- Email: [email protected]
Reversible inhibition of acetylcholinesterase and butyrylcholinesterase by 4,4′-bipyridine and by a coumarin derivative
Chemico-Biological Interactions 119 – 120 (1999) 119 – 128
Reversible inhibition of acetylcholinesterase and butyrylcholinesterase by 4,4%-bipyridine and by a coumarin derivative Vera Simeon-Rudolf a,*, Zrinka Kovarik a, Zoran Radic´ b, Elsa Reiner a b
a Institute for Medical Research and Occupational Health, P.O. Box 291, 10001 Zagreb, Croatia 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 evaluated from 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, was consistent with inhibitor binding to both catalytic and peripheral sites. The dissociation constants for the peripheral site were about 3.5-times higher than for the catalytic site. The competition between CHMC and substrate displayed two binding sites on the AChE mutants Y72N, Y124Q, W286A and W286R, and on the atypical and fluoride-resistant BChE variants. The dissociation constants for the peripheral site were on average two-times higher than 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 usual BChE. Modelling of the 4,4%-BP and CHMC binding to wild type mouse AChE substantiated the difference between the inhibitors in their mode of binding which was revealed in the kinetic 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: S 0 0 0 9 - 2 7 9 7 ( 9 9 ) 0 0 0 2 0 - 4
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1. Introduction It was shown earlier that the coumarin derivative 3-chloro-7-hydroxy-4-methylcoumarin (CHMC) is a peripheral site ligand of acetylcholinesterase (AChE; EC 3.1.1.7) 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 the catalytic and peripheral sites of the enzymes [1–4]. The aim of the present study was to evaluate the role of amino acid residues in the peripheral and choline binding sites of two enzymes on inhibition by CHMC and 4,4%-BP. The enzyme preparations were recombinant mouse AChE, BChE and AChE site-directed mutants, and human serum BChE and its native variants. The dissociation constants of the compounds for the enzymes were determined from the effect of substrate upon the degree of inhibition. A comparison of the constants with those determined previously for other AChE and BChE preparations has been made. Interaction of mouse recombinant wild type AChE and the inhibitors were further studied using computational 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] with acetylthiocholine (ATCh) as substrate. The enzyme sources were usual human serum BChE and its variants, the recombinant mouse wild type AChE and BChE, and site-directed mutants of AChE with amino acid substitutions in the peripheral and choline binding sites. The recombinant enzymes were prepared as described by Radic´ 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 the absence 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 the following equation [7 – 9]: Vm 1 + b · S/Kss V= (1) 1 + Km/S 1 +S/Kss This equation was derived on the assumption of substrate binding on two sites on the enzyme. The Km is the Michaelis constant and Kss is the substrate-inhibition constant. The parameter b reflects the efficiency of hydrolysis of the ternary complex of the enzyme and two substrate molecules.
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121
Catalytic constants of BChE were determined by using the following equation [10,11]: V=
V1 · S + (V2 · S 2/K2) K1 +S + (S 2/K2)
(2)
This equation was derived on the assumption of substrate binding on two sites on the enzyme. K1 and K2 are the enzyme-substrate dissociation constants and V1 and V2 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 concentrations between 0.01 and 10 mM. At each substrate concentration inhibition was determined with two to three different inhibitor concentrations: 25–300 mM for CHMC and 1 – 10 mM for 4,4%-BP. Dissociation constants of the enzyme-inhibitor complex K(I) were calculated using the Hunter–Downs plot [9]. The apparent enzyme-inhibitor dissociation constants (Kapp) were calculated from Kapp =
V ·i K = K(I) + (I) S (V0 −V) K(S)
(3)
where V0 and V are the enzyme activities at a given substrate concentration (S) in the absence and the presence of the inhibitor (i ). When Kapp is a linear function of substrate concentration, the intercept of the line on the abscissa K(S) corresponds either to the Michaelis constant of the substrate (Km) or to the substrate-inhibition constant (Kss) (Eq. (1)). By analogy these intercepts should correspond to the enzyme-substrate constants in Eq. (2). The intercept on the ordinate K(i) is either the enzyme-ligand dissociation constant for the catalytic site (Ka) or the dissociation constant 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 was calculated 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] using Insight II software package (MSI, San Diego). The starting position of inhibitor was systematically varied within the coordinates of mouse AChE w.t. by increasing its distance from W86 of AChE, while at the same time decreasing the distance to W286. Coordinates of AChE backbone and sidechains were kept constant during the simulation, except for sidechains of F295, F297, Y124, W286, Y337, Y72 and W86. The inhibitor molecule was free to move.
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3. Results and discussion The catalytic constants for the hydrolysis of ATCh by the cholinesterase preparations used in this study are summarised in Table 1. Hydrolysis by AChE is inhibited by substrate concentrations above 1.0 mM while the hydrolysis by BChE increases over the total range of substrate concentrations studied. It has been shown that Eqs. (1) and (2) fitted the concentration dependency for substrate hydrolysis of AChE 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 second substrate molecule to a peripheral site of the enzyme, resulting in inhibition of AChE catalyzed substrate hydrolysis [1,3] or activation of BChE catalyzed substrate hydrolysis [10,11]. Binding of a second substrate molecule to a peripheral, substrate-inhibition site, was suggested for AChE by kinetic measurements and confirmed by competition with peripheral site ligands whose binding to the peripheral site of AChE could be detected directly [1,3,14]. The kinetics of inhibition of AChE and BChE by 4,4%-BP was biphasic for inhibition of mouse AChE w.t. and its triple mutant (Fig. 1). The derived K(I) and K(S) constants are given in Table 2. The two K(I) constants derived for each Table 1 Catalytic constants for the hydrolysis of acetylthiocholine calculated from cholinesterase activities in the absence of inhibitors Acetylcholinesterase
Km (mM)
Kss (mM)
b
References
Bovine erythrocytes Human erythrocytes T. californica
0.11 0.14 0.06
14 29 17
– – –
[15] [6] [3]
Mouse recombinant: Wild type Y72N/Y124Q/W286A D74N Y72N Y124Q W286R W286A
0.05 0.18 1.3 0.11 0.12 0.42 0.06
7 1.5 530 35 25 23 46
0.32 0.46 0 0.18 0.35 0.24 0.26
This paper This paper [7] [7] [7] [7] [16]
Butyrylcholinesterase
K1 (mM)
K2 (mM)
V2/V1
References
Horse serum
0.23
0.66
1.2
[15]
Human serum: Usual (UU) Fluoride-resistant (FS) Atypical (AA)
0.03 0.06 0.08
6.1 9.0 4.9
2.4 2.9 3.8
This paper This paper This paper
Mouse recombinant: Wild type
0.05
1.9
3.2
This paper
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123
Fig. 1. Reversible inhibition of mouse recombinant w.t. AChE and its triple mutant by the indicated inhibitors.
cholinesterase preparation were attributed to binding of the inhibitor to the catalytic site and to a peripheral site of the enzyme. Consequently, the K(I) values should represent the Ka and Ki enzyme-inhibitor dissociation constants. The derived K(S) values should therefore correspond to Km and Kss, of AChE or K1 and K2 of BChE. The kinetics of inhibition by CHMC (Fig. 1 and Table 3) displayed binding to only one site on the enzyme in eight of the 15 different mutant and wildtype cholinesterases studied. Comparing the derived K(S) with the catalytic constants given in Table 1 it follows that the K(S) constants for AChE are higher than Km, but lower than Kss, while for BChE they are much higher than K2. For the other seven cholinesterase preparations the kinetics of inhibition was biphasic, but the two
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phases were not very pronounced which follows from the fact that the two derived K(I) constants differed only by a factor of two. CHMC had the highest affinity for the AChE choline binding site mutant D74N while affinity of both CHMC and 4,4%-BP was the lowest for the triple mutant Y72N/Y124N/W286A. Single mutations at the peripheral site (Y72N, Y124N, W286R and W286A) revealed a biphasic inhibition by coumarin similar to that of fluoride-resistant and atypical BChE variants. The above results suggest that both 4,4%-BP and CHMC bind well to the AChE peripheral 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 peripheral site residues are substituted and is also seen for BChE which lacks the aromatic residues in the peripheral site. Steric or electrostatic barriers may therefore exclude CHMC 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 one in the AChE active centre and another one in the peripheral site. The energies of the two clusters appear more confined for CHMC than for 4,4%-BP suggesting the possibility of effective trapping of CHMC at the peripheral site on its way into the active centre gorge, or an insufficient energetic drive to pull the molecule into the active centre, whereas this is not the case for 4,4%-BP. The results of modelling are in agreement with kinetic studies which display preferential binding of CHMC to the peripheral site of AChE w.t., and of 4,4%-BP to both sites. From the results of the 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 2 Inhibition of cholinesterases by 4,4%-bipyridinea Enzyme
K(I)/mM
K(S)/mM
References
Acetylcholinesterase Human erythrocytes
1.0 & 9.0
0.10 &
[2]
Mouse recombinant: Wild type Y72N/Y124Q/W286A
0.35 & 1.8 6.4 & 15
0.20 & 5.1 0.40 & 26
This paper This paper
Butyrylcholinesterase Human serum: Usual (UU) Fluoride-resistant (FS) Atypical (AA)
1.6 & 5.7 1.3 & 4.8 1.8 & 3.2
0.28 & 12 0.17 & 11 0.40 & 6.3
[17] [17] [17]
Mouse recombinant: Wild type
0.92 & 3.2
0.70 & 8.0
This paper
a
Enzyme-inhibitor K(I) and enzyme-substrate K(S) dissociation constants derived from the kinetics of cholinesterase inhibition; each constant was calculated from activities measured with 8–13 substrate-inhibitor concentration pairs.
V. Simeon-Rudolf et al. / Chemico-Biological Interactions 119–120 (1999) 119–128
Fig. 2. Stereo three dimensional plots resulting from molecular modelling of inhibitor binding into the wild type mouse AChE. Correlation between position of inhibitor and the corresponding total nonbonding energy (Etot-nonb) is shown for 4,4%-bipyridine and coumarin. Position of inhibitors are described by distances 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 and 4,4%-bipyridine or coumarin in the starting conformations are shown by a set of symbols displayed at the bottom of the plots.
125
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V. Simeon-Rudolf et al. / Chemico-Biological Interactions 119–120 (1999) 119–128
Fig. 2. (Continued)
V. Simeon-Rudolf et al. / Chemico-Biological Interactions 119–120 (1999) 119–128
127
Table 3 Inhibition of cholinesterases by the coumarin derivativea Enzyme
K(I)/mM
K(S)/mM
References
Acetylcholinesterase Bovine erythrocytes Human erythrocytes T. californica
28 51 116
2.3 4.0 0.49
[1,5] [18] [3]
Mouse recombinant: Wild type Y72N/Y124Q/W286A D74N Y72N Y124Q W286R W286A
27 100 13 45 & 59 & 25 & 28 &
2.6 0.72 1.4 0.72 & 13 1.1 & 9.8 0.63 & 5.3 0.39 & 5.3
This This This This This This This
Butyrylcholinesterase Horse serum
17
2.4
[15]
Human serum: Usual (UU) Fluoride-resistant (FS) Atypical (AA)
150 64 & 120 54 & 93
0.51 & 44 1.3 & 19
This paper This paper This paper
Mouse recombinant: Wild type
20 & 34
0.92 & 4.2
This paper
100 100 55 77
paper paper paper paper paper paper paper
a
Enzyme-inhibitor K(I) and enzyme-substrate K(S) constants derived from the kinetics of cholinesterase inhibition; each constant was calculated from activities measured with 8–13 substrate-inhibitor concentration pairs.
reversible inhibition of the single residue mutants at the peripheral site, mouse BChE w.t. and human serum BChE variants revealed binding to both sites. CHMC binds preferably to the peripheral site but binding to the active centre is possible upon substitution of the peripheral site residues.
Acknowledgements This work was supported in part by the Ministry of Science and Technology of the Republic of Croatia (Grant No. 00220104) and by the DAMDC Grant 17-98-1-8014, USA.
References [1] W.N. Aldridge, E. Reiner, Acetylcholinesterase. Two types of inhibition by an organophosphorus compound: one the formation of phosphorylated enzyme and the other analogous to inhibition by substrate, Biochem. J. 115 (1969) 147 – 162.
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[2] E. Reiner, Inhibition of acetylcholinesterase by 4,4%-bipyridine and its effect upon phosphylation of the enzyme, Croat. Chem. Acta 59 (1986) 925 – 931. [3] Z. Radic´, E. Reiner, P. Taylor, Role of the peripheral anionic site on acetylcholinesterase: Inhibition by substrates and coumarin derivatives, Mol. Pharmacol. 39 (1991) 98 – 104. [4] E. Reiner, M. S& krinjaric´-S& poljar, V. Simeon-Rudolf, Binding sites on acetylcholinesterase and butyrylcholinesterase for pyridinium and imidazolium oximes, and other reversible ligands, Period. Biol. 98 (1996) 325–329. [5] E. Reiner, V. Simeon, Kinetic study of the effect of substrates on reversible inhibition of cholinesterase and acetylcholinesterase by two coumarin derivatives, Croat. Chem. Acta 47 (1975) 321–331. [6] G.L. Ellman, K.D. Courtney, V. Andres Jr, R.M. Featherstone, A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol. 7 (1961) 88 – 95. [7] Z. Radic´, N.A. Pickering, D.C. Vellom, S. Camp, P. Taylor, Three distinct domains in the cholinesterase molecule confer selectivity for acetyl- and butyrylcholinesterase inhibitors, Biochemistry 32 (1993) 12074–12084. [8] J.L. Webb, Enzyme and Metabolic Inhibitors, vol. 1, Academic Press, New York, 1963, pp. 46 – 47. [9] W.N. Aldridge, E. Reiner, Enzyme Inhibitors as Substrates. Interaction of Esterases with Esters of Organophosphorus and Carbamic Acids, XV +328, North Holland, Amsterdam, 1972. [10] G. Cauet, A. Friboulet, D. Thomas, Horse serum butyrylcholinesterase kinetics: a molecular mechanism based on inhibition studies with dansylaminoethyltrimethylammonium, Biochem. Cell. Biol. 65 (1987) 529–535. [11] P. Masson, S. Adkins, P. Gouet, O. Lockridge, Recombinant human butyrylcholinesterase G390V, the fluoride-2 variant, expressed in Chinese hamster ovary cells, is a low affinity variant, J. Biol. Chem. 268 (1993) 14329–14341. [12] Y. Ashani, Z. Radic´, I. Tsigelny, D.C. Vellom, N.A. Pickering, D.M. Quinn, B.P. Doctor, P. Taylor, Amino acid residues controlling reactivation of organophosphonyl conjugates of acetylcholinesterase by mono- and bisquaternary oximes, J. Biol. Chem. 270 (1995) 6370 – 6380. [13] V. Simeon-Rudolf, E. Reiner, R.T. Evans, P.M. George, H.C. Potter, Catalytic parameters for the hydrolysis of butyrylthiocholine by human serum butyrylcholinesterase variants. Chem.-Biol. Interact., this volume [14] P. Taylor, S. Lappi, Interaction of fluorescent probes with acetylcholinesterase: the site and specificity of propidium binding, Biochemistry 14 (1975) 1989 – 1997. [15] V. Simeon, Michaelis constants and substrate inhibition constants for the reaction of acetylthiocholine with acetylcholinesterase and cholinesterase, Croat. Chem. Acta 46 (1974) 137 – 144. [16] N.A. Hosea, Z. Radic´, I. Tsigelny, H.A. Berman, D.M. Quinn, P. Taylor, Aspartate 74 as a primary determinant in acetylcholinesterase governing specificity to cationic organophosphonates, Biochemistry 35 (1996) 10995–11004. [17] E. Reiner, V. Simeon-Rudolf, M. S& krinjaric´-S& poljar, Catalytic properties and distribution profiles of paraoxonase and cholinesterase phenotypes in human sera, Toxicol. Lett. 82/83 (1995) 447 – 452. [18] Z. Radic´, E. Reiner, V. Simeon, Binding sites on acetylcholinesterase for reversible ligands and phosphorylating agents, Biochem. Pharmacol. 33 (1984) 671 – 677.
.
Reversible inhibition of acetylcholinesterase and butyrylcholinesterase by 4,4%-bipyridine and by a coumarin derivative Vera Simeon-Rudolf a,*, Zrinka Kovarik a, Zoran Radic´ b, Elsa Reiner a b
a Institute for Medical Research and Occupational Health, P.O. Box 291, 10001 Zagreb, Croatia 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 evaluated from 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, was consistent with inhibitor binding to both catalytic and peripheral sites. The dissociation constants for the peripheral site were about 3.5-times higher than for the catalytic site. The competition between CHMC and substrate displayed two binding sites on the AChE mutants Y72N, Y124Q, W286A and W286R, and on the atypical and fluoride-resistant BChE variants. The dissociation constants for the peripheral site were on average two-times higher than 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 usual BChE. Modelling of the 4,4%-BP and CHMC binding to wild type mouse AChE substantiated the difference between the inhibitors in their mode of binding which was revealed in the kinetic 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: S 0 0 0 9 - 2 7 9 7 ( 9 9 ) 0 0 0 2 0 - 4
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1. Introduction It was shown earlier that the coumarin derivative 3-chloro-7-hydroxy-4-methylcoumarin (CHMC) is a peripheral site ligand of acetylcholinesterase (AChE; EC 3.1.1.7) 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 the catalytic and peripheral sites of the enzymes [1–4]. The aim of the present study was to evaluate the role of amino acid residues in the peripheral and choline binding sites of two enzymes on inhibition by CHMC and 4,4%-BP. The enzyme preparations were recombinant mouse AChE, BChE and AChE site-directed mutants, and human serum BChE and its native variants. The dissociation constants of the compounds for the enzymes were determined from the effect of substrate upon the degree of inhibition. A comparison of the constants with those determined previously for other AChE and BChE preparations has been made. Interaction of mouse recombinant wild type AChE and the inhibitors were further studied using computational 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] with acetylthiocholine (ATCh) as substrate. The enzyme sources were usual human serum BChE and its variants, the recombinant mouse wild type AChE and BChE, and site-directed mutants of AChE with amino acid substitutions in the peripheral and choline binding sites. The recombinant enzymes were prepared as described by Radic´ 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 the absence 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 the following equation [7 – 9]: Vm 1 + b · S/Kss V= (1) 1 + Km/S 1 +S/Kss This equation was derived on the assumption of substrate binding on two sites on the enzyme. The Km is the Michaelis constant and Kss is the substrate-inhibition constant. The parameter b reflects the efficiency of hydrolysis of the ternary complex of the enzyme and two substrate molecules.
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121
Catalytic constants of BChE were determined by using the following equation [10,11]: V=
V1 · S + (V2 · S 2/K2) K1 +S + (S 2/K2)
(2)
This equation was derived on the assumption of substrate binding on two sites on the enzyme. K1 and K2 are the enzyme-substrate dissociation constants and V1 and V2 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 concentrations between 0.01 and 10 mM. At each substrate concentration inhibition was determined with two to three different inhibitor concentrations: 25–300 mM for CHMC and 1 – 10 mM for 4,4%-BP. Dissociation constants of the enzyme-inhibitor complex K(I) were calculated using the Hunter–Downs plot [9]. The apparent enzyme-inhibitor dissociation constants (Kapp) were calculated from Kapp =
V ·i K = K(I) + (I) S (V0 −V) K(S)
(3)
where V0 and V are the enzyme activities at a given substrate concentration (S) in the absence and the presence of the inhibitor (i ). When Kapp is a linear function of substrate concentration, the intercept of the line on the abscissa K(S) corresponds either to the Michaelis constant of the substrate (Km) or to the substrate-inhibition constant (Kss) (Eq. (1)). By analogy these intercepts should correspond to the enzyme-substrate constants in Eq. (2). The intercept on the ordinate K(i) is either the enzyme-ligand dissociation constant for the catalytic site (Ka) or the dissociation constant 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 was calculated 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] using Insight II software package (MSI, San Diego). The starting position of inhibitor was systematically varied within the coordinates of mouse AChE w.t. by increasing its distance from W86 of AChE, while at the same time decreasing the distance to W286. Coordinates of AChE backbone and sidechains were kept constant during the simulation, except for sidechains of F295, F297, Y124, W286, Y337, Y72 and W86. The inhibitor molecule was free to move.
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3. Results and discussion The catalytic constants for the hydrolysis of ATCh by the cholinesterase preparations used in this study are summarised in Table 1. Hydrolysis by AChE is inhibited by substrate concentrations above 1.0 mM while the hydrolysis by BChE increases over the total range of substrate concentrations studied. It has been shown that Eqs. (1) and (2) fitted the concentration dependency for substrate hydrolysis of AChE 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 second substrate molecule to a peripheral site of the enzyme, resulting in inhibition of AChE catalyzed substrate hydrolysis [1,3] or activation of BChE catalyzed substrate hydrolysis [10,11]. Binding of a second substrate molecule to a peripheral, substrate-inhibition site, was suggested for AChE by kinetic measurements and confirmed by competition with peripheral site ligands whose binding to the peripheral site of AChE could be detected directly [1,3,14]. The kinetics of inhibition of AChE and BChE by 4,4%-BP was biphasic for inhibition of mouse AChE w.t. and its triple mutant (Fig. 1). The derived K(I) and K(S) constants are given in Table 2. The two K(I) constants derived for each Table 1 Catalytic constants for the hydrolysis of acetylthiocholine calculated from cholinesterase activities in the absence of inhibitors Acetylcholinesterase
Km (mM)
Kss (mM)
b
References
Bovine erythrocytes Human erythrocytes T. californica
0.11 0.14 0.06
14 29 17
– – –
[15] [6] [3]
Mouse recombinant: Wild type Y72N/Y124Q/W286A D74N Y72N Y124Q W286R W286A
0.05 0.18 1.3 0.11 0.12 0.42 0.06
7 1.5 530 35 25 23 46
0.32 0.46 0 0.18 0.35 0.24 0.26
This paper This paper [7] [7] [7] [7] [16]
Butyrylcholinesterase
K1 (mM)
K2 (mM)
V2/V1
References
Horse serum
0.23
0.66
1.2
[15]
Human serum: Usual (UU) Fluoride-resistant (FS) Atypical (AA)
0.03 0.06 0.08
6.1 9.0 4.9
2.4 2.9 3.8
This paper This paper This paper
Mouse recombinant: Wild type
0.05
1.9
3.2
This paper
V. Simeon-Rudolf et al. / Chemico-Biological Interactions 119–120 (1999) 119–128
123
Fig. 1. Reversible inhibition of mouse recombinant w.t. AChE and its triple mutant by the indicated inhibitors.
cholinesterase preparation were attributed to binding of the inhibitor to the catalytic site and to a peripheral site of the enzyme. Consequently, the K(I) values should represent the Ka and Ki enzyme-inhibitor dissociation constants. The derived K(S) values should therefore correspond to Km and Kss, of AChE or K1 and K2 of BChE. The kinetics of inhibition by CHMC (Fig. 1 and Table 3) displayed binding to only one site on the enzyme in eight of the 15 different mutant and wildtype cholinesterases studied. Comparing the derived K(S) with the catalytic constants given in Table 1 it follows that the K(S) constants for AChE are higher than Km, but lower than Kss, while for BChE they are much higher than K2. For the other seven cholinesterase preparations the kinetics of inhibition was biphasic, but the two
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phases were not very pronounced which follows from the fact that the two derived K(I) constants differed only by a factor of two. CHMC had the highest affinity for the AChE choline binding site mutant D74N while affinity of both CHMC and 4,4%-BP was the lowest for the triple mutant Y72N/Y124N/W286A. Single mutations at the peripheral site (Y72N, Y124N, W286R and W286A) revealed a biphasic inhibition by coumarin similar to that of fluoride-resistant and atypical BChE variants. The above results suggest that both 4,4%-BP and CHMC bind well to the AChE peripheral 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 peripheral site residues are substituted and is also seen for BChE which lacks the aromatic residues in the peripheral site. Steric or electrostatic barriers may therefore exclude CHMC 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 one in the AChE active centre and another one in the peripheral site. The energies of the two clusters appear more confined for CHMC than for 4,4%-BP suggesting the possibility of effective trapping of CHMC at the peripheral site on its way into the active centre gorge, or an insufficient energetic drive to pull the molecule into the active centre, whereas this is not the case for 4,4%-BP. The results of modelling are in agreement with kinetic studies which display preferential binding of CHMC to the peripheral site of AChE w.t., and of 4,4%-BP to both sites. From the results of the 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 2 Inhibition of cholinesterases by 4,4%-bipyridinea Enzyme
K(I)/mM
K(S)/mM
References
Acetylcholinesterase Human erythrocytes
1.0 & 9.0
0.10 &
[2]
Mouse recombinant: Wild type Y72N/Y124Q/W286A
0.35 & 1.8 6.4 & 15
0.20 & 5.1 0.40 & 26
This paper This paper
Butyrylcholinesterase Human serum: Usual (UU) Fluoride-resistant (FS) Atypical (AA)
1.6 & 5.7 1.3 & 4.8 1.8 & 3.2
0.28 & 12 0.17 & 11 0.40 & 6.3
[17] [17] [17]
Mouse recombinant: Wild type
0.92 & 3.2
0.70 & 8.0
This paper
a
Enzyme-inhibitor K(I) and enzyme-substrate K(S) dissociation constants derived from the kinetics of cholinesterase inhibition; each constant was calculated from activities measured with 8–13 substrate-inhibitor concentration pairs.
V. Simeon-Rudolf et al. / Chemico-Biological Interactions 119–120 (1999) 119–128
Fig. 2. Stereo three dimensional plots resulting from molecular modelling of inhibitor binding into the wild type mouse AChE. Correlation between position of inhibitor and the corresponding total nonbonding energy (Etot-nonb) is shown for 4,4%-bipyridine and coumarin. Position of inhibitors are described by distances 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 and 4,4%-bipyridine or coumarin in the starting conformations are shown by a set of symbols displayed at the bottom of the plots.
125
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Fig. 2. (Continued)
V. Simeon-Rudolf et al. / Chemico-Biological Interactions 119–120 (1999) 119–128
127
Table 3 Inhibition of cholinesterases by the coumarin derivativea Enzyme
K(I)/mM
K(S)/mM
References
Acetylcholinesterase Bovine erythrocytes Human erythrocytes T. californica
28 51 116
2.3 4.0 0.49
[1,5] [18] [3]
Mouse recombinant: Wild type Y72N/Y124Q/W286A D74N Y72N Y124Q W286R W286A
27 100 13 45 & 59 & 25 & 28 &
2.6 0.72 1.4 0.72 & 13 1.1 & 9.8 0.63 & 5.3 0.39 & 5.3
This This This This This This This
Butyrylcholinesterase Horse serum
17
2.4
[15]
Human serum: Usual (UU) Fluoride-resistant (FS) Atypical (AA)
150 64 & 120 54 & 93
0.51 & 44 1.3 & 19
This paper This paper This paper
Mouse recombinant: Wild type
20 & 34
0.92 & 4.2
This paper
100 100 55 77
paper paper paper paper paper paper paper
a
Enzyme-inhibitor K(I) and enzyme-substrate K(S) constants derived from the kinetics of cholinesterase inhibition; each constant was calculated from activities measured with 8–13 substrate-inhibitor concentration pairs.
reversible inhibition of the single residue mutants at the peripheral site, mouse BChE w.t. and human serum BChE variants revealed binding to both sites. CHMC binds preferably to the peripheral site but binding to the active centre is possible upon substitution of the peripheral site residues.
Acknowledgements This work was supported in part by the Ministry of Science and Technology of the Republic of Croatia (Grant No. 00220104) and by the DAMDC Grant 17-98-1-8014, USA.
References [1] W.N. Aldridge, E. Reiner, Acetylcholinesterase. Two types of inhibition by an organophosphorus compound: one the formation of phosphorylated enzyme and the other analogous to inhibition by substrate, Biochem. J. 115 (1969) 147 – 162.
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[2] E. Reiner, Inhibition of acetylcholinesterase by 4,4%-bipyridine and its effect upon phosphylation of the enzyme, Croat. Chem. Acta 59 (1986) 925 – 931. [3] Z. Radic´, E. Reiner, P. Taylor, Role of the peripheral anionic site on acetylcholinesterase: Inhibition by substrates and coumarin derivatives, Mol. Pharmacol. 39 (1991) 98 – 104. [4] E. Reiner, M. S& krinjaric´-S& poljar, V. Simeon-Rudolf, Binding sites on acetylcholinesterase and butyrylcholinesterase for pyridinium and imidazolium oximes, and other reversible ligands, Period. Biol. 98 (1996) 325–329. [5] E. Reiner, V. Simeon, Kinetic study of the effect of substrates on reversible inhibition of cholinesterase and acetylcholinesterase by two coumarin derivatives, Croat. Chem. Acta 47 (1975) 321–331. [6] G.L. Ellman, K.D. Courtney, V. Andres Jr, R.M. Featherstone, A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol. 7 (1961) 88 – 95. [7] Z. Radic´, N.A. Pickering, D.C. Vellom, S. Camp, P. Taylor, Three distinct domains in the cholinesterase molecule confer selectivity for acetyl- and butyrylcholinesterase inhibitors, Biochemistry 32 (1993) 12074–12084. [8] J.L. Webb, Enzyme and Metabolic Inhibitors, vol. 1, Academic Press, New York, 1963, pp. 46 – 47. [9] W.N. Aldridge, E. Reiner, Enzyme Inhibitors as Substrates. Interaction of Esterases with Esters of Organophosphorus and Carbamic Acids, XV +328, North Holland, Amsterdam, 1972. [10] G. Cauet, A. Friboulet, D. Thomas, Horse serum butyrylcholinesterase kinetics: a molecular mechanism based on inhibition studies with dansylaminoethyltrimethylammonium, Biochem. Cell. Biol. 65 (1987) 529–535. [11] P. Masson, S. Adkins, P. Gouet, O. Lockridge, Recombinant human butyrylcholinesterase G390V, the fluoride-2 variant, expressed in Chinese hamster ovary cells, is a low affinity variant, J. Biol. Chem. 268 (1993) 14329–14341. [12] Y. Ashani, Z. Radic´, I. Tsigelny, D.C. Vellom, N.A. Pickering, D.M. Quinn, B.P. Doctor, P. Taylor, Amino acid residues controlling reactivation of organophosphonyl conjugates of acetylcholinesterase by mono- and bisquaternary oximes, J. Biol. Chem. 270 (1995) 6370 – 6380. [13] V. Simeon-Rudolf, E. Reiner, R.T. Evans, P.M. George, H.C. Potter, Catalytic parameters for the hydrolysis of butyrylthiocholine by human serum butyrylcholinesterase variants. Chem.-Biol. Interact., this volume [14] P. Taylor, S. Lappi, Interaction of fluorescent probes with acetylcholinesterase: the site and specificity of propidium binding, Biochemistry 14 (1975) 1989 – 1997. [15] V. Simeon, Michaelis constants and substrate inhibition constants for the reaction of acetylthiocholine with acetylcholinesterase and cholinesterase, Croat. Chem. Acta 46 (1974) 137 – 144. [16] N.A. Hosea, Z. Radic´, I. Tsigelny, H.A. Berman, D.M. Quinn, P. Taylor, Aspartate 74 as a primary determinant in acetylcholinesterase governing specificity to cationic organophosphonates, Biochemistry 35 (1996) 10995–11004. [17] E. Reiner, V. Simeon-Rudolf, M. S& krinjaric´-S& poljar, Catalytic properties and distribution profiles of paraoxonase and cholinesterase phenotypes in human sera, Toxicol. Lett. 82/83 (1995) 447 – 452. [18] Z. Radic´, E. Reiner, V. Simeon, Binding sites on acetylcholinesterase for reversible ligands and phosphorylating agents, Biochem. Pharmacol. 33 (1984) 671 – 677.
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