J BIOCHEM MOLECULAR TOXICOLOGYVolume 23, Number 5, 2009

Comparison of Active Sites of Butyrylcholinesterase andAcetylcholinesterase Based on Inhibition by GeometricIsomers of Benzene-di-N-Substituted CarbamatesShyh-Ying Chiou,1,2 Chuan-Fu Huang,1,2 Mei-Ting Hwang,3 and Gialih Lin3

1Department of Neurosurgery, Chung Shan Medical University Hospital, Taichung 402, Taiwan2School of Medicine, Chung Shan Medical University, Taichung 402, Taiwan3Department of Chemistry, National Chung-Hsing University, Taichung 402, Taiwan; E-mail: gilin@dragon.nchu.edu.tw

Received 14 October 2008; revised 8 January 2009; accepted 2 February 2009

ABSTRACT: We have reported that benzene-1,2-,1,3-, and 1,4-di-N-substituted carbamates (1–15) arecharacterized as the conformationally constrained in-hibitors of acetylcholinesterase and mimic gauche,eclipsed, and anti-conformations of acetylcholine, re-spectively (J Biochem Mol Toxicol 2007;21:348–353). Wefurther report the inhibition of butyrylcholinesteraseby these inhibitors. Carbamates 1–15 are also charac-terized as the pseudosubstrate inhibitors of butyryl-cholinesterase as in the acetylcholinesterase catalysis.Benzene-1,4-di-N-n-hexylcarbamate (12) and benzene-1,4-di-N-n-octylcarbamate (13) are the two most potentinhibitors of butyrylcholinesterase among inhibitors1–15. These two para compounds, with the angle of 180◦between two C(benzene) O bonds, mimic the prefer-able anti C O/C N conformers for the choline ethy-lene backbone of butyrylcholine during the butyryl-cholinesterase catalysis. The second n-hexylcarbamylor n-octylcarbamyl moiety of inhibitors 12 and 13 isproposed to bind tightly to the peripheral anionicsite of butyrylcholinesterase from molecular mod-eling. Butyrylcholinesterase prefers para-carbamatesto ortho- and meta-carbamates, whereas acetyl-cholinesterase prefers para- and meta-carbamates toortho-carbamates. This result implies that the anionicsite of butyrylcholinesterase is relatively smaller thanthat of acetylcholinesterase because meta-carbamates,which may bind to the anionic sites of both enzymes,are not potent inhibitors of butyrylcholinesterase.C© 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol23:303–308, 2009; Published online in Wiley InterScience(www.interscience.wiley.com). DOI 10:1002/jbt.20286

Correspondence to: Gialih Lin.Contract Grant Sponsor: National Council of Taiwan.

c© 2009 Wiley Periodicals, Inc.

KEYWORDS: Butyrylcholinesterase; Acetylcholineste-rase; Carbamate; Conformation; Inhibitor

INTRODUCTION

Two forms of cholinesterase coexist ubiquitouslythroughout the body, acetylcholinesterase (AChE; EC3.1.1.7) [1–3] and butyrylcholinesterase (BChE; EC3.1.1.8) [4–7], and although highly homologous, >65%,they are products of different genes on chromosomes7 and 3 in humans, respectively. Both subtype unse-lective cholinesterase and human AChE-selective in-hibitors have been used in Alzheimer’s disease toamplify the action of acetylcholine at remaining cholin-ergic synapses within the Alzheimer’s disease brain.The X-ray crystal structures of Torpedo californica AChEhave revealed that the enzyme contains a catalytic triadsimilar to that present in other serine hydrolases. It hasalso revealed that this triad is located near the bottomof a deep and narrow gorge about 20 A in depth [2].The X-ray crystal structure of human BChE has beenrecently reported [4,5,7]. Torpedo californica AChE andhuman BChE have a common catalytic triad, Ser–His–Glu. The active sites of both enzymes are located atthe bottom of a cavity and act as nucleophiles to attackthe carbonyl groups of substrates or pseudosubstrateinhibitors.

Carbamate inhibitors, such as Alzheimer’s dis-ease drug Rivastigmine (Exelon) and aryl carbamates,are characterized as the pseudosubstrate inhibitors ofAChE, BChE, cholesterol esterase, and lipase [3,8–15].In the presence of substrate, the kinetic schemes forpseudosubstrate inhibitions of serine hydrolases bycarbamate inhibitors have been illustrated (Figure 1)[8]. These reactions are going on simultaneously, withthe inhibitor and substrate competing for the active

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304 CHIOU ET AL. Volume 23, Number 5, 2009

E + SKm

ES E + Pkcat

E + I

KiEI E + Q

k2

P'

EI'k3

FIGURE 1. Kinetic scheme for the pseudosubstrate inhibition of bu-tyrylcholinesterase by carbamates 1–15 in the presence of substrate.E, enzyme; S, substrate butyrylthiocholine; ES, acylenzyme interme-diate; I, carbamate, EI, enzyme-inhibitor tetrahedral intermediate;EI′, carbamyl enzyme intermediate; P, thiocholine from the substratereaction; P′, alcohol-leaving groups from the pseudosubstrate inhibi-tion; Q, carbamic acids.

site of the enzyme. In addition, reactivation of the en-zyme is insignificant when compared to carbamylationof the enzyme and therefore the k3 values can be ig-nored (k2 � k3). Equation (1) is the solution of differen-tial equation that describes the set of reactions depictedin Figure 1. In Eq. (1), the kapp values are first-order rateconstants, which are obtained by Hosie’s method.

kapp = k2[I]/(Ki (1 + [S]/Km) + [I]) (1)

The Km value of BChE-catalyzed hydrolysis of bu-tyrylthiocholine in the presence of 5,5′-dithio-bis(2-nitrobenzoic acid) is determined to be 0.10 ± 0.02 mMfrom the Michaelis–Menten equation. Thus, Eq. (1) be-comes Eq. (2) when [S] = 0.1 mM.

kapp = k2[I]/(2Ki + [I]) (2)

Therefore, the Ki and k2 values are obtained as param-eters from the nonlinear least squares of curve fittingsof kapp vs. inhibitor concentration [I] following Eq. (2).The bimolecular rate constant, ki = k2/Ki , is defined asthe overall inhibitory potency.

Benzene-1,2-, 1,3-, and 1,4-di-N-substituted carba-mates (1–15) are synthesized as the conformationallyconstrained substrate (or inhibitor) analogs of phos-pholipase A2 [16,17], acetylcholinesterase [18], lipase[19], and cholesterol esterase [20] since these com-pounds mimic gauche, eclipsed, and anti-conformationsof the ethylene backbones of the substrates, respec-tively (Figure 2). Thus, with the angle of 60◦, 120◦, and180◦ between two C(benzene)–O bonds, benzene-1,2-di-N- substituted carbamates (1–5), benzene-1,3-di-N-substituted carbamates (6–10), and benzene-1,4-di-N-substituted carbamates (11–15) mimic gauche, eclipsed,and anti-C O/C N conformers of the choline ethy-lene backbone of butyrylcholine during the butyryl-cholinesterase catalysis, respectively.

MATERIALS AND METHODS

Materials

All chemicals were of the highest grade avail-able. Silica gel used in liquid chromatography andthin-layer chromatography plates were obtained fromMerck (Whitehouse Station, NJ). Horse serum BChE,butyrylthiocholine, and 5,5′-dithio-bis (2-nitrobenzoicacid) (DTNB) were obtained from Sigma (St. Louis,MO).

Chemistry

Benzene-1,2-di-N-substituted carbamates (1–5),benzene-1,3-di-N-substituted carbamates (6–10), andbenzene-1,4-di-N-substituted carbamates (11–15)(Figure 2) were synthesized from the condensation ofcatechol, resorcinol, and hydroquinone, respectively,with three equivalents of the corresponding isocyanatein triethylamine at 25◦C for 24 h (65%–90% yield) asdescribed previously [18–20].

Instrumental Methods

All steady-state kinetic data were obtained froma UV–visible spectrometer (Agilent 8453) with a cellholder circulated with a water bath.

Data Reduction and Molecular Modeling

Origin (version 6.0) was used for the linear andnonlinear least-squares curve fittings. Molecular struc-tures of para-13, meta-8, and ortho-3 shown in Figure 4were depicted from the molecular structures afterMM-2 energy minimization (minimum root meansquare gradient was set to be 0.01) by CS Chem 3D(version 6.0).

BChE Inhibition

The inhibition reactions of BChE were determinedby the Ellman assay [21]. The BChE-catalyzed hydroly-sis of butyrylthiocholine (0.1 mM) in the presence of5,5′-dithio-bis(2-nitrobenzoic acid) (0.1 mM) and in-hibitors 1–15 were followed continuously at 410 nmon a UV–visible spectrometer at 25◦C, pH 7.1. The Ki

and k2 values were obtained from the nonlinear leastsquares of curve fittings of the kapp values vs. inhibi-tion concentration [I] plot following Eq. (2) (Figure 3and Table 1).

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Volume 23, Number 5, 2009 BENZENE-DI-CARBAMATES INHIBIT BCHE AND AChE 305

O

O

OO

O

O

NHR

O

O

RNH

NHR

O

H

H

O

N+

H H

H

O

H

N+

H H

HH

H

O

N+

H

O

C3H7

C3H7

O

O

C3H7

O

N+O

ϕ=60o

ϕ=120o

ϕ=180o

Ψ=120o

Ψ=60o

Ψ=180o

NHR

O

RNH

O

O

NHR

1: R = n-C4H9

2: R = n-C6H13

3: R = n-C8H17

4: R = t-C4H9

5: R = CH2Ph

gauche eclipsed

anti

6: R = n-C4H9

7: R = n-C6H13

8: R = n-C8H17

9: R = t-C4H9

10: R = CH2Ph11: R = n-C4H9

12: R = n-C6H13

13: R = n-C8H17

14: R = t-C4H9

15: R = CH2Ph

FIGURE 2. Possible conformations of the choline ethylene glycol backbone of butyrylcholine and chemical structures of inhibitors 1–15.

TABLE 1. Inhibition of BChE by Benzene-di-N-Substituted Carbamates (1–15) and the Selectivity of AChE over BChEa

Inhibitors Ki (nM)b k2(10−3 s−1)b ki (103 M−1s−1)c ki (AChE)/ki (BChE)a

1 240 ± 60 5.8 ± 0.2 24 ± 6 600 ± 2002 130 ± 40 6.5 ± 0.3 50 ± 20 1000 ± 6003 21 ± 7 5.7 ± 0.2 280 ± 90 (3 ± 1) × 104

4 740 ± 60 6.56 ± 0.06 8.8 ± 0.8 320 ± 705 11000 ± 3000 9.4 ± 0.3 0.8 ± 0.2 100 ± 506 260 ± 30 7.3 ± 0.1 28 ± 3 100 ± 407 50 ± 10 6.5 ± 0.1 140 ± 30 1300 ± 5008 11 ± 3 4.4 ± 0.1 400 ± 100 (2.5 ± 0.6) × 104

9 21000 ± 6000 7.16 ± 0.07 0.33 ± 0.09 6 ± 210 34000 ± 5000 13.09 ± 0.03 0.39 ± 0.05 700 ± 10011 5.3 ± 0.6 2.43 ± 0.03 440 ± 50 900 ± 20012 0.7 ± 0.2 2.61 ± 0.06 4000 ± 1000 (2.5 ± 0.6) × 104

13 2.5 ± 0.5 13 ± 2 5000 ± 1000 (1.3 ± 0.5) × 105

14 80 ± 30 7.16 ± 0.09 90 ± 40 600 ± 30015 4600 ± 800 6.28 ± 0.08 1.4 ± 0.3 50 ± 20

a The selectivity of AChE over BChE is defined as ki (AChE)/ki (BChE), where the ki (AChE) values are obtained from [18].b Obtained from parameters of the nonlinear least-squares curve fittings of kapp vs. [I] plot following Eq. (2).c Defined as k2/Ki .

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306 CHIOU ET AL. Volume 23, Number 5, 2009

A

−0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

0.000

0.001

0.002

0.003

0.004

0.005

0.006

k ap

p (

s)

[I] (μM)

B

0.0 0.5 1.0 1.5 2.0 2.5

0.000

0.001

0.002

0.003

0.004

0.005

k app(s

)

[I] (μM)

C

0 1 2 3 4 5−0.002

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

k ap

p(s

)

[I] (μM)

FIGURE 3. Nonlinear least-squares curve fittings of kapp vs. inhibitorconcentration [I] plots against Eq. (1) for the pseudosubstrate inhi-bitions [8] of BChE by benzene-1,2-di-N-n-butylcarbamate (1) (A),benzene-1,3-di-N-n-octylcarbamate (8) (B), and benzene-1,4-di-N-n-butylcarbamate (13) (C).

Statistics

Errors of Ki and k2 values (Table 1) were depictedas the errors of parameters from the nonlinear leastsquares of curve fittings of the kapp values vs. inhibi-tion concentration [I] plot following Eq. (2) by Origin(version 6.0). In general, the number of independent

experiments (n) was from 10 to 15. Errors of ki values(Table 1) were depicted as the followings:

Error of ki = ((Error of Ki)2 + (Error of k2)2)1/2

Errors of selectivity of AChE over BChE (Table 1) werealso depicted similarly.

RESULTS

Benzene-1,2-di-N-substituted carbamates (1–5),benzene-1,3-di-N-substituted carbamates (6–10), andbenzene-1,4-di-N-substituted carbamates (11–15) [16–20] were synthesized to mimic three conformations atthe choline ethylene backbone of butyrylcholine duringthe BChE catalysis (Figure 2).

Inhibitors 1–15 were all characterized as pseudo-substrate inhibitors (Figure 1) [8–15] of BChE (Figure 3and Table 1) as those of AChE [18]. For a seriesof geometric isomers, para compounds (benzene-1,4-di-N-substituted carbamates) are the most potent in-hibitors (Table 1). Comparison of the carbamyl sub-stituents of inhibitors 1–15 indicates that inhibitorswith n-hexylcarbamyl or n-octylcarbamyl group aremuch more potent than the other carbamate inhibitors(Table 1). The inhibitor selectivity for BChE over AChEis low (Table 1).

DISCUSSION

Inhibitory Potencies for VariedDi-Substituted Geometries ofCarbamates (1–15)

Benzene-1,2-di-N-substituted carbamates (1–5),benzene-1,3-di-N-substituted carbamates (6–10), andbenzene-1,4-di-N-substituted carbamates (11–15)(Figure 2) are characterized as pseudosubstrate in-hibitors (Figure 1) of BChE (Figure 3 and Table 1) andAChE [18]. Therefore, one of two carbamyl groupsof the inhibitors covalently binds to the acyl-bindingsite of BChE (Figure 4). Then, the second carbamylgroup of the inhibitor is in different binding sites ofBChE (Figure 4). For a series of geometric isomers,for example, benzene-1,2-di-N-n-octylcarbamate (3),benzene-1,3-di-N-n-octylcarbamate (8), and benzene-1,4-di-N-n-octylcarbamate (13), para compound 13 isa more potent BChE inhibitor than meta and orthocompounds (Table 1). Therefore, para-carbamates 11–15, with the angle of 180◦ between two C(benzene)–Obonds, mimic the preferable anti C O/C N conform-ers of choline ethylene backbone of butyrylcholinein the butyrylcholinesterase catalysis (Figure 2). In

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Volume 23, Number 5, 2009 BENZENE-DI-CARBAMATES INHIBIT BCHE AND AChE 307

O

O

N

O

O

N

H

H

O ON

O

O

NH

H

O

O

N

OO

N

H

H

Oxyanion hole

Anionic site

OH

Ser198

Catalytic site

Entrance

His438

Glu325Trp82

Gly116 Gly117

Ala199

Peripheral anionic site

Acyl group binding site

FIGURE 4. Superimposition of para-13, meta-8, and ortho-3 into the active site of BChE. Molecular structures of para-13, meta-8, and ortho-3 aredepicted from the molecular structures after MM-2 energy minimization. One of two carbamyl groups of the inhibitors covalently is fit into theacyl-binding site of BChE [7] manually. Then, the second carbamyl group of the inhibitor is in different binding sites of BChE.

other words, the conformation for the butyryl andquaternary ammonium groups at the choline ethylenebackbone of butyrylcholine (Figure 2) may fullyextend as the anti-conformation during the BChEcatalysis. Furthermore, the second carbamyl groupsof para-carbamates 11–15 are fit into the peripheralanionic site of BChE from molecular modeling, but thesecond carbamyl groups of ortho- and meta- carbamatesare far away from the peripheral anionic site of BChEfrom molecular modeling (Figure 4).

Meta-carbamates 6–10 are potent inhibitors ofAChE [18] but are less potent inhibitors of BChE(Table 1). The second carbamyl groups of meta-carbamates 6–10 are fit into the anionic binding sitesof both BChE and AChE (Figure 4). The X-ray crystalstructures of both enzymes indicate that three residues,Trp84, Glu199, and Phe330, in the anionic binding siteof AChE represent the interaction between the positivecharge of the substrate and π electrons of these residues[2] but only one residue, Trp82, in the anionic bindingsite of BChE does this interaction [4,5,7]. Therefore, thesecond carbamyl groups of meta-carbamates 6–10 bindtightly to the anionic binding site of AChE but bindloosely to the anionic binding site of BChE due to theabove-mentioned interaction.

Inhibitory Potencies for Varied Substituentsof Carbamates 1–15

When different carbamate substituents in 1–15are compared, compounds with long alkylcarbamylgroups such as n-hexyl- and n-octyl-carbamates 2, 3,7, 8, 12, and 13 are more potent than those with shortalkylcarbamyl groups such as n-butylcarbamates 1,5, and 10 and those with bulky substituents such as

t-butyl- and benzyl-carbamates 4, 5, 9, 10, 14, and 15(Table 1). The possible reason for this is because theacyl-binding site of BChE [4,5,7] is relatively large whencompared with that of AChE [2] and therefore is suit-able for long alkylcarbamyl substituents [2].

Selectivity of AChE over BChE

High selectivity of AChE over BChE indicates thatthe inhibitors 1–15 are more potent inhibitors of AChEthan inhibitors of BChE (Table 1). This may be due to thefact that the active site gorge of BChE is large enoughto generate tight bindings between the inhibitors andthe enzyme.

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