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Analysis of cholinesterase inactivation and reactivation by systematic structural modification and enantiomeric selectivity
Chemico-Biological Interactions 119 – 120 (1999) 3 – 15
Analysis of cholinesterase inactivation and reactivation by systematic structural modification and enantiomeric selectivity Palmer Taylor a,*, Lilly Wong a, Zoran Radic´ a, Igor Tsigelny a, Roger Bru¨ggemann a, Natilie A. Hosea a, Harvey A. Berman b a
Department of Pharmacology, 0636, Uni6ersity of California, San Diego, La Jolla, CA 92093 -0636, USA b Department of Biochemical Pharmacology, State Uni6ersity of New York at Buffalo, Buffalo, NY 14620 -0001, USA
Abstract We show here with a congeneric series of Rp- and Sp-alkoxymethyl phosphonothiolates of known absolute stereochemistry that chiral selectivity in their reaction with acetylcholinesterase can be described in terms of discrete orientational and steric requirements. Stereoselectivity depends on acyl pocket dimensions, which govern leaving group orientation and a productive association of the phosphonyl oxygen in the oxyanion hole. Overall geometry is consistent with a pentavalent intermediate where the attacking serine and leaving group are at apical positions. Oxime reactivation of the phosphonylated enzyme occurs through a similar associative intermediate presumably forming an oxime phosphonate. The oximes of differing structure show distinct angles of attacking the phosphate where the attack angles and access to the phosphorus are constrained in the sterically impacted gorge. Hence, efficacy of oxime reactivation is dependent on both oxime and conjugated phosphonate structures. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Acetylcholinesterase; Organophosphate enantiomers; Alkoxy phosphonothiolates; Cholinesterase stereoselectivity; Cholinesterase inactivation; Cholinesterase reactivation; Oximes; 2-PAM; HI-6
* Corresponding author. Fax: + 1-619-534-8248. Presented at the Third International Meeting on Esterases Reacting with Organophosphorus Compounds, Dubrovnik, Croatia, April 15 – 18, 1998. 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 0 9 - 5
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P. Taylor et al. / Chemico-Biological Interactions 119–120 (1999) 3–15
1. Introduction The organophosphates may best be considered as cholinesterase hemisubstrates for the large disparity between the rate of acylation of the active site serine and deacylation with these agents results in stable conjugation of the organophosphate at the active center serine and enzyme inactivation [1–3]. Nucleophiles such as oximes have long been known to enhance deacylation [1,4], and more recently certain modifications of side chains in the active center gorge have been shown to increase spontaneous deacylation rates [5]. The overall reaction of inactivation of the active center serine and reactivation with an oxime is shown in Scheme 1. Although the first organophosphate was synthesized in 1854 [6], it was not until the mid-1930s that their mechanism of action and the basis for their extreme toxicity were understood. Since that time these agents have been administered to enhance peripheral autonomic and motor function in various diseases, administered systemically and locally to eliminate parasites and applied as insecticides, all with varying degrees of success [7]. A more recent application is the augmentation of central nervous system function in the treatment of Alzheimer dementias. The toxicity of the more volatile organophosphates has been explored in the insidious development of various gases of chemical warfare. With their potential for multifaceted use, questions of selectivity, duration of action and recovery from inhibition continue to be prominent considerations for the organophosphates. A systematic approach to these questions, even when simplified with in vitro systems, reveals an extensive variety of structural variables with which we must contend. Recombinant DNA techniques which led to the cloning of the cholinesterases [1] and the eventual determination of the three-dimensional structures [8,9] provided the critical template to establish the arrangement of the atoms in the structure. However, crystal packing may constrain potential global conformations and local configurations of side chains limiting our understanding of details of AChE structure in solution. Site-specific mutagenesis has opened the dimension of structural modification of the macromolecule, yet simple side chain
Scheme 1.
P. Taylor et al. / Chemico-Biological Interactions 119–120 (1999) 3–15
5
substitutions can produce mass changes comparable to mass of the substrate size and, perhaps, unpredictable structural perturbations. Analysis of organophosphate reactions is rendered more complex by virtue of the phosphorus being tetrahedral and chirality often entering the reaction mechanisms. Hence enantiomer isolation emerges as a consideration of great importance to reactivity and structure of the compounds. Finally, the oxime reactivators are a rather eclectic group of agents presenting additional challenges to detailed structure-activity considerations. Herein, we rely on the prior development of a series of chiral methylphosphonates [10,11], in which the leaving group and retained alkoxy group have been systematically modified, to analyze inactivation and oxime reactivation of wild-type and mutant cholinesterases. The experimental approaches involving chemical syntheses and recombinant DNA techniques have been described in previous publications [12,13]. Fig. 1 shows a listing of the organophosphates and oximes employed to date in our studies. Chiral syntheses were undertaken and the absolute stereochemistry of the organophosphates ascertained [10,11,14]. Analyses of their reactions with the wild-type enzyme shows several features. First, the Sp-phosphonates are up to
Fig. 1. Structures of the organophosphates and oximes used in this study.
6
Leaving group
Thiocholine
Alkoxy group
Cycloheptyl
Chirality
Sp
Rp
Sp/Rp
Sp
Rp
Sp/Rp
Sp
Rp
Sp/Rp
Sp
Rp
Sp/Rp
AChE (wild-type) Phe 295 Leu Phe 297 Ile
190 000 66 000 16 000
820 8700 62 000
230 7.6 0.3
16 000 3400 950
140 1200 1200
110 3 0.8
360 000 140 000 56 000
19 000 10 000 12 000
19 14 5
74 160 55
0.16 1 26
460 160 2.1
a
Ethanethiol Isopropyl
3,3-Dimethylbutyl
Cycloheptyl
Rate constants given as 103/M min; standard error of the mean from typically three experiments was 10–15%. Data were obtained from refs. [12,13].
P. Taylor et al. / Chemico-Biological Interactions 119–120 (1999) 3–15
Table 1 Rate constantsa of transphosphonylation by Rp- and Sp-alkyl methylphosphonyl thioates with wild-type and acyl pocket mutations of mouse acetylcholinesterase
Enzyme
Leaving group SCH2CH2N+(CH3)3
AChE (Wild-type) Asp 74 Asn Glu 202 Gln Glu 450 Gln a
SCH3
SCH2CH3
kthiocholine
kthiocholine
kmethanethiol
kethanethiol
Sp
Rp
Sp/Rp
Sp
Rp
Sp/Rp
Sp
Rp
Sp/Rp
Sp
Rp
Sp
Rp
190 000 1400 21 000 1400
820 8.0 130 23
230 180 160 61
310 530 14 9
1.7 2.3 0.060 0.050
180 230 230 180
74 190 2.3 14
0.16 0.41 0.014 0.018
460 460 160 780
610 2.6 1500 160
480 3.5 2200 460
2600 7.4 9100 100
5100 19 9300 1300
Rate constants given as 103/M min; the standard error of the mean from typically three experiments was 10–15%. Data were obtained from refs. [12,13].
P. Taylor et al. / Chemico-Biological Interactions 119–120 (1999) 3–15
Table 2 Rate constantsa of transphosphonylation by Rp- and Sp-cycloheptyl methylphosphonyl thioates possessing charged and uncharged leaving groups
7
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P. Taylor et al. / Chemico-Biological Interactions 119–120 (1999) 3–15
Fig. 2. Free energy relationships for mutant cycle analysis of charged and uncharged analogs of Rp- and Sp-Alkoxy methyl phosphonates with Wild Type and Mutant Acetylcholinesterase. The free energy change in the respective cycle is obtained from DDG‡c =RTlnc.
250-fold more reactive with the greatest enantiomeric selectivity seen with the more bulky alkoxy group. Second, the agents with the cationic thiocholine leaving group are far more reactive than the neutral thioates (Table 1). Third, modification of the phenylalanines residing in the acyl pocket of the active center to smaller and more flexible side chains results in a loss of enantiomeric selectivity. In fact, chiral selectivity is actually inverted where the Rp phosphonate is more reactive in the F297I mutation. Enantiomeric selectivity is maintained for congeneric compounds where neutral thioate leaving groups are substituted for thiocholine. Mutant cycle analysis provides a means for identifying the precise groups interacting in a ligand-macromolecule complex by delineating free energy linkages between interactive pairs. For the enantiomeric phosphonates, we can assign interaction energy giving rise to both charge and enantiomeric specificity by using the appropriately paired methyl phosphonates and residue modifications in the active site gorge of acetylcholinesterase. Equations which describe the equilibrium and free energy relationships are shown in Fig. 2. The corresponding graphs for free energy changes (DDG‡c) associated with the rate constants shown in Tables 1 and 2 are given in Fig. 3. The magnitude of DDG‡ clearly shows the involvement of the acyl pocket in governing enantiomer selectivity and the charge on D74, but not that on E202 or E450, in dictating the preferential reactivity of organophosphates containing the cationic thiocholine leaving group. The observations that acyl pocket dimensions and a charged side chain near the gorge entry govern, respectively, chiral preference and charge specificity lead to a consideration of the requirements for stereospecific acylation by the phosphonates. In the early 1930s, Eason and Stedman [15] proposed that stereospecific interactions require a three-point interaction between the enantiomer and a dissymmetric surface on the macromolecule. Although Eason and Stedman’s studies on stereospecificity did not include cholinesterase, in separate studies they characterized the activity of the enzyme in plasma and various tissues and coined the term ‘choline esterase’ [16].
P. Taylor et al. / Chemico-Biological Interactions 119–120 (1999) 3–15
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Fig. 3. Relationship between free energy differences DDG‡ for the reaction rate of various alkoxymethyl phosphonates and mutant acetylcholinesterases. The respective three-dimensional graphs show the relationships between: (A) the change in free energy of activation for phosphonylation (Y axis), chirality (X axis) and mutations (Z axis); and (B) the change in free energy of activation for phosphonylation (Y axis) charge versus uncharged (X axis) and mutations (Z axis). Modified from ref. [13].
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P. Taylor et al. / Chemico-Biological Interactions 119–120 (1999) 3–15
In the case of the AChE interaction, its surface is at the base of a narrow gorge and the three-point attachment comes from: (a) a conjugating bond distance between the active site serine and the phosphonyl moiety; (b) entry of the phosphonyl oxygen into the oxyanion hole; and (c) the thiocholine leaving group directed towards the gorge exit (Fig. 5). In the case of the Sp enantiomer, the large alkoxy group can fit into the space accommodating choline subsite; whereas for the Rp enantiomer, a similar placement of the alkoxy group requires that either the
Fig. 4. Stereoviews of Sp- and Rp-cycloheptyl methylphosphonothiocholine docked in the active center of mouse acetylcholinesterase. Residues of the acyl pocket (Phe 295 and 297), choline binding subsite (Trp 86, Tyr 337, Glu 202) are shown (c.f.: refs. [12,13] for details). The phosphonate is positioned for attack by serine 203 and the phosphonyl oxygen is positioned in the oxyanion hole.
P. Taylor et al. / Chemico-Biological Interactions 119–120 (1999) 3–15
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Fig. 5. Stereoview of Sp-cycloheptyl methylphosphonothiocholine docked in the active center of acetylcholinesterase. The view is a side view from Fig. 4 to show the orientation of the thiocholine moiety with respect to the gorge exit and Asp 74. A portion of the cholinesterase molecule on the acyl pocket side is cut away in order to show an unobstructed view of the inhibitor (c.f.: refs. [9,12,13] for details).
phosphonyl oxygen be out of the oxyanion hole or the leaving group be directed away from the gorge exit. By enlarging the acyl pocket so that the alkoxy group orients in that direction, both of these criteria can be satisfied [17] (Figs. 4 and 5). This arrangement of substitutent groups is depicted in the molecular dynamics analysis shown in Fig. 6. The XY plane represents the phosphorus to g-serine hydroxyl interatomic distance and would be one of the dimensions of the threepoint attachment. The Sp compound allows for the orientation of the phosphonyl oxygen in the oxyanion hole consistent with hydrogen bonding distances from the three-donor hydrogens of the amide backbone in AChE [12,13]. By contrast, steric occlusion of the alkoxy group with the phenylalanines in the acyl pocket (F297 and F295) precludes the three-point attachment for the Rp phosphonate; the latter can only be achieved by mutation of F295 or preferably F297 to the smaller and more flexible aliphatic moieties. As shown in Fig. 6, only two of the three favored orientations of the substituent groups is achieved with the Rp compound in the absence of mutations. These considerations also establish a discrete orientation for the transition state of the organophosphate-active center serine interaction. Interestingly, polarization of the phosphonyl bond and an orientation of the leaving group to approximate a trigonal bipyramidal intermediate appear essential for an efficient reaction. In the latter intermediate, the attacking serine and the leaving group would assume the apical positions.
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P. Taylor et al. / Chemico-Biological Interactions 119–120 (1999) 3–15
2. Reactivation of inhibited acetylcholinesterase The respective diastereomeric conjugates formed from the chiral phosphonates can be used advantageously to examine reactivation with oximes. The well studied oximes of potential use in man are structurally a rather eclectic group, and a systematic examination of structure-activity relationships has not been undertaken. We show here some representative data for pralidoxime (2-PAM) and HI-6 reactivation (Table 3). Some general principles emerge from an examination of the data. First, the more reactive Sp compounds are also the most susceptible to oxime reactivation. All of the Sp compounds reactivate fully, whereas only the isopropoxy Rp compound can be reactivated at appreciable rates (data not shown). The same applies for AChEs with mutations in the acyl pocket (data not shown). This implies that the oxyanion hole induced polarization of the phosphonyl oxygen is also
Fig. 6. Molecular dynamics simulation followed by energy minimization of a docked Sp () and Rp () cycloheptyl methylphosphonothiocholine in a reversible complex with acetylcholinesterase. The phosphorus group is docked within bonding distance with the g-oxygen of serine 203 in the enzyme. This reaction position along the Z axis then becomes defined by the plane X, Y. A productive conformation is assumed to require: (a) the appropriate Ser-O-P distance on the Z axis (i.e. the X, Y plane); (b) insertion of the phosphonyl oxygen in the oxyanion hole (Y axis): a mean hydrogen bonding distance of ˚ from the amide backbone hydrogens of Gly 121, Gly 122 and Ala 204; and (c) an orientation of 3–4 A the leaving group directed towards gorge entrance. The ideal position is assumed to be 180° from the attacking serine oxygen placing the serine oxygen and the leaving group in apical positions and the remaining three groups in equatorial positions. The deviations reflect the difference in distances for the quaternary nitrogen between the energy minimized position and that expected for apical positioning (180°) of the serine g-oxygen and the leaving group. Modified from ref. [17].
P. Taylor et al. / Chemico-Biological Interactions 119–120 (1999) 3–15
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Table 3 Rate constants determined for oxime reactivation of mouse acetylcholinesterase inhibited with chiral Sp alkoxy methylphosphonates Oxime
Enzyme
Alkoxy substitution
kr (M−1 min−1)
% Reactivation
2-PAM
Sp-AChE Sp (F295L) AChE Sp (F297I) AChE
Cycloheptyl
0.46 1.39 9.2
70 76 74
2-PAM
Sp-AChE Sp (F295L) AChE Sp (F297I) AChE
3,3-Dimethylbutyl
0.24 1.11 30
103 103 102
2-PAM
Sp-AChE Sp (F295L) AChE Sp (F297I) AChE
Isopropyl
155 116 1260
99 107 92
HI-6
Sp-AChE Sp (F295L) AChE Sp (F297I) AChE
Cycloheptyl
186 2520 353
96 94 127
HI-6
Sp-AChE Sp (F295L) AChE Sp (F297I) AChE
3,3-Dimethylbutyl
181 1230 254
97 93 120
HI-6
Sp-AChE Sp (F295L) AChE Sp (F297I) AChE
Isopropyl
2160 3320 2400
91 104 102
Fig. 7. Structure of the active center gorge with the Sp-cycloheptyl methyl phosphonyl moiety conjugated to the serine. The likely angles of attack for 2-PAM and HI-6 are shown for the respective mutant enzyme. Critical residues within and Connolly surfaces of the active center gorge are shown.
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P. Taylor et al. / Chemico-Biological Interactions 119–120 (1999) 3–15
required for efficient reactivation by nucleophiles. Second, reactivation, even with the preferred Sp enantiomer, is a far slower process than inactivation. Here we find bimolecular rate constants of 103 M − 1 min − 1 rather than 108 M − 1 min − 1. Third, mutations in the acyl pocket greatly enhance reactivation rates. What these basic observations point to is formation of an associative intermediate of rather similar orientation to that expected for the acylation reaction. Formation of a phosphonyl-oxime appears to be an initial step in the reaction and this reaction is facilitated by polarization of the phosphonyl oxygen bond in the oxyanion hole. Less than optimal geometry for interaction is achieved with the oximes for two reasons: oxime entry to the point of optimal reaction is impacted by the spatial constraints of the gorge and steric limitations intrinsic to the oxime itself. Accordingly, the gorge dimensions preclude the attacking nucleophile and scissile bond being apically positioned. These factors are likely to account for reactions with oximes being far slower than the phosphonylation reaction. Mutation of the phenylalanines 295 and 297 in the acyl pocket to smaller groups enhances reactivation some ten- to 20-fold indicating that mutation facilitates an avenue of entry for the oxime (Table 3). It is also noteworthy that opening the 295 position enhances HI-6 reactivation, whereas mutation at the 297 position is most influential for 2-PAM reactivation. Hence the two nucleophiles appear to show distinctive preferred entry routes for attack of the phosphonyl moiety (Fig. 7). 2-PAM being the smaller molecule is likely to have additional degrees of freedom and finds entry near the exit side of the acyl pocket. By contrast, HI-6 appears to have its oxime attack from deeper in the gorge. This may arise from the two quaternary ammoniums in HI-6 and its distal end being tethered to a locus near the gorge mouth. The face of attack of the conjugated phosphonate may be described in terms of the four faces of the tetrahedron defined by the tetrahedral phosphorus. The geometric confines of the gorge and steric factors intrinsic to the oxime itself are likely to dictate the preferential face of reactivity. Hence, optimization of the reaction rate will be both phosphonate and oxime dependent and details on optimizing this reaction are not yet fully understood. The analyses presented here are being extended for a large number of oximes and phosphonates, and will be a subject of future report.
References [1] P. Taylor, Z. Radic´, The cholinesterases: from genes to proteins, Ann. Rev. Pharmacol. Toxicol. 34 (1993) 281–320. [2] D.M. Quinn, Acetylcholinesterase: enzyme structure reaction dynamics and virtual transition states, Chem. Rev. 87 (1987) 955–979. [3] W.N. Aldridge, E. Reiner, Enzyme Inhibitors as Substrates, Elsevier, Amsterdam, 1972, p. 328. [4] I.B. Wilson, Molecular complementarity and antidotes for alkyl phosphate poisoning, Fed. Proc. 18 (1959) 752–758. [5] O. Lockridge, R.M. Blong, P. Masson, M.-T. Froment, C.B. Millard, C.A. Broomfield, A single amino acid substitution, Gly 117 His, confers phosphotriesterase activity in human butyrylcholinesterase, Biochemistry 36 (1997) 786 – 795.
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[6] P. de Clermont, Chimie organique— note sur la pre´paration de quelques e´thers, C.R. Hebd. Seanc. Acad. Sci. Paris 39 (1854) 338–341. [7] P. Taylor, Anticholinesterase agents, in: J.G. Hardman, L.E. Limbird (Eds.), Goodman & Gilman’s Pharmacological Basis of Therapeutics, McGraw – Hill, New York, 1995, pp. 161 – 176. [8] J.L. Sussman, M. Harel, F. Frolow, C. Oefner, A. Goldman, L. Toker, I. Silman, Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein, Science 253 (1991) 872–879. [9] Y. Bourne, P. Taylor, P. Marchot, Acetylcholinesterase inhibition by fasciculin: crystal structure of the complex, Cell 83 (1995) 503–512. [10] H.A. Berman, M.M. Decker, Kinetic, equilibrium and spectroscopic studies on dealkylation of alkyl organophosphonyl acetylcholinesterase, J. Biol. Chem. 261 (1986) 10646 – 10652. [11] H.A. Berman, K. Leonard, Chiral reactions of acetylcholinesterase probed with enantiomeric methylphosphonothioates: non-covalent determinants of enzyme chirality, J. Biol. Chem. 264 (1989) 3942–3950. [12] N.A. Hosea, H.A. Berman, P. Taylor, Specificity and orientation of trigonal carboxyl esters and tetrahedral alkylphosphonyl esters in cholinesterases, Biochemistry 34 (1995) 11528 – 11536. [13] 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. [14] H.A. Berman, M.M. Decker, Chiral nature of covalent methylphosphonyl conjugates of acetylcholinesterase, J. Biol. Chem. 264 (1989) 3951 – 3956. [15] L.M. Eason, E. Stedman, Studies on the relation between chemical constitution and physiological action, Biochem. J. 27 (1933) 1257– 1266. [16] E. Stedman, E. Stedman, L.M. Eason, ‘‘Choline esterase’’ and enzymes present in the blood-serum of the horse, Biochem. J. 25 (1932) 2056 – 2066. [17] P. Taylor, N.A. Hosea, I. Tsigelny, Z. Radic, H.A. Berman, Determining ligand orientation and transphosphonylation mechanisms in acetylcholinesterases by Rp, Sp enantiomer selectivity and site-specific mutagenesis, Enantiomer 2 (1997) 249 – 260.
.
Analysis of cholinesterase inactivation and reactivation by systematic structural modification and enantiomeric selectivity Palmer Taylor a,*, Lilly Wong a, Zoran Radic´ a, Igor Tsigelny a, Roger Bru¨ggemann a, Natilie A. Hosea a, Harvey A. Berman b a
Department of Pharmacology, 0636, Uni6ersity of California, San Diego, La Jolla, CA 92093 -0636, USA b Department of Biochemical Pharmacology, State Uni6ersity of New York at Buffalo, Buffalo, NY 14620 -0001, USA
Abstract We show here with a congeneric series of Rp- and Sp-alkoxymethyl phosphonothiolates of known absolute stereochemistry that chiral selectivity in their reaction with acetylcholinesterase can be described in terms of discrete orientational and steric requirements. Stereoselectivity depends on acyl pocket dimensions, which govern leaving group orientation and a productive association of the phosphonyl oxygen in the oxyanion hole. Overall geometry is consistent with a pentavalent intermediate where the attacking serine and leaving group are at apical positions. Oxime reactivation of the phosphonylated enzyme occurs through a similar associative intermediate presumably forming an oxime phosphonate. The oximes of differing structure show distinct angles of attacking the phosphate where the attack angles and access to the phosphorus are constrained in the sterically impacted gorge. Hence, efficacy of oxime reactivation is dependent on both oxime and conjugated phosphonate structures. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Acetylcholinesterase; Organophosphate enantiomers; Alkoxy phosphonothiolates; Cholinesterase stereoselectivity; Cholinesterase inactivation; Cholinesterase reactivation; Oximes; 2-PAM; HI-6
* Corresponding author. Fax: + 1-619-534-8248. Presented at the Third International Meeting on Esterases Reacting with Organophosphorus Compounds, Dubrovnik, Croatia, April 15 – 18, 1998. 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 0 9 - 5
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P. Taylor et al. / Chemico-Biological Interactions 119–120 (1999) 3–15
1. Introduction The organophosphates may best be considered as cholinesterase hemisubstrates for the large disparity between the rate of acylation of the active site serine and deacylation with these agents results in stable conjugation of the organophosphate at the active center serine and enzyme inactivation [1–3]. Nucleophiles such as oximes have long been known to enhance deacylation [1,4], and more recently certain modifications of side chains in the active center gorge have been shown to increase spontaneous deacylation rates [5]. The overall reaction of inactivation of the active center serine and reactivation with an oxime is shown in Scheme 1. Although the first organophosphate was synthesized in 1854 [6], it was not until the mid-1930s that their mechanism of action and the basis for their extreme toxicity were understood. Since that time these agents have been administered to enhance peripheral autonomic and motor function in various diseases, administered systemically and locally to eliminate parasites and applied as insecticides, all with varying degrees of success [7]. A more recent application is the augmentation of central nervous system function in the treatment of Alzheimer dementias. The toxicity of the more volatile organophosphates has been explored in the insidious development of various gases of chemical warfare. With their potential for multifaceted use, questions of selectivity, duration of action and recovery from inhibition continue to be prominent considerations for the organophosphates. A systematic approach to these questions, even when simplified with in vitro systems, reveals an extensive variety of structural variables with which we must contend. Recombinant DNA techniques which led to the cloning of the cholinesterases [1] and the eventual determination of the three-dimensional structures [8,9] provided the critical template to establish the arrangement of the atoms in the structure. However, crystal packing may constrain potential global conformations and local configurations of side chains limiting our understanding of details of AChE structure in solution. Site-specific mutagenesis has opened the dimension of structural modification of the macromolecule, yet simple side chain
Scheme 1.
P. Taylor et al. / Chemico-Biological Interactions 119–120 (1999) 3–15
5
substitutions can produce mass changes comparable to mass of the substrate size and, perhaps, unpredictable structural perturbations. Analysis of organophosphate reactions is rendered more complex by virtue of the phosphorus being tetrahedral and chirality often entering the reaction mechanisms. Hence enantiomer isolation emerges as a consideration of great importance to reactivity and structure of the compounds. Finally, the oxime reactivators are a rather eclectic group of agents presenting additional challenges to detailed structure-activity considerations. Herein, we rely on the prior development of a series of chiral methylphosphonates [10,11], in which the leaving group and retained alkoxy group have been systematically modified, to analyze inactivation and oxime reactivation of wild-type and mutant cholinesterases. The experimental approaches involving chemical syntheses and recombinant DNA techniques have been described in previous publications [12,13]. Fig. 1 shows a listing of the organophosphates and oximes employed to date in our studies. Chiral syntheses were undertaken and the absolute stereochemistry of the organophosphates ascertained [10,11,14]. Analyses of their reactions with the wild-type enzyme shows several features. First, the Sp-phosphonates are up to
Fig. 1. Structures of the organophosphates and oximes used in this study.
6
Leaving group
Thiocholine
Alkoxy group
Cycloheptyl
Chirality
Sp
Rp
Sp/Rp
Sp
Rp
Sp/Rp
Sp
Rp
Sp/Rp
Sp
Rp
Sp/Rp
AChE (wild-type) Phe 295 Leu Phe 297 Ile
190 000 66 000 16 000
820 8700 62 000
230 7.6 0.3
16 000 3400 950
140 1200 1200
110 3 0.8
360 000 140 000 56 000
19 000 10 000 12 000
19 14 5
74 160 55
0.16 1 26
460 160 2.1
a
Ethanethiol Isopropyl
3,3-Dimethylbutyl
Cycloheptyl
Rate constants given as 103/M min; standard error of the mean from typically three experiments was 10–15%. Data were obtained from refs. [12,13].
P. Taylor et al. / Chemico-Biological Interactions 119–120 (1999) 3–15
Table 1 Rate constantsa of transphosphonylation by Rp- and Sp-alkyl methylphosphonyl thioates with wild-type and acyl pocket mutations of mouse acetylcholinesterase
Enzyme
Leaving group SCH2CH2N+(CH3)3
AChE (Wild-type) Asp 74 Asn Glu 202 Gln Glu 450 Gln a
SCH3
SCH2CH3
kthiocholine
kthiocholine
kmethanethiol
kethanethiol
Sp
Rp
Sp/Rp
Sp
Rp
Sp/Rp
Sp
Rp
Sp/Rp
Sp
Rp
Sp
Rp
190 000 1400 21 000 1400
820 8.0 130 23
230 180 160 61
310 530 14 9
1.7 2.3 0.060 0.050
180 230 230 180
74 190 2.3 14
0.16 0.41 0.014 0.018
460 460 160 780
610 2.6 1500 160
480 3.5 2200 460
2600 7.4 9100 100
5100 19 9300 1300
Rate constants given as 103/M min; the standard error of the mean from typically three experiments was 10–15%. Data were obtained from refs. [12,13].
P. Taylor et al. / Chemico-Biological Interactions 119–120 (1999) 3–15
Table 2 Rate constantsa of transphosphonylation by Rp- and Sp-cycloheptyl methylphosphonyl thioates possessing charged and uncharged leaving groups
7
8
P. Taylor et al. / Chemico-Biological Interactions 119–120 (1999) 3–15
Fig. 2. Free energy relationships for mutant cycle analysis of charged and uncharged analogs of Rp- and Sp-Alkoxy methyl phosphonates with Wild Type and Mutant Acetylcholinesterase. The free energy change in the respective cycle is obtained from DDG‡c =RTlnc.
250-fold more reactive with the greatest enantiomeric selectivity seen with the more bulky alkoxy group. Second, the agents with the cationic thiocholine leaving group are far more reactive than the neutral thioates (Table 1). Third, modification of the phenylalanines residing in the acyl pocket of the active center to smaller and more flexible side chains results in a loss of enantiomeric selectivity. In fact, chiral selectivity is actually inverted where the Rp phosphonate is more reactive in the F297I mutation. Enantiomeric selectivity is maintained for congeneric compounds where neutral thioate leaving groups are substituted for thiocholine. Mutant cycle analysis provides a means for identifying the precise groups interacting in a ligand-macromolecule complex by delineating free energy linkages between interactive pairs. For the enantiomeric phosphonates, we can assign interaction energy giving rise to both charge and enantiomeric specificity by using the appropriately paired methyl phosphonates and residue modifications in the active site gorge of acetylcholinesterase. Equations which describe the equilibrium and free energy relationships are shown in Fig. 2. The corresponding graphs for free energy changes (DDG‡c) associated with the rate constants shown in Tables 1 and 2 are given in Fig. 3. The magnitude of DDG‡ clearly shows the involvement of the acyl pocket in governing enantiomer selectivity and the charge on D74, but not that on E202 or E450, in dictating the preferential reactivity of organophosphates containing the cationic thiocholine leaving group. The observations that acyl pocket dimensions and a charged side chain near the gorge entry govern, respectively, chiral preference and charge specificity lead to a consideration of the requirements for stereospecific acylation by the phosphonates. In the early 1930s, Eason and Stedman [15] proposed that stereospecific interactions require a three-point interaction between the enantiomer and a dissymmetric surface on the macromolecule. Although Eason and Stedman’s studies on stereospecificity did not include cholinesterase, in separate studies they characterized the activity of the enzyme in plasma and various tissues and coined the term ‘choline esterase’ [16].
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Fig. 3. Relationship between free energy differences DDG‡ for the reaction rate of various alkoxymethyl phosphonates and mutant acetylcholinesterases. The respective three-dimensional graphs show the relationships between: (A) the change in free energy of activation for phosphonylation (Y axis), chirality (X axis) and mutations (Z axis); and (B) the change in free energy of activation for phosphonylation (Y axis) charge versus uncharged (X axis) and mutations (Z axis). Modified from ref. [13].
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In the case of the AChE interaction, its surface is at the base of a narrow gorge and the three-point attachment comes from: (a) a conjugating bond distance between the active site serine and the phosphonyl moiety; (b) entry of the phosphonyl oxygen into the oxyanion hole; and (c) the thiocholine leaving group directed towards the gorge exit (Fig. 5). In the case of the Sp enantiomer, the large alkoxy group can fit into the space accommodating choline subsite; whereas for the Rp enantiomer, a similar placement of the alkoxy group requires that either the
Fig. 4. Stereoviews of Sp- and Rp-cycloheptyl methylphosphonothiocholine docked in the active center of mouse acetylcholinesterase. Residues of the acyl pocket (Phe 295 and 297), choline binding subsite (Trp 86, Tyr 337, Glu 202) are shown (c.f.: refs. [12,13] for details). The phosphonate is positioned for attack by serine 203 and the phosphonyl oxygen is positioned in the oxyanion hole.
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Fig. 5. Stereoview of Sp-cycloheptyl methylphosphonothiocholine docked in the active center of acetylcholinesterase. The view is a side view from Fig. 4 to show the orientation of the thiocholine moiety with respect to the gorge exit and Asp 74. A portion of the cholinesterase molecule on the acyl pocket side is cut away in order to show an unobstructed view of the inhibitor (c.f.: refs. [9,12,13] for details).
phosphonyl oxygen be out of the oxyanion hole or the leaving group be directed away from the gorge exit. By enlarging the acyl pocket so that the alkoxy group orients in that direction, both of these criteria can be satisfied [17] (Figs. 4 and 5). This arrangement of substitutent groups is depicted in the molecular dynamics analysis shown in Fig. 6. The XY plane represents the phosphorus to g-serine hydroxyl interatomic distance and would be one of the dimensions of the threepoint attachment. The Sp compound allows for the orientation of the phosphonyl oxygen in the oxyanion hole consistent with hydrogen bonding distances from the three-donor hydrogens of the amide backbone in AChE [12,13]. By contrast, steric occlusion of the alkoxy group with the phenylalanines in the acyl pocket (F297 and F295) precludes the three-point attachment for the Rp phosphonate; the latter can only be achieved by mutation of F295 or preferably F297 to the smaller and more flexible aliphatic moieties. As shown in Fig. 6, only two of the three favored orientations of the substituent groups is achieved with the Rp compound in the absence of mutations. These considerations also establish a discrete orientation for the transition state of the organophosphate-active center serine interaction. Interestingly, polarization of the phosphonyl bond and an orientation of the leaving group to approximate a trigonal bipyramidal intermediate appear essential for an efficient reaction. In the latter intermediate, the attacking serine and the leaving group would assume the apical positions.
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2. Reactivation of inhibited acetylcholinesterase The respective diastereomeric conjugates formed from the chiral phosphonates can be used advantageously to examine reactivation with oximes. The well studied oximes of potential use in man are structurally a rather eclectic group, and a systematic examination of structure-activity relationships has not been undertaken. We show here some representative data for pralidoxime (2-PAM) and HI-6 reactivation (Table 3). Some general principles emerge from an examination of the data. First, the more reactive Sp compounds are also the most susceptible to oxime reactivation. All of the Sp compounds reactivate fully, whereas only the isopropoxy Rp compound can be reactivated at appreciable rates (data not shown). The same applies for AChEs with mutations in the acyl pocket (data not shown). This implies that the oxyanion hole induced polarization of the phosphonyl oxygen is also
Fig. 6. Molecular dynamics simulation followed by energy minimization of a docked Sp () and Rp () cycloheptyl methylphosphonothiocholine in a reversible complex with acetylcholinesterase. The phosphorus group is docked within bonding distance with the g-oxygen of serine 203 in the enzyme. This reaction position along the Z axis then becomes defined by the plane X, Y. A productive conformation is assumed to require: (a) the appropriate Ser-O-P distance on the Z axis (i.e. the X, Y plane); (b) insertion of the phosphonyl oxygen in the oxyanion hole (Y axis): a mean hydrogen bonding distance of ˚ from the amide backbone hydrogens of Gly 121, Gly 122 and Ala 204; and (c) an orientation of 3–4 A the leaving group directed towards gorge entrance. The ideal position is assumed to be 180° from the attacking serine oxygen placing the serine oxygen and the leaving group in apical positions and the remaining three groups in equatorial positions. The deviations reflect the difference in distances for the quaternary nitrogen between the energy minimized position and that expected for apical positioning (180°) of the serine g-oxygen and the leaving group. Modified from ref. [17].
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Table 3 Rate constants determined for oxime reactivation of mouse acetylcholinesterase inhibited with chiral Sp alkoxy methylphosphonates Oxime
Enzyme
Alkoxy substitution
kr (M−1 min−1)
% Reactivation
2-PAM
Sp-AChE Sp (F295L) AChE Sp (F297I) AChE
Cycloheptyl
0.46 1.39 9.2
70 76 74
2-PAM
Sp-AChE Sp (F295L) AChE Sp (F297I) AChE
3,3-Dimethylbutyl
0.24 1.11 30
103 103 102
2-PAM
Sp-AChE Sp (F295L) AChE Sp (F297I) AChE
Isopropyl
155 116 1260
99 107 92
HI-6
Sp-AChE Sp (F295L) AChE Sp (F297I) AChE
Cycloheptyl
186 2520 353
96 94 127
HI-6
Sp-AChE Sp (F295L) AChE Sp (F297I) AChE
3,3-Dimethylbutyl
181 1230 254
97 93 120
HI-6
Sp-AChE Sp (F295L) AChE Sp (F297I) AChE
Isopropyl
2160 3320 2400
91 104 102
Fig. 7. Structure of the active center gorge with the Sp-cycloheptyl methyl phosphonyl moiety conjugated to the serine. The likely angles of attack for 2-PAM and HI-6 are shown for the respective mutant enzyme. Critical residues within and Connolly surfaces of the active center gorge are shown.
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required for efficient reactivation by nucleophiles. Second, reactivation, even with the preferred Sp enantiomer, is a far slower process than inactivation. Here we find bimolecular rate constants of 103 M − 1 min − 1 rather than 108 M − 1 min − 1. Third, mutations in the acyl pocket greatly enhance reactivation rates. What these basic observations point to is formation of an associative intermediate of rather similar orientation to that expected for the acylation reaction. Formation of a phosphonyl-oxime appears to be an initial step in the reaction and this reaction is facilitated by polarization of the phosphonyl oxygen bond in the oxyanion hole. Less than optimal geometry for interaction is achieved with the oximes for two reasons: oxime entry to the point of optimal reaction is impacted by the spatial constraints of the gorge and steric limitations intrinsic to the oxime itself. Accordingly, the gorge dimensions preclude the attacking nucleophile and scissile bond being apically positioned. These factors are likely to account for reactions with oximes being far slower than the phosphonylation reaction. Mutation of the phenylalanines 295 and 297 in the acyl pocket to smaller groups enhances reactivation some ten- to 20-fold indicating that mutation facilitates an avenue of entry for the oxime (Table 3). It is also noteworthy that opening the 295 position enhances HI-6 reactivation, whereas mutation at the 297 position is most influential for 2-PAM reactivation. Hence the two nucleophiles appear to show distinctive preferred entry routes for attack of the phosphonyl moiety (Fig. 7). 2-PAM being the smaller molecule is likely to have additional degrees of freedom and finds entry near the exit side of the acyl pocket. By contrast, HI-6 appears to have its oxime attack from deeper in the gorge. This may arise from the two quaternary ammoniums in HI-6 and its distal end being tethered to a locus near the gorge mouth. The face of attack of the conjugated phosphonate may be described in terms of the four faces of the tetrahedron defined by the tetrahedral phosphorus. The geometric confines of the gorge and steric factors intrinsic to the oxime itself are likely to dictate the preferential face of reactivity. Hence, optimization of the reaction rate will be both phosphonate and oxime dependent and details on optimizing this reaction are not yet fully understood. The analyses presented here are being extended for a large number of oximes and phosphonates, and will be a subject of future report.
References [1] P. Taylor, Z. Radic´, The cholinesterases: from genes to proteins, Ann. Rev. Pharmacol. Toxicol. 34 (1993) 281–320. [2] D.M. Quinn, Acetylcholinesterase: enzyme structure reaction dynamics and virtual transition states, Chem. Rev. 87 (1987) 955–979. [3] W.N. Aldridge, E. Reiner, Enzyme Inhibitors as Substrates, Elsevier, Amsterdam, 1972, p. 328. [4] I.B. Wilson, Molecular complementarity and antidotes for alkyl phosphate poisoning, Fed. Proc. 18 (1959) 752–758. [5] O. Lockridge, R.M. Blong, P. Masson, M.-T. Froment, C.B. Millard, C.A. Broomfield, A single amino acid substitution, Gly 117 His, confers phosphotriesterase activity in human butyrylcholinesterase, Biochemistry 36 (1997) 786 – 795.
P. Taylor et al. / Chemico-Biological Interactions 119–120 (1999) 3–15
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[6] P. de Clermont, Chimie organique— note sur la pre´paration de quelques e´thers, C.R. Hebd. Seanc. Acad. Sci. Paris 39 (1854) 338–341. [7] P. Taylor, Anticholinesterase agents, in: J.G. Hardman, L.E. Limbird (Eds.), Goodman & Gilman’s Pharmacological Basis of Therapeutics, McGraw – Hill, New York, 1995, pp. 161 – 176. [8] J.L. Sussman, M. Harel, F. Frolow, C. Oefner, A. Goldman, L. Toker, I. Silman, Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein, Science 253 (1991) 872–879. [9] Y. Bourne, P. Taylor, P. Marchot, Acetylcholinesterase inhibition by fasciculin: crystal structure of the complex, Cell 83 (1995) 503–512. [10] H.A. Berman, M.M. Decker, Kinetic, equilibrium and spectroscopic studies on dealkylation of alkyl organophosphonyl acetylcholinesterase, J. Biol. Chem. 261 (1986) 10646 – 10652. [11] H.A. Berman, K. Leonard, Chiral reactions of acetylcholinesterase probed with enantiomeric methylphosphonothioates: non-covalent determinants of enzyme chirality, J. Biol. Chem. 264 (1989) 3942–3950. [12] N.A. Hosea, H.A. Berman, P. Taylor, Specificity and orientation of trigonal carboxyl esters and tetrahedral alkylphosphonyl esters in cholinesterases, Biochemistry 34 (1995) 11528 – 11536. [13] 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. [14] H.A. Berman, M.M. Decker, Chiral nature of covalent methylphosphonyl conjugates of acetylcholinesterase, J. Biol. Chem. 264 (1989) 3951 – 3956. [15] L.M. Eason, E. Stedman, Studies on the relation between chemical constitution and physiological action, Biochem. J. 27 (1933) 1257– 1266. [16] E. Stedman, E. Stedman, L.M. Eason, ‘‘Choline esterase’’ and enzymes present in the blood-serum of the horse, Biochem. J. 25 (1932) 2056 – 2066. [17] P. Taylor, N.A. Hosea, I. Tsigelny, Z. Radic, H.A. Berman, Determining ligand orientation and transphosphonylation mechanisms in acetylcholinesterases by Rp, Sp enantiomer selectivity and site-specific mutagenesis, Enantiomer 2 (1997) 249 – 260.
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