analysed according to the kinetic model in
second-order rate constant kcat ⁄ Km with saturating [I] Scheme 3, as ESI was assumed not to be formed in
to that in the absence of inhibitor, and b is the quo- both cases (see above). Our investigations on the basis
tient of the first-order rate constants kcat for substrate of Eqns (10) and (14) revealed increased KI values for
conversion by EI (at saturating [I]) and E (Scheme 1) the two inhibitors compared with the studies without
[46]. According to Eqns (6) and (11), a and b represent MeCN. For tacrine and gallamine, 9.6-fold and 7.5-
the relative efficiency of EI (Scheme 1) if [S] << Km fold increases in the dissociation constant were
and [S]  Km, respectively. Thus, the efficiency of the observed, which resulted in KI = 0.26 lm and
complex AChE–gallamine to hydrolyse ATCh is 1.7– KI = 2020 lm, respectively (Table 1). The factors for
25% of that of free AChE. the increase in KI are in good agreement with data
from a previous study [76], where a sevenfold increase
in KI was determined for the inhibition of AChE by
Influence of MeCN on the inhibition of AChE
neostigmine iodide in the presence of 6.5% MeCN and
The inhibition of AChE by tacrine, gallamine and [S] = 20Km. In addition, a considerable loss of inhibi-
compound 1 was investigated in the presence of 6% tion of immobilized AChE by dichlorvos and parao-
v ⁄ v MeCN (corresponding to a concentration of xon in the presence of increasing amounts of MeCN
1.15 m), and the results are shown in Figs 2B,3B,4 and (0–15%) was reported [79]. This loss was suggested to
Table 1. Even without the addition of inhibitors, our be caused by the denaturing effect of the organic
research showed that the presence of MeCN reduced solvent [79,81], which can also be considered as a
the rate of enzyme-catalysed substrate conversion, pseudo-inhibition process [80]. One reason for enzyme
which is in accordance with several literature reports denaturation in the presence of water-miscible solvents,
on soluble and immobilized AChE from E. electricus such as MeCN, might be the removal of essential
[75–80]. At the highest substrate concentration used in water molecules from the enzyme, necessary for mani-
our experiments, [S] = 2250 lm, the absolute enzyme festing the catalytic activity [80,82].
activity without MeCN was 0.251 ± 0.052 min)1 The inhibition of AChE by tacrine was characterized
(n = 8), whereas the addition of 6% MeCN resulted by values of a¢ = 1.1 and b = 0 (Table 1), i.e. tacrine
in a decrease in the rate to 0.089 ± 0.020 min)1 (in the presence of 6% MeCN) acts as a mixed-type
(n = 8), i.e. to 36% (data not shown). As depicted in inhibitor with a strong noncompetitive component and
Table 1, MeCN also had an influence on the Km value completely blocks deacylation of EAI (Scheme 3) This
of enzymatic substrate conversion, which increased behaviour is identical to that in the absence of MeCN.
from 101–135 to 607–699 lm when 6% MeCN was In contrast with tacrine, the PAS ligand gallamine
present in the assay. A similar result was found in a tends to inhibit AChE in a competitive manner. During

FEBS Journal 276 (2009) 2292–2307 ª 2009 The Authors Journal compilation ª 2009 FEBS 2301
Inhibition kinetics of acetylcholinesterase M. Pietsch et al.

the course of the corresponding analysis, the parameter PAS. In so doing, the inhibitor is proposed to simply
b was assumed to be unity (Table 1), i.e. gallamine act as a ‘permeable cork’ at the entrance of the active
does not affect the deacylation of the acyl-enzyme. site gorge [46]. In contrast, compound 1 is proposed to
Under these conditions, the parameter a¢ becomes bind along this gorge to both the active site at the bot-
irrelevant for the inhibition process and is therefore tom and PAS, which should prevent ligand access to
nondeterminable (Table 1). Comparison of AChE inhi- the active site [83]. Recently, an alternative route to
bition by gallamine in the absence and presence of and ⁄ or from the active site gorge that may be involved
MeCN revealed several similarities. The inhibitor in substrate ⁄ product traffic was found on the basis of
either acts with a pronounced competitive component kinetic crystallography [84]. In that study, opening of
or behaves in a purely competitive manner. Binding of a hole adjacent to the choline-binding locus of the
gallamine to PAS does not alter the deacylation rate anionic site was observed to be particularly caused by
constant of EAI (b = 1) when MeCN is present. This rotation of Trp84 (TcAChE numbering [8]). Hints of a
has already been calculated for gallamine inhibition in ‘back door’ exit were also obtained by mutagenesis
the absence of MeCN under nonequilibrium conditions experiments [85], molecular dynamics simulations [86]
(see above [46]). On the basis of the conclusions drawn and from the absence of bulky leaving groups in crys-
from this calculation, we propose that gallamine not tal structures of TcAChE conjugated with more or less
only slows down substrate association with and sub- gorge-filling inhibitors covalently linked to Ser200
strate ⁄ product dissociation from the active site, as [83,87]. In this context, the usage of inhibitors, such as
assumed in the absence of MeCN [19,46], but blocks compound 1, might be helpful to further elucidate
these events if the organic solvent is present. alternative substrate access to the active site.
As a result of these findings, it can be concluded
that the organic solvent has an influence on the inhibi-
Conclusions
tory potency rather than on the mode of inhibition.
Such behaviour is found regardless of whether the Our investigations with the prototype AChE inhibitors
inhibitor is active site directed, such as tacrine, or tacrine and gallamine have shown that defined amounts
binds to PAS, such as gallamine. of MeCN alter the KI value of the inhibitors (by a
The inhibition of AChE by compound 1 was exclu- certain factor), but not their principal mode of inhibi-
sively investigated in the presence of 6% MeCN tion. This is a new finding which requires further investi-
because of solubility issues associated with com- gation. The presence of MeCN, however, repressed the
pound 1 without organic solvent. An analysis of the catalytic activity of the ternary complex formed by
enzyme–inhibitor interaction, according to Eqn (7), AChE, ATCh and the PAS inhibitor gallamine. On the
revealed a hyperbolic mixed-type inhibition character- basis of the ‘steric blockade hypothesis’ [46], where PAS
ized by KI = 0.59 lm, a¢ = 1.1 and b¢ = b = 0.096; inhibitors are proposed to act as a ‘permeable cork’ at
a = 0.087 was calculated as the quotient of b and a¢ the entrance of the active site gorge, we conclude that
(Table 1). A value of a¢  1 implies that compound 1 the ‘permeability’ of gallamine simply disappears in the
exhibits the same dissociation constant towards AChE presence of the organic solvent. In other words, the PAS
regardless of whether the free enzyme or an enzyme– inhibitor blocks ligand access to the active site. The
substrate intermediate ES¢ (Scheme 2) is involved in kinetic analysis performed in this study was based on
the interaction [49]. However, no statements can be the simultaneous determination of the inhibitory param-
made regarding the dissociation constants of ESI and eters KI, a¢, b or b, as well as Km and Vmax, all obtained
EAI (Scheme 1), as a¢KI (Scheme 2) reflects a compos- by a single calculation. We also demonstrated two
ite of these two constants [14]. Nevertheless, the algorithms for the estimation of kinetic parameters to
observed kinetic behaviour supports the proposed successfully perform kinetic analysis in such circum-
binding mode of compound 1 to act as a dual-site stances. Finally, compound 1 was demonstrated to be
inhibitor and bind along the active site gorge [44], as an effective dual-site inhibitor of AChE.
similarly concluded for heterobivalent tacrine inhibi-
tors [52]. The a and b values obtained indicate that the
Experimental procedures
relative efficiency of EI to catalyse ATCh cleavage (at
saturating [I]) ranges from 8.7% (if [S] << Km) to
Materials
9.6% (if [S]  Km).
The residual activity of EI cannot be explained by ATCh, Nbs2, gallamine triethiodide and tacrine hydrochlo-
the ‘steric blockade hypothesis’, as this hypothesis was ride were obtained from Sigma (Steinheim, Germany).
constructed for inhibitors that exclusively interact with AChE from E. electricus and MeCN (HPLC grade) were

2302 FEBS Journal 276 (2009) 2292–2307 ª 2009 The Authors Journal compilation ª 2009 FEBS
M. Pietsch et al. Inhibition kinetics of acetylcholinesterase

purchased from Fluka (Deisenhofen, Germany). MeCN included in the calculation of the inhibition constant [44]
was dried using phosphorus pentoxide, distilled and stored using Eqn (15):
over molsieve 3 Å. Compound 1 was synthesized as
ðv0  v   Þ
described elsewhere [44]. All measurements were performed I !1
v ¼     þ v  ð15Þ
on a Varian (Darmstadt, Germany) Cary 50 Bio UV ⁄ VIS I I !1
1 þ
spectrometer with a cell holder equipped with a Julabo IC50
(Seelbach, Germany) UC-5B constant temperature water
where v0 is the velocity in the absence of the inhibitor and
bath. Data were analysed using Grafit v5.0 (R. J. Leather-
IC50 is the concentration of the inhibitor which reduces the
barrow, Erithacus Software Ltd, Horley, Surrey, UK).
velocity of the enzyme-catalysed reaction to a value halfway
between v0 and v[I] fi ¥. To determine IC50 values for gall-
AChE inhibition assay amine and tacrine (in the presence and absence of 6%
MeCN), v[I] fi ¥ was set to zero in Eqn (15).
AChE activity was assayed spectrophotometrically at 25 C
according to the method of Ellman et al. [88] in the absence
or presence of 6% v ⁄ v MeCN. Assay buffer was 100 mm Acknowledgements
sodium phosphate, 100 mm NaCl, pH 7.3. A stock solution
This work was supported by grants from the Research
of AChE (100 UÆmL)1) in assay buffer was kept at 0 C. A
1 : 30 dilution of AChE stock was prepared in ice-cold
Training Group 804 ‘Analysis of cellular functions by
assay buffer immediately before starting each measurement. combinatorial chemistry and biochemistry’ (to L.C.
ATCh (0.5–45 mm) and Nbs2 (7 mm) were dissolved in and M.G.). M.P. gratefully acknowledges financial
assay buffer and kept at 0 C. Stock solutions of tacrine, support from Professor Andrew Abell (School of
gallamine and compound 1 were prepared in distilled water, Chemistry and Physics, The University of Adelaide,
assay buffer and MeCN, respectively, and kept at room Adelaide, Australia), ARC grant DP0771901 and a fel-
temperature. Inhibition of enzyme activity was determined lowship within the ‘Postdoc-Programm’ of the German
with 12 (gallamine and tacrine: 25–2250 lm) or 10 (com- Academic Exchange Service (DAAD). The authors
pound 1: 125–2250 lm) different substrate concentrations wish to thank Megan Garvey for critical reading of
in the presence of six (gallamine: 500–5000 lm for measure- the manuscript.
ments without MeCN, 750–7500 lm for measurements in
the presence of 6% MeCN; tacrine: 0.025–0.25 lm for mea-
surements without MeCN, 0.125–1.25 lm for measurements
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2306 FEBS Journal 276 (2009) 2292–2307 ª 2009 The Authors Journal compilation ª 2009 FEBS
M. Pietsch et al. Inhibition kinetics of acetylcholinesterase

88 Ellman GL, Courtney KD, Andres V & Featherstone RM Fig. S2. (A) Modified specific velocity plot for the inhi-
(1961) A new rapid colorimetric determination of acetyl- bition of AChE by gallamine in the presence of 6%
cholinesterase activity. Biochem Pharmacol 7, 88–95. MeCN. (B) Plot of int0 ⁄ (int0)1) versus the reciprocal
concentrations of gallamine.
Supporting information This supplementary material can be found in the
online version of this article.
The following supplementary material is available: Please note: Wiley-Blackwell is not responsible for
Doc. S1. A complete list of equations, including those the content or functionality of any supplementary
that are not part of the manuscript, together with cor- materials supplied by the authors. Any queries (other
responding annotations. than missing material) should be directed to the corre-
Fig. S1. (A) Specific velocity plot for the inhibition of sponding author for the article.
AChE by gallamine in the absence of MeCN. (B) Plot
of int0 ⁄ (int0)1) and int1 ⁄ (int1)1) versus the reciprocal
concentrations of gallamine.

FEBS Journal 276 (2009) 2292–2307 ª 2009 The Authors Journal compilation ª 2009 FEBS 2307

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