reversible inhibition of acetylcholinesterase and butyrylcholinesterase by 4,4′-bipyridine and by...

Chemico-Biological Interactions 119–120 (1999) 119–128

Reversible inhibition of acetylcholinesterase andbutyrylcholinesterase by 4,4%-bipyridine and by a

coumarin derivative

Vera Simeon-Rudolf a,*, Zrinka Kovarik a, Zoran Radic b,Elsa Reiner a

a Institute for Medical Research and Occupational Health, P.O. Box 291, 10001 Zagreb, Croatiab Department of Pharmacology, Uni6ersity of California at San Diego, La Jolla, CA 92093-0636, USA

Abstract

Inhibition of recombinant mouse wild type AChE (EC 3.1.1.7) and BChE (EC 3.1.1.8),and AChE peripheral site-directed mutants and human serum BChE variants by 4,4%-bipyridine (4,4%-BP) and the coumarin derivative 3-chloro-7-hydroxy-4-methylcoumarin(CHMC) was studied. The enzyme activity was measured with acetylthiocholine as substrate.Enzyme-inhibitor dissociation constants for the catalytic and peripheral sites were evaluatedfrom the apparent dissociation constants as a function of the substrate concentration.Inhibition by 4,4%-BP of AChE, BChE and the AChE mutant Y72N/Y124Q/W286A, wasconsistent with inhibitor binding to both catalytic and peripheral sites. The dissociationconstants for the peripheral site were about 3.5-times higher than for the catalytic site. Thecompetition between CHMC and substrate displayed two binding sites on the AChE mutantsY72N, Y124Q, W286A and W286R, and on the atypical and fluoride-resistant BChEvariants. The dissociation constants for the peripheral site were on average two-times higherthan for the catalytic site. CHMC displayed binding only to the catalytic site of Y72N/Y124Q/W286A mutant and only to the peripheral site of w.t. AChE and the human usualBChE. Modelling of the 4,4%-BP and CHMC binding to wild type mouse AChE substanti-ated the difference between the inhibitors in their mode of binding which was revealed in thekinetic studies. © 1999 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Acetylcholinesterase; Butyrylcholinesterase; 4,4%-Bipyridine; Coumarin derivative;Reversible inhibition; Molecular modelling

* Corresponding author. Tel.: +385-1-4673188; fax: +385-1-4673303.E-mail address: [email protected] (V. Simeon-Rudolf)

0009-2797/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved.

PII: S0009 -2797 (99 )00020 -4

V. Simeon-Rudolf et al. / Chemico-Biological Interactions 119–120 (1999) 119–128120

1. Introduction

It was shown earlier that the coumarin derivative 3-chloro-7-hydroxy-4-methyl-coumarin (CHMC) is a peripheral site ligand of acetylcholinesterase (AChE; EC3.1.1.7) and butyrylcholinesterase (BChE; EC 3.1.1.8) while inhibition by 4,4%-bipyridine (4,4%-BP) was consistent with binding of the inhibitor to both thecatalytic and peripheral sites of the enzymes [1–4]. The aim of the present studywas to evaluate the role of amino acid residues in the peripheral and cholinebinding sites of two enzymes on inhibition by CHMC and 4,4%-BP. The enzymepreparations were recombinant mouse AChE, BChE and AChE site-directed mu-tants, and human serum BChE and its native variants. The dissociation constantsof the compounds for the enzymes were determined from the effect of substrateupon the degree of inhibition. A comparison of the constants with those determinedpreviously for other AChE and BChE preparations has been made. Interaction ofmouse recombinant wild type AChE and the inhibitors were further studied usingcomputational molecular modelling techniques.

2. Experimental procedure

All experiments were performed in 0.1 M phosphate buffer, pH 7.4 at 25°C.Details of the experimental procedure were described earlier [5].

2.1. Enzyme acti6ity assay

The enzyme activity was measured by the method of Ellman et al. [6] withacetylthiocholine (ATCh) as substrate. The enzyme sources were usual humanserum BChE and its variants, the recombinant mouse wild type AChE and BChE,and site-directed mutants of AChE with amino acid substitutions in the peripheraland choline binding sites. The recombinant enzymes were prepared as described byRadic et al. [7].

2.2. Catalytic constants

Catalytic constants for the hydrolysis of ATCh were evaluated for BChE w.t.,AChE w.t. and the AChE triple mutant from enzyme activities measured in theabsence of the inhibitors over the range of ATCh from 0.02 to 10 mM.

Catalytic constants of AChE and their mutants were determined by using thefollowing equation [7–9]:

V=Vm

1+Km/S�1+b · S/Kss

1+S/Kss

�(1)

This equation was derived on the assumption of substrate binding on two sites onthe enzyme. The Km is the Michaelis constant and Kss is the substrate-inhibitionconstant. The parameter b reflects the efficiency of hydrolysis of the ternarycomplex of the enzyme and two substrate molecules.

V. Simeon-Rudolf et al. / Chemico-Biological Interactions 119–120 (1999) 119–128 121

Catalytic constants of BChE were determined by using the following equation[10,11]:

V=V1 · S+ (V2 · S2/K2)

K1+S+ (S2/K2)(2)

This equation was derived on the assumption of substrate binding on two sites onthe enzyme. K1 and K2 are the enzyme-substrate dissociation constants and V1 andV2 the respective maximum velocities for substrate hydrolysis. The ratio V2/V1

corresponds, but is not formally identical, to the b value from Eq. (1).Catalytic constants were calculated by a nonlinear fitting of Eq. (1) or Eq. (2)

using Sigma Plot (Jandel Scientific) computer programme.

2.3. Enzyme-inhibitor dissociation constants

Enzyme inhibition by CHMC and 4,4%-BP was measured with substrate concen-trations between 0.01 and 10 mM. At each substrate concentration inhibition wasdetermined with two to three different inhibitor concentrations: 25–300 mM forCHMC and 1–10 mM for 4,4%-BP. Dissociation constants of the enzyme-inhibitorcomplex K(I) were calculated using the Hunter–Downs plot [9]. The apparentenzyme-inhibitor dissociation constants (Kapp) were calculated from

Kapp=V · i

(V0−V)=K(I)+

K(I)

K(S)

S (3)

where V0 and V are the enzyme activities at a given substrate concentration (S) inthe absence and the presence of the inhibitor (i ). When Kapp is a linear function ofsubstrate concentration, the intercept of the line on the abscissa K(S) correspondseither to the Michaelis constant of the substrate (Km) or to the substrate-inhibitionconstant (Kss) (Eq. (1)). By analogy these intercepts should correspond to theenzyme-substrate constants in Eq. (2). The intercept on the ordinate K(i) is either theenzyme-ligand dissociation constant for the catalytic site (Ka) or the dissociationconstant for the peripheral site (Ki). If a ligand binds to both sites on the enzyme,Kapp is a non-linear function of the substrate concentration. In that case, Ka wascalculated from Kapp values measured with substrate concentrations below 1.0 mM,and Ki from Kapp values measured with ATCh above 1.0 mM.

2.4. Molecular modelling

Molecular modelling was performed essentially as described earlier [12] usingInsight II software package (MSI, San Diego). The starting position of inhibitorwas systematically varied within the coordinates of mouse AChE w.t. by increasingits distance from W86 of AChE, while at the same time decreasing the distance toW286. Coordinates of AChE backbone and sidechains were kept constant duringthe simulation, except for sidechains of F295, F297, Y124, W286, Y337, Y72 andW86. The inhibitor molecule was free to move.


3. Results and discussion

The catalytic constants for the hydrolysis of ATCh by the cholinesterase prepara-tions used in this study are summarised in Table 1. Hydrolysis by AChE is inhibitedby substrate concentrations above 1.0 mM while the hydrolysis by BChE increasesover the total range of substrate concentrations studied. It has been shown thatEqs. (1) and (2) fitted the concentration dependency for substrate hydrolysis ofAChE and BChE better than either the Haldane or Michaelis–Menten equation[7,10,11,13]. The equations were derived by assuming the binding of a secondsubstrate molecule to a peripheral site of the enzyme, resulting in inhibition ofAChE catalyzed substrate hydrolysis [1,3] or activation of BChE catalyzed sub-strate hydrolysis [10,11]. Binding of a second substrate molecule to a peripheral,substrate-inhibition site, was suggested for AChE by kinetic measurements andconfirmed by competition with peripheral site ligands whose binding to the periph-eral site of AChE could be detected directly [1,3,14].

The kinetics of inhibition of AChE and BChE by 4,4%-BP was biphasic forinhibition of mouse AChE w.t. and its triple mutant (Fig. 1). The derived K(I) andK(S) constants are given in Table 2. The two K(I) constants derived for each

Table 1Catalytic constants for the hydrolysis of acetylthiocholine calculated from cholinesterase activities inthe absence of inhibitors

ReferencesAcetylcholinesterase Km (mM) Kss (mM) b

14 – [15]0.11Bovine erythrocytes[6]Human erythrocytes 0.14 29 –

0.06 17T. californica – [3]

Mouse recombinant :Wild type This paper0.05 7 0.32

0.18 1.5Y72N/Y124Q/W286A 0.46 This paperD74N 1.3 530 0 [7]

[7]0.1835Y72N 0.1125Y124Q [7]0.350.12

W286R 230.42 0.24 [7]46 0.26 [16]W286A 0.06

ReferencesV2/V1Butyrylcholinesterase K2 (mM)K1 (mM)

0.66 1.2 [15]Horse serum 0.23

Human serum :0.03Usual (UU) 6.1 This paper2.4

2.90.06 This paper9.0Fluoride-resistant (FS)4.9 3.8 This paper0.08Atypical (AA)

Mouse recombinant :This paper3.21.9Wild type 0.05


Fig. 1. Reversible inhibition of mouse recombinant w.t. AChE and its triple mutant by the indicatedinhibitors.

cholinesterase preparation were attributed to binding of the inhibitor to thecatalytic site and to a peripheral site of the enzyme. Consequently, the K(I) valuesshould represent the Ka and Ki enzyme-inhibitor dissociation constants. The derivedK(S) values should therefore correspond to Km and Kss, of AChE or K1 and K2 ofBChE.

The kinetics of inhibition by CHMC (Fig. 1 and Table 3) displayed binding toonly one site on the enzyme in eight of the 15 different mutant and wildtypecholinesterases studied. Comparing the derived K(S) with the catalytic constantsgiven in Table 1 it follows that the K(S) constants for AChE are higher than Km, butlower than Kss, while for BChE they are much higher than K2. For the other sevencholinesterase preparations the kinetics of inhibition was biphasic, but the two


phases were not very pronounced which follows from the fact that the two derivedK(I) constants differed only by a factor of two.

CHMC had the highest affinity for the AChE choline binding site mutant D74Nwhile affinity of both CHMC and 4,4%-BP was the lowest for the triple mutantY72N/Y124N/W286A. Single mutations at the peripheral site (Y72N, Y124N,W286R and W286A) revealed a biphasic inhibition by coumarin similar to that offluoride-resistant and atypical BChE variants.

The above results suggest that both 4,4%-BP and CHMC bind well to the AChEperipheral site. In addition, 4,4%-BP binds even better within the active centre gorge.Binding of CHMC to two sites is seen when some of the aromatic AChE peripheralsite residues are substituted and is also seen for BChE which lacks the aromaticresidues in the peripheral site. Steric or electrostatic barriers may therefore excludeCHMC from binding to the active centre in mouse AChE w.t.

Molecular modelling of mouse w.t. AChE and the bound inhibitors (Fig. 2)shows two clusters of stable conformations for both compounds: a more stable onein the AChE active centre and another one in the peripheral site. The energies ofthe two clusters appear more confined for CHMC than for 4,4%-BP suggesting thepossibility of effective trapping of CHMC at the peripheral site on its way into theactive centre gorge, or an insufficient energetic drive to pull the molecule into theactive centre, whereas this is not the case for 4,4%-BP. The results of modelling arein agreement with kinetic studies which display preferential binding of CHMC tothe peripheral site of AChE w.t., and of 4,4%-BP to both sites. From the results ofthe kinetic studies the binding of CHMC to the triple mutant and to D74N mutant,could be attributed to the binding of CHMC to the active centre whereas the

Table 2Inhibition of cholinesterases by 4,4%-bipyridinea

K(I)/mM ReferencesEnzyme K(S)/mM

AcetylcholinesteraseHuman erythrocytes [2]1.0 & 9.0 0.10 & �

Mouse recombinant :This paper0.20 & 5.1Wild type 0.35 & 1.8

Y72N/Y124Q/W286A 0.40 & 26 This paper6.4 & 15

ButyrylcholinesteraseHuman serum :

1.6 & 5.7 0.28 & 12 [17]Usual (UU)1.3 & 4.8Fluoride-resistant (FS) 0.17 & 11 [17]

Atypical (AA) 1.8 & 3.2 0.40 & 6.3 [17]

Mouse recombinant :0.92 & 3.2 0.70 & 8.0Wild type This paper

a Enzyme-inhibitor K(I) and enzyme-substrate K(S) dissociation constants derived from the kinetics ofcholinesterase inhibition; each constant was calculated from activities measured with 8–13 substrate-in-hibitor concentration pairs.

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Interactions119

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125

Fig. 2. Stereo three dimensional plots resulting from molecular modelling of inhibitor binding into the wild type mouse AChE. Correlation between positionof inhibitor and the corresponding total nonbonding energy (Etot-nonb) is shown for 4,4%-bipyridine and coumarin. Position of inhibitors are described bydistances between atom C5 of 4,4%-bipyridine or atom C4A of coumarin and CD2 atoms of AChE residues W86 and W286. The distances between W86 and4,4%-bipyridine or coumarin in the starting conformations are shown by a set of symbols displayed at the bottom of the plots.

V.

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Interactions119

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(1999)119

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126

Fig. 2. (Continued)


Table 3Inhibition of cholinesterases by the coumarin derivativea

ReferencesEnzyme K(I)/mM K(S)/mM

Acetylcholinesterase2.3Bovine erythrocytes 28 [1,5]4.0 [18]51Human erythrocytes

116 0.49 [3]T. californica

Mouse recombinant :This paper2.627Wild type

0.72Y72N/Y124Q/W286A This paper100This paperD74N 13 1.4

0.72 & 13Y72N 45 & 100 This paper1.1 & 9.8 This paper59 & 100Y124Q0.63 & 5.3W286R 25 & 55 This paper

28 & 77 0.39 & 5.3W286A This paper

Butyrylcholinesterase[15]2.417Horse serum

Human serum :� This paper150Usual (UU)

This paperFluoride-resistant (FS) 64 & 120 0.51 & 441.3 & 19Atypical (AA) 54 & 93 This paper

Mouse recombinant :This paperWild type 20 & 34 0.92 & 4.2

a Enzyme-inhibitor K(I) and enzyme-substrate K(S) constants derived from the kinetics of cholinesteraseinhibition; each constant was calculated from activities measured with 8–13 substrate-inhibitor concen-tration pairs.

reversible inhibition of the single residue mutants at the peripheral site, mouseBChE w.t. and human serum BChE variants revealed binding to both sites. CHMCbinds preferably to the peripheral site but binding to the active centre is possibleupon substitution of the peripheral site residues.

Acknowledgements

This work was supported in part by the Ministry of Science and Technology ofthe Republic of Croatia (Grant No. 00220104) and by the DAMDC Grant17-98-1-8014, USA.

References

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[2] E. Reiner, Inhibition of acetylcholinesterase by 4,4%-bipyridine and its effect upon phosphylation ofthe enzyme, Croat. Chem. Acta 59 (1986) 925–931.

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reversible inhibition of acetylcholinesterase and butyrylcholinesterase by 4,4′-bipyridine and by...

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