The kinetic inhibition of acetylcholinesterase from human herytrocyte by tacrine and some tacrine derivatives

Article abstract of DOI:10.1006/bioo.1998.1121

The kinetics of the reaction catalyzed by human erythrocyte Acetylcholinesterase (ACHE) is studied in the presence of its inhibitor Tacrine (1,2,3,4-tetrahydro-9-acridinamine), and of two newly synthesized compounds, 6-metoxy-Tacrine and N-eptyl-Tacrine. The proposed kinetic model describes the rate of the enzymatic reaction in terms of competitive and uncompetitive mixed inhibition of the three different inhibitors. The kinetic parameters describing the rate of the reaction are obtained. The competitive and uncompetitive inhibition constants of the different inhibitors are reported and the mixed competitive-uncompetitive inhibition is discussed.

Full text of DOI:10.1006/bioo.1998.1121

Bioorganic Chemistry 27, 197–205 (1999)
Article ID bioo.1998.1121, available online at http://www.idealibrary.com on
The Kinetic Inhibition of Acetylcholinesterase from Human
Herytrocyte by Tacrine and Some Tacrine Derivatives1
Fernando Porcelli,2 Maurizio Delfini, and Maria Rosaria Del Giudice
`
Cattedra di Chimica Fisica, Dipartimento di Scienze Ambientali, Universita della Tuscia,
`
Via S. Camillo de Lellis-01100, Viterbo, Italy; Dipartimento di Chimica, Universita “La Sapienza,”
P.le Aldo Moro 5, 00185, Roma, Italy; and Laboratorio Chimica del Farmaco,
`
Istituto Superiore di Sanita, V.le Regina Elena 299-00161, Roma, Italy
Received June 23, 1998
The kinetics of the reaction catalyzed by human erythrocyte Acetylcholinesterase (AChE)
is studied in the presence of its inhibitor Tacrine (1,2,3,4-tetrahydro-9-acridinamine), and of
two newly synthesized compounds, 6-metoxy-Tacrine and N-eptyl-Tacrine. The proposed
kinetic model describes the rate of the enzymatic reaction in terms of competitive and uncompeti-
tive mixed inhibition of the three different inhibitors. The kinetic parameters describing the
rate of the reaction are obtained. The competitive and uncompetitive inhibition constants of
the different inhibitors are reported and the mixed competitive–uncompetitive inhibition is
discussed.
᭧ 1999 Academic Press
Key Words: kinetics; enzyme inhibition; acetylcholinesterase; tacrine; Alzheimer’s disease.
INTRODUCTION
Acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7, AChE) is an ex-
trinsic membrane-bound enzyme projecting into the synapse. It promotes the hydroly-
sis of the neurotransmitter acetylcholine (ACh) at the cholinergic synapses with
liberation of choline (1–7). Because of its central role in the neurotransmission system,
the AChE, has been an attractive target for the rational design of mechanism-based
inhibitors (4). The inhibition of AchE via phosphorylation of the serine at the active
site has been used in chemical warfare agents such as “sarin” and “soman.” Also,
other AchE inhibitors such as physostigmine and neostigmine have been used as
therapeutic agents in the treatment of glaucoma and “myasthenia gravis.” Over the
last decade inhibitors of AChE have been employed in allaying the symptoms of
Alzheimer’s disease. In fact, a topic of the disease is a developing cholinergic deficit
that severely impairs the neuromodulatory function of this transmitter. Thus, it may
1 This research was supported by Grant COFIN MURST 97 CFSIB.
2 To whom correspondence and reprint requests should be addressed. Fax: ϩ39-0761-357179.
E-mail: porcelli@unitus.it.
197
0045-2068/99 $30.00
Copyright ᭧ 1999 by Academic Press
All rights of reproduction in any form reserved.

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PORCELLI, DELFINI, AND DEL GIUDICE
be possible to increase ACh levels in affected regions of the brain by inhibiting AChE.
Tacrine, 1,2,3,4-tetrahydro-9-aminoacridine (for a Pharmacological review see (8)) is
a potent inhibitor of AChE and has become the first drug specifically approved by
FDA (Food and Drug Administration) in 1993 for the treatment of Alzheimer’s
symptoms. Therefore, Tacrine derivatives have become the main scaffold to obtain
more active and better tolerated drugs for Alzheimer treatment by modulating their
pharmacokinetics and pharmacodynamics.
Although several kinetic models describing the hydrolysis of Ach in the absence
and presence of inhibitors have been proposed (9–16), there is a fundamental lack
of agreement in the literature on the type of inhibition caused by Tacrine.
In this paper the inhibition of AChE from human erythrocytes by three different
inhibitors, Tacrine and two recently synthesized Tacrine derivatives (Fig. 1), is studied.
A new kinetic inhibition model is proposed to describe the reaction rate in term of
noncompetitive and mixed competitive–uncompetitive behavior of the inhibitors.
Further, the kinetic and thermodynamic constants obtained are used to distinguish
between the two different categories of inhibition.
MATERIALS AND METHODS
Enzymes and substrates. The lyophilized powder of AChE type XII from human
erythrocytes (0.62 units/mg solid) containing phosphate buffer salts was purchased
from Sigma Chemical Co. (St. Louis, MO; by definition, one unit of AChE hydrolyzes
FIG. 1. Molecular structures of synthesized Tacrine darivatives: 1a, Tacrine; 1b, 6-metoxyacrine; 1c,
N-epthltacrine.

INHIBITION OF ACETYLCHOLINESTERASE FROM HUMAN HERYTROCYTE
199
1 mM of acetylcholine to choline and acetate per minute at pH 8.0 and 37ЊC. Acetyl-
thiocholine iodide, Dithiobisnitrobenzoic acid (DTNB), and sodium phosphate were
reagent grade also from Sigma Chemical Co. The inhibitors Tacrine, 6-metoxytacrine,
and N-eptyltacrine were prepared by M. R. Del Giudice according to a procedure
already described elsewhere (23).
Spectrophotometric measurements were performed by a Varian DMS 90 UV-VIS
spectrophotometer, equipped with a thermostated cell holder. Stock enzyme-buffered
solutions (2.0 units per milliliter) were stored at 5ЊC and prepared daily to avoid the
loss of enzyme activity after a freeze–thaw cycle (24). The amount of enzyme used
in the kinetic assays was in the order of 0.02 units per milliliter.
Initial velocity measurements. The reaction rate was determined according to the
modified Ellmann’s method (25), which assumes the following reactions:
AChe
Acetylthiocholine —→ Thiocholine ϩ Acetate
fast
Thiocholine ϩ DTNB —→ Yellow color,
[1]
where the formation of the yellow anion of 5-thio-2-nitro-benzoic anion is a function
of hydrolase activity (␧ ϭ 13600 MϪ1 cmϪ1 at ␭ ϭ 412 nm).
Reaction mixtures were made up to a 1.00 ml containing sodium phosphate buffer
0.1 M, pH 8.00, Acetylthiocholine 1.0 ϫ 10Ϫ4 Ϫ 5.0 ϫ 10Ϫ4 M, DTNB 5.0 ϫ 10Ϫ4
M, and inhibitors, Tacrine, 6-metoxytacrine, and N-eptyltacrine 1.5 ϫ 10Ϫ7 Ϫ 7.0 ϫ
10Ϫ7 M.
The reaction was initiated by adding 0.010 ml of standardized enzyme solution.
Initial velocities were based upon the early linear portions of the reaction curves
usually involving the first few percent of the total reaction and were corrected for
background spontaneous hydrolysis of acetylthiocholine.
The experiments of inhibition kinetics were performed after constant preincubation
time (15 min) and inhibitor concentration while varying substrate concentration.
KINETIC EQUATIONS
The rate equations. In presence of inhibitor like Tacrine and in absence of substrate
inhibition, the kinetic mechanism can be described by the following equations:
k
k
k
p
EA
1
3
E ϩ S i ES → ϩ → E ϩ A
[2a]
[2b]
[2c]
[2d]
k
C
Ϫ1
k
2
E ϩ I i EI
k
Ϫ2
k
4
ES ϩ I i ESI
k
Ϫ4
k
5
EA ϩ I i EAI.
k
Ϫ5

200
PORCELLI, DELFINI, AND DEL GIUDICE
The symbols S, C, and A refer to concentrations of acethylcholine, choline, and acetic
acid, respectively. A similar approach, although with different enzyme and substrates,
has been successfully used (26) to describe the kinetics of the GABA (␥-aminobutyric
acid)—glutamate transformation in presence of inhibitors of the ␥-aminobutyrate
aminotransferase.
The Eq. [2a] involves two enzyme–substrate intermediates: the first one, ES, may
be considered a Michaelis addition complex, while the second, EA, may be considered
the acylenzyme derived from the Michaelis complex when the acetyl group of the
acetylcholine is transferred from the substrate to a group of the active site of the
enzyme (E). Since we are interested in studying the competitive and uncompetitive
inhibitions by Tacrine and its derivatives, Scheme 2 does not account for the inhibition
by the substrate. Of course the proposed equations are then valid for substrate concen-
trations below the range of substrate inhibition.
The total enzyme concentrations [Eo] is given by:
[Eo] ϭ [E] ϩ [EA] ϩ [ES] ϩ [EI] ϩ [ESI] ϩ [EAI].
[3]
Because of the extremely fast acylation and deacylation rates, the kinetic analyses
involve the steady-state approximation, that is (d[E]/dt) ϭ 0 during initial velocity
measurements, under the condition [Eo] ¿[S]. The reciprocal form of the forward
rate equation is then given by:
Kapp
vm
v
[I]
ϭ 1 ϩ
1 ϩ
ϩ Kunc [I],
[4]
΂ ΃
[S]
KI
with
vm ϭ kp [Eo],
[5]
[6]
(kϪ1 ϩ k3)kp
kϪ2
kp
1
Kapp
ϭ
;
KI ϭ
;
Kunc
ϭ
ϩ
,
΂ ΃
k1k3
k2
k3KI KA
being
kϪ4
kϪ5
KIЈ ϭ
;
KA ϭ
.
[7]
k4
k5
In the equations KI , KIЈ, and KA are the dissociation constants of the various enzyme–
inhibitor complexes and the different quantities are expressed in the following units:
(1/vm) in [min/mol], kϪ1, kϪ2, kϪ4, kϪ5, k3, kp in [minϪ1], k1, k2, k4, k5, in [1/mol min],
KI , KIЈ, and KA in [mol/1] (i.e., M), Kunc in [1/mol] (i.e., MϪ1), [Eo] and [S] in [mol/
1] (i.e., M). The constant Kapp expressed in [mol/1] can be considered as the apparent
Michaelis constant. Rate Eq. [4] assumes the deacylation as the rate determining step
and is then formally similar to that proposed by Rosenberry (3,15). However, in

INHIBITION OF ACETYLCHOLINESTERASE FROM HUMAN HERYTROCYTE
201
Scheme 2 the uncompetitive inhibition is attributed to the kinetic interactions given
in the Eqs. [2c] and [2d].
RESULTS AND DISCUSSION
Kinetic predictions. Equation [4] allows the predictions summarized hereafter.
1. A double reciprocal plot of the initial velocity 1/v versus the concentration of
the substrate 1/[S] in the presence and absence of inhibitors yields a set of straight
lines with the intercepts on the ordinates H(1, 0) and slopes T(1, 0) given by
1
vm
H(1, 0) ϭ
T(1, 0) ϭ
(1 ϩ Kunc[I])
[8]
[9]
Kapp
vm
[I]
1 ϩ
.
΂ ΃
KI
2. The intercepts H(1, 0) should be equal to the reciprocal of vm in the absence of
the inhibitor, but should increase with the inhibitors concentration [I]. The slopes
T(1, 0) should increase with [I]. The value of Kapp, representing the apparent Michaelis
constant, can be determined from Eq. (9) in absence of inhibitor.
3. The plot of the intercepts H(1, 0) versus the concentration of the inhibitor [I]
is represented by a straight line whose slope is equal to the constant Kunc given in
Eqs. [6] and [7] in terms of the dissociation constants of the enzyme–inhibitor
complexes (KI , KA) and of the acylated enzyme k3 as described in Eqs. [6] and [7].
4. The plot of the slopes T(1, 0) versus [I] is a straight line whose slope allows
to determine the constant KI , which describes the competitive inhibition in terms of
associations of the inhibitor with the free enzyme.
Results. The predictions reported above were experimentally observed as shown
by the data reported in Figs. 2–4, in Table 1, and summarized hereafter.
1. The reciprocal plots (Fig. 2 for Tacrine) 1/v versus 1/[S] in the absence and in
the presence of increasing concentrations of inhibitors are straight lines with different
values of the intercept and slope as predicted from Eq. [4]. This behavior is typical
of a mixed competitive–uncompetitive inhibition pattern (15,27). In fact the terms
uncompetitive and competitive inhibition used in this paper have been recommended
by the IUB (International Union of Biochemistry). The uncompetitive inhibition has
TABLE 1
The KI and Kunc constants
KI (␮M)
1/Kunc (␮M)
Tacrine
a 0.034 Ϯ 0.002
a* 0.040 Ϯ 0.010
0.056 Ϯ 0.003
b 0.14 Ϯ 0.01
b* 0.13 Ϯ 0.05
0.16 Ϯ 0.02
6-metoxytacrine
N-eptyltacrine
0.039 Ϯ 0.005
0.05 Ϯ 0.01
Note. a and b this paper, a* and b* from reference (28).

202
PORCELLI, DELFINI, AND DEL GIUDICE
FIG. 2. Double reciprocal plot of initial velocities against acetylthiocholine concentration at T ϭ
25ЊC, pH 8.0, in a sodium phosphate buffer in the presence and absence of Tacrine. The numbers by the
curves indicate the concentration of Tacrine.
FIG. 3. Linear plot of intercepts H(1, 0) against concentrations of inhibitors. (Triangle is 6-metoxyta-
crine; square is N-eptyltacrine; circle is Tacrine.)

INHIBITION OF ACETYLCHOLINESTERASE FROM HUMAN HERYTROCYTE
203
FIG. 4. Linear plot of slopes T(1, 0) against concentrations of inhibitors. (Triangle is 6-metoxytacrine;
square is N-eptyltacrine; circle is Tacrine.)
pictured as the complex formation between inhibitor and some enzyme–substrate
intermediates and has no effect on the apparent value of (vm /KM). The competitive
inhibition is the binding between the free enzyme and the inhibitor and has no effect
on the apparent value of vm. The values of 1/vm ϭ 1.18(Ϯ0.1) ϫ 105 min/mol (the
intercept), and of Kapp ϭ Km ϭ 0.11 Ϯ 0.05 mM (the slope), were calculated from
the straight line obtained in the absence of the inhibitor and are in good agreement
with the same reported in literature (25).
2. As predicted by Eq. [8] the experimental plots of H(1, 0) vs [I] (Fig. 3) are
straight lines whose slopes are related to the values of the constant Kunc, which
accounts for the uncompetitive inhibition of different Tacrine derivatives on AChE.
The values of 1/Kunc for the three inhibitors were determined from these plots and
reported in Table 1. It is worth noting that those regarding Tacrine are in good
agreement with values reported in the literature (16,28).
3. The plots of the slopes T(1, 0) vs [I] (Fig. 4) are straight lines as predicted by
Eq. [9]. The analysis of the curves allows to determine the values of the constant
KI from the slopes and of the constant Kapp from the intercepts. The intercepts on the
abscissa (T ϭ 0) are the constants KI ϭ [I] expressed in mol/1. As for the
1/Kunc, the value of KI for Tacrine is in good agreement with values reported in the
literature (28).
Discussion. In the absence of substrate inhibition (28) the new kinetic model fits
very well the experimental inhibition of AChE by Tacrine. The kinetic constants
values for Tacrine and 6-metoxytacrine (see Table 1) indicate that there is a mixed
competitive–uncompetitive inhibition mechanism. In agreement with data reported

204
PORCELLI, DELFINI, AND DEL GIUDICE
in the literature for Tacrine (16,28), the values of KI are smaller than those of 1/Kunc.
Moreover, the values of KI for 6-metoxy-tacrine, which account for the competitive
inhibition, (KI ϭ 0.056 Ϯ 0.003 ␮M) show that it has about the same inhibitory
power of Tacrine (KI ϭ 0.034 Ϯ 0.002 ␮M). For N-eptyltacrine derivative, the values
of the inhibition constant KI ϭ 0.039 Ϯ 0.005 ␮M is very similar to Tacrine but the
(1/Kunc) ϭ 0.05 Ϯ 0.01 ␮M has much lower value showing a competitive inhibition
pattern. In this case, the inhibitor effects only the values of Km but does not change
vm. The obtained results show that 6-metoxytacrine does not have any significant
increase in the affinity for the AChE and has the identical inhibition pattern of Tacrine.
On the contrary, N-eptylacrine shows a significant different inhibitory pattern respect
to Tacrine. These behaviors may be due to the steric hindrance of the eptyl group
which may disrupt the planarity of the aminoacridine ring system. According to the
X-ray structures of the complex Tacrine–AchE (6), the active site of AChE contains
a catalytic triad (Ser-Hys-Glu) located at the bottom of a narrow gorge (5) and Tacrine
is stacked between the rings of Phe-330 and Trp-84. Thus, the planarity of the
aminoacridine ring is very crucial in order to obtain the interaction between the ␲
lectrons of Phe330 and Trp84 and the aminoacridine ring. Solution NMR studies are
in progress to verify these hypothesis.
CONCLUSIONS
From the systematic analysis of the initial velocity kinetics of the enzymatic reaction
in the presence and absence of added inhibitors (Tacrine or 6-metoxy-tacrine or
N-eptyltacrine) the following informations have been obtained.
1. In presence of inhibitors, and when the substrate inhibition can be neglected,
the kinetics of the reaction catalyzed by AChE are well described by the rate equations
reported under Kinetic Equations.
2. The kinetics parameters which account for the Tacrine inhibition of the reaction
are in good agreement with other reported values (28).
3. The inhibition constants values for Tacrine and 6-metoxy-tacrine are consistent
with the previous data for Tacrine, KI Ͻ (1/Kunc) (16). For both the inhibitors the
inhibition pattern is mixed competitive-uncompetitive (20–22). Further, given the
values of the Kunc, both derivatives seen to have similar uncompetitive inhibitory
power.
4. The inhibition constants for N-eptyl-tacrine, KI and (1/Kunc) can be considered,
within the error, the same so that the inhibition pattern is competitive.
5. Finally, the metoxy group in position 6 of the aminoacridine ring, aimed to
increase the electron density on the nitrogen in position 9, did not show remarkable
effects on the inhibitory power. On the contrary the presence of the eptyl chain on
the nitrogen in position 9 shown a drastic change in the inhibition, pattern, suggesting
a key role of this position in modulating the compound activity.
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