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Acceleration of Drosophila melanogaster acetylcholinesterase methanesulfonylation: peripheral ligand d -tubocurarine enhances the affinity for small methanesulfonylfluoride
Chemico-Biological Interactions 139 (2002) 145– 157
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Acceleration of Drosophila melanogaster acetylcholinesterase methanesulfonylation: peripheral ligand D-tubocurarine enhances the affinity for small methanesulfonylfluoride Marko Golicˇnik a, Didier Fournier b, Jure Stojan a,* a
Institute of Biochemistry, Medical Faculty, Uni6ersity of Ljubljana, Vrazo6 trg 2, 1000 Ljubljana, Slo6enia b Laboratoire d’Entomologie et Groupe de Chimie Biologie, Uni6ersite` Paul Sabatier, 31062 Toulouse, France Received 14 May 2001; received in revised form 28 May 2001; accepted 3 November 2001
Abstract D-Tubocurarine, a reversible peripheral inhibitor of cholinesterases accelerates methanesulfonylation of Drosophila melanogaster wild type and W359L mutant. The kinetic evaluation of the process was performed in a step-by-step analysis. The second order overall sulfonylation rate constants, determined from classical residual activity measurements, were used in the subsequent analysis of progress curves. The latter were obtained by measuring the hydrolysis of acetylthiocholine in a complex reaction system of enzyme, substrate, irreversible and reversible inhibitor. The underlying kinetic mechanisms, from such a complex data, could only be untangled by targeted inspection and successive incorporation of reaction steps for which experimental evidence existed. The study showed that the peripheral ligand D-tubocurarine, by binding at the entrance into the active site of the two investigated enzymes (Golicˇnik et al., Biochemistry 40 (2001) 1214), enhances the affinity for small methanesulfonylfluoride, rather to speeding up the formation of a stable covalent enzyme-inhibitor complex. The specific arrangements at the rim of the active site of each individual
Abbre6iations: AChE, acetylcholinesterase; ATCh, acetylthiocholine; BChE, butyrylcholinesterase; ChE, cholinesterase; DmAChE, Drosophila melanogaster acetylcholinesterase; DTNB, 5,5%-dithio-bisnitrobenzoic acid; MSF, methanesulfonylfluoride; TC, D-tubocurarine. * Corresponding author. Tel.: +386-1-5437651; fax: +386-1-5437641. E-mail address: [email protected] (J. Stojan). 0009-2797/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 2 7 9 7 ( 0 1 ) 0 0 2 9 4 - 0
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enzyme dictate the actual events which can be detected by kinetic means. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Acetylcholinesterase; Kinetic models; curves
D-tubocurarine;
Methanesulfonylfluoride; Progress
1. Introduction The catalytic mechanism and inhibition of ChEs have been extensively investigated [1] during the past decades. Recently, the crystal structure determination of AChEs from vertebrates and from Drosophila melanogaster [2–5] revealed the active site, :20 A, deep inside the core of the molecule, at the bottom of a narrow gorge. It seems that such penetrated catalytic site is responsible for their peculiar kinetic behavior. All ChEs, either native or various specifically mutated enzymes, exhibit deviations from Michaelis–Menten hyperbolic kinetics. While moderate positive homotropic pseudo-cooperativity at intermediate substrate concentrations is present only in certain enzymes, the inhibition by excess substrate is seldom missing [6– 8]. Several kinetic models were proposed to describe non Michaelis– Menten behavior of ChEs [9,10], but they were able only to explain one deviation. Recently, a six parameter kinetic model valid for all ChEs was introduced [11]. It can describe both mentioned deviations and by an appropriate extension, also the data in the presence of various reversible or irreversible active site directed inhibitors [7,12– 14]. It was also found that D-tubocurarine (TC), an inhibitor, whose shape and size prevent the interaction with the active site at the bottom of the gorge, can enhance the substrate hydrolysis at high substrate concentrations in the case of Drosophila W359L AChE mutant [14]. This paradox has been explained as inhibition, less effective than the substrate inhibition itself. To obtain further information on the behavior of relatively narrow active site gorge in Drosophila AChE (50% less space as in Torpedo enzyme [5]) we investigated the methanesulfonylation of wild type DmAChE and the W359L mutant in the absence and presence of TC. Methanesulfonylfluoride (MSF), a poweful irreversible anticholinesterase agent, sulfonylates insect AChE with faster rates than vertebrate enzymes or BChEs [15–17]. It is also known that the action of this tiny sulfonate on vertebrate AChEs is accelerated by active site directed inhibitors like tetramethylammonium and decamethonium [16] but TC is an inhibitor of methanesulfonylation [18]. The fact, that TC can also activate W359L DmAChE, lead us to investigate its action on the methanesulfonylation of wild type DmAChE and the W359L mutant. We have performed the experiments, where the substrate hydrolysis was followed in the presence of two inhibitors, TC and MSF. Such experiments represent a very complex system, and the obtained data require sophisticated step-by-step kinetic analysis [19]. In the present paper, a two level description of the underlying events is given: (i) a logical explanation based on thorough inspection of raw experimental data and (ii) a profound successive kinetic analysis using modern mathematical modelling principles.
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2. Materials and methods
2.1. Chemicals Acetylthiocholine (ATCh) and 5,5%-dithio-bis-nitrobenzoic acid (DTNB) were purchased from Sigma Chemicals Co. (St. Louis, USA), D-tubocurarine chloride (TC) from Fluka Chemie AG (Buchs, Switzerland) and methanesulfonylfluoride (MSF) was a product of SERVA Biochemica (Heidelberg, Germany). MSF stock solution was prepared in spectroquality acetone. Other substances were reagent grade. All experiments were done at 25 °C in 25 mM sodium phosphate buffer with pH 7.0.
2.2. Source of enzymes Truncated cDNA encoding soluble AChE of D. Melanogaster was expressed with the baculovirus system [20]. Secreted enzymes were purified and stabilized with 1 mg/ml BSA according to Estrada-Mondaca and Fournier [21]. The titration of the enzyme’s active sites was carried out using 7-(methylethoxyphosphinyloxy)-1methylquinolinium iodide (MEPQ) synthesized as described by Levy and Ashani [22]. Residue numbering follows that of the precursor [23].
2.3. Kinetic experiments 2.3.1. Dilution experiments The time course of irreversible AChE inhibition by methanesulfonylfluoride was determined by measuring residual enzyme activity after various times of incubation in the absence and presence of TC, according to the dilution method of Aldridge [24]. The enzyme, in the presence of seven TC concentrations (0–500 mM), was incubated for 0.5– 20 min (eight time points on the average) with five different MSF concentrations (62.5– 750 mM). After a dilution of 300 times, the remaining enzyme activity was determined spectrophotometrically according to Ellman et al. [25]. Each measurement was repeated at least twice to obtain : 560 data points for the wild type enzyme and the W359L mutant, respectively. We added in the incubation mixture 5% of acetone. 2.3.2. Progress cur6e measurements In other different experiments, sulfonylation was followed on a stopped-flow apparatus in order to get important diagnostic information on the early stages of this process. Aliquots of two solutions, one containing only the enzyme and the other one ATCh, TC, DTNB, various concentrations of MSF and 6% of acetone were mixed together in the mixing chamber of the apparatus. The absorbance of the reaction mixture was recorded spectrophotometrically [25] at 412 nm. The measurements were performed in the presence of 1 mM substrate and with final MSF concentrations between 5.5 and 60 mM. The concentration of DTNB was 1 mM. In order to avoid possible product modulation, we stopped the measurement when : 60 mM concentration of the product was formed.
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Scheme 1.
2.4. Data analysis 2.4.1. Dilution experiments The sulfonylation of AChEs by MSF may be represented by Scheme 1 [16], where E is free enzyme, EMSF represents a reversible complex between the enzyme and MSF and E* is the inactivated enzyme. Ki is the equilibrium constant and k2 is the first order sulfonylation rate constant. It has been shown that Scheme 1 gives the following equation [16]: ln
[E]t k2·[MSF]·t =− [E]0 [MSF] +Ki
(1)
where [E]0 is the total enzyme concentration and [E]t is the remaining enzyme concentration at any time. Due to the relatively rapid sulfonylation of Drosophila AChE in the investigated MSF concentration range, sulfonylation appeared as a single step process. Since higher MSF concentrations are technically not applicable we used, for the analysis, the following simplified reaction scheme which permits the same flux through the step as the full mechanism [26] as in Scheme 2, where ks is the second order sulfonylation rate constant. In this case, Eq. (1) also simplifies: ln
[E]t = −ks·[MSF]·t [E]0
(2)
To describe the methanesulfonylation of DmAChE in the presence of TC, the appropriate steps should be added in the reaction scheme. According to previously collected kinetic data [14], the binding of one TC molecule to the wild type DmAChE can be detected, but two molecules of TC bind to the W359L mutant. Therefore, we constructed a general kinetic scheme for the methanesulfonylation of DmAChE in the presence of TC, which foresees a double successive TC binding as in Scheme 3 where I represents TC. According to this scheme, we derived the equation for residual activity, assuming instantaneous establishment of equilibria that involve the binding of TC and slow sulfonylation of all enzyme species. Under the conditions where [MSF], [I] [E] the following equation for residual activity was obtained: [E]t =[E]0·e − ks[MSF]st where
[I] [I]2 +q K3 K3K7 [I] [I]2 1+ + K3 K3K7
1+p
k=
(3)
(4)
Scheme 2.
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Scheme 3.
The kinetic parameters ks, p and q (Scheme 3) were determined by fitting Eq. (3) simultaneously to all experimental data obtained in dilution experiments, with each individual tested enzyme (:560 data points, see Section 2). K3 and K7 were set as previously evaluated [14] (for values see Table 1).
2.4.2. Progress cur6es measurements It should be emphasized that in these experiments the enzyme in the reaction vessel can react with the substrate (ATCh), TC and MSF. The kinetic representation of such a complex reaction system must include all these interactions, but it should also be in accordance with the data from the precedent dilution experiments. We included therefore, the interaction of MSF with all non-acylated enzyme species. We also included fast forming reversible MSF complexes with these species, since the rapid stopped-flow technique provided us the relevant information on these steps (Scheme 4).
Scheme 4.
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In this scheme, M denotes MSF, the symbol EA stands for acylated enzyme, while S, P1 and P2 are acetylthiocholine, thiocholine and acetate, respectively. The progress curve equation valid for this reaction scheme was derived essentially as described previously [27], again assuming that concentrations of MSF and TC were several grades of magnitude higher than the enzyme concentration (the derivation is given in Appendix A).
Table 1 Characteristic constants for the interactions of Drosophila wild type and W359L mutant acetylcholinesterases with ATCh, TC and MSF according to Scheme 4 Wild type
W359L
k Pi k P3 K P1 K P2 aP bP K P3 K P4 cP dP K P5 K P6 eP fP K P7
1.1×108/M/s 978/s 4.16 mM 24.1 mM 0.067 0.193 0.252 mM 46.7 mM 0.365 0.037 1.16 mM 1.27 mM 0 0.00461 –
8.23×107/M/s 5527/s 8.79 mM 2.29 mM 0.015 0.00040 165 mM 487 mM 0.302 0.884 – – – – 32.1 mM
ks pks qks gk2/K9 ik2/K11 k2 K8 K9 K10 K11 K12 gk2 hk2 ik2 jk2
13.2/M/s* 15.6/M/s* – 45.8/M/sa 49.8/M/sa ? ? ? ? ? – ? ? ? –
14.9/M/s* 14.7/M/s* 17.8/M/s* 7.5/M/s – ? ? 62.9 9 1.1 mM 32.4 9 25.5 mM – 17.9 9 6.0 mM 0.47 9 0.02/s 0.48 9 0.01/s 0.32 9 0.02/s
Superscript P denotes the previously determined constants (Ref. [14]); (?) not possible to determine (alone standing equilibrium constants were set to , the ratios in Eq. (5) were substituted by the relation in Eq. (6)); (–) unneeded in the model (discarded in the corresponding equations). * From dilution experiments (SDB1%). a Not possible to separate constants.
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[Pt ]=
V0(1 − e − t)
151
(5)
The expressions for V0 and are also given in Appendix A. The evaluation of ten missing kinetic parameters in Scheme 4 (K8, K9, K10, K11, K12, k2, gk2, hk2, ik2, jk2) was performed by fitting the progress curve equation (Eq. (5)) to all experimental curves, setting previously determined parameters as constants (Table 1). In order to do this we used the relation [26] ks =
k2 Ki
(6)
It follows from dilution experiments and Scheme 4 that ks = k2/K8, pks = hk2/K10 and qks =jk2/K12. So, instead of ten, we had to evaluate only seven missing parameters (K9, K11, k2, gk2, hk2, ik2, jk2) from progress curves data. All calculations were performed on IBM-PC compatible computer running LINUX, using C translation of non-linear regression program, originaly written by Duggleby [28].
3. Results Dilution experiments are the basic approach for the determination of acylation rate constants of irreversible cholinesterases’ inhibitors. Briefly, initial rates measured at various preincubation times yield pseudo first order rate constants which upon plotting vs. MSF concentration give second order sulfonylation rate constant. Subsequently, the effect of various TC concentrations on this second-order sulfonyl-
Fig. 1. Simulation of the dependence of second order sulfonylation rate constant on the concentration of D-tubocurarine for the wild type ( ) and the W359L mutant () of D. melanogaster AChEs. ksim represents the product between sulfonylation rate constant ks and s (see Eq. (4)). Concentrations on X-axis are linear from 0 to 20 mM, and logarithmic from 20 to 500 mM.
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Fig. 2. Progress curves for the hydrolysis of acetylthiocholine catalyzed by the wild type and the W359L mutant of D. melanogaster AChEs in the absence and presence of D-tubocurarine and methanesulfonylfluoride. Measurements were performed at 1 mM substrate concentration in the presence of 3% acetone. In panels A and C the concentrations of D-tubocurarine are 0, in panels B and D are 0.2 mM. Methanesulfonylfluoride concentrations are 0, 5.5, 11.1, 30 and 60 mM.
ation rate constant is inspected. We, however, performed a simultaneous analysis of all residual activity data using equation with three independent variables: time, [MSF] and [TC] (Eq. (3)). For clarity, only final dependence, the second order sulfonylation rate constants vs. TC concentration, is simulated in Fig. 1, according to the evaluated parameters ks, p and q. Although the curves for both studied enzymes describe acceleration of methanesufonylation, one can clearly distinguish different shapes of the two curves. For WT DmAChE, it shows a hyperbolic increase of second order overall sulfonylation rate constant (ks in Scheme 2) as a function of TC concentration. Such a pattern points to a simple Michaelis–Menten like saturation mechanism. It should be emphasized that the shape of hyperbolic curve provides the information for the high affinity binding of only one TC molecule (K3) and consequently, the analysis yields only the corresponding parameters (see Table 1, parameters denoted with ‘– ’ were omitted). In contrast, the acceleration of methanesulfonylation in the W359L mutant follows a sigmoidal curve, which can only be explained by multiple binding of TC (K3, K7). Therefore, we have included in the analysis the successive binding of two TC molecules (Scheme 3). The results of progress curve experiments with the wild type and the W359L mutant
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of DmAChE are presented in Fig. 2. A targeted inspection of the curves for the two enzymes reveals three very important differences in the effect of TC on the time course of product formation during the sulfonylation. (i) The initial slopes of the curves in panels A and B, representing WT DmAChE are MSF independent, but the progress curves for the W359L mutant (panels C and D) show clear MSF dependence of initial slopes. (ii) The next clearly visible characteristic of progress curves is the opposite effect of TC on the two enzymes, that can be recognized from the slope of the uppermost lines (the curves in the absence of MSF are linear). While TC inhibits WT DmAChE, it increases the initial rate of the W359L mutant. (iii) Finally, the sulfonylation is more accelerated by TC in the WT enzyme, but goes faster in the W359L mutant in the absence of TC. These can be seen by comparing the curvatures of the corresponding progress curves (faster sulfonylation, more concave progress curves) and is reflected by the values of the kinetic parameters in Table 1.
4. Discussion Methanesulfonylation and the influence of various anti-cholinesterase agents on this process have been extensively studied with various cholinesterases. Usually, the dilution method proposed by Aldridge [24] which excludes the interference of the substrate was used to determine the reversible Michaelis–Menten complex formation and irreversible sulfonylation rate constant. With vertebrate AChEs as well as with BChEs, the acceleration of methanesulfonylation by various active site-directed ligands is due to increased affinity of the enzyme for a small inhibitor rather to speeding up the formation of a stable covalent enzyme-inhibitor complex [16]. In Drosophila AChE, however, dilution experiments do not distinguish the two steps in the reaction. Our data agree completely with Krupka’s results [15] for AChE from the brain of the house fly: very low affinity of MSF for these enzymes and rather fast sulfonylation rate do not permit to use saturating MSF concentration. Surprisingly, TC turned to be an accelerator rather an inhibitor of methanesulfonylation in the WT enzyme and the biphasic activation is seen in the W359L mutant (Fig. 1). To be able to explain these findings, we measured the simultaneous influence of MSF and TC on the hydrolysis of ATCh by both enzymes. Indeed, these experiments also confirmed a very low affinity of the WT enzyme for MSF, characterized by non-dependent initial rates in Fig. 2, panels A, B [29]. On the other hand, the data for the W359L mutant show MSF dependent initial rates, allowing us to discriminate two steps in the process of sulfonylation: rapid initial complex formation is followed by slow irreversible inactivation. Of course, the enlargement of the kinetic model required for the analysis is such, that several sets of parameters apply unless some are fixed. This is reasonable, since Scheme 4 includes reaction intermediates which theoretically appear as the reaction system gets more and more complex. Therefore, in the analysis we fixed those parameters that we had determined in the analysis of dilution measurements but also in an independent study in the absence of MSF [14]. As mentioned in Section 2.4, there were seven parameters to be evaluated from progress curves. According to the conclusions based on the
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inspection of the data in Fig. 1 (Section 3), only six parameters for the WT enzyme and five for the W359L mutant remained to be determined. Unfortunately, our progress curves for the WT enzyme could only provide the information for two parameters and for the W359L mutant four of five. The experiments in the presence of the substrate do not discriminate k2 and K8 (Scheme 4), because the high substrate affinities of free enzymes prevent their accumulation (see the values of K1). The dissociation constants for the initial binding of MSF to the WT enzyme also cannot be evaluated as the initial rates are MSF independent. However, from the values of dissociation constants for the binding of MSF to the W359L mutant and the corresponding first order sulfonylation rate constants we can anticipate that K8 is of the same order as K9, K10 or K12. In this case it is evident that with dilution experiments it is technically impossible to evaluate K8. We can speculate that a similar situation happens when the substrate reacts with the enzyme: high affinity at the modulation site masks low affinity formation of the MichaelisMenten complex. Additionally, a continuous affinity gradient in the main catalytic pathway results kinetically in an overall second order acylation step (ki ) in Scheme 4. We have shown that by using rapid kinetic equipment and in the presence of TC it is possible to follow early stages in methanesulfonylation of W359L Drosophila AChE. The pattern of progress curves revealed the existence of an initial addition complex which gradually accumulates with the rising MSF concentrations or by the addition of reversible peripheral ligand TC. An appropriate analysis enabled us to evaluate dissociation constants for enzyme–MSF complexes but unfortunately, these study and all our previous attempts did not provide direct evidence for the existence of an analogous enzyme-substrate complex (ES). At the end, an important virtual discrepancy should be discussed. From our experimental data it is evident that methanesulfonylation is accelerated in both tested enzymes (Fig. 1), but the activity towards ATCh is only increased in the W359L mutant (uppermost lines in Fig. 2 and see also Ref. [14]). The explanation most probably lays in the size of the ligand entering the gorge of various DmAChEs. Small ligands are enhanced in their action under the influence of peripheral inhibitors and the ligands which exceed a certain size are hindered. The actual sign of the effect, however, depends upon specific arrangements at the rim of the active site of each individual enzyme. A similar conclusion can be drawn from the action of triton X-100, another peripheral inhibitor of WT Drosophila AChE: small carbamates and organophosphates are accelerated in their action but the large are inhibited [30].
Acknowledgements This work was supported by the Ministry of Science and Technology of the Republic of Slovenia, Grant No. P3-8720-0381, and by grants from DRET (94084), IFREMER and CNRS (GRD 1105 and ACS-SV3). The authors are indebted to Mrs Nevenka Klenovsˇek-S& pat for her valuable and skillful technical assistance.
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Appendix A. Derivation of Eq. (5) in the main text Scheme 4 can be written according to Cha [31]:
n
ZX X Y x
Assuming realistically, rapid equilibria in all reversible steps, mass conservation law and n, x it follows: [X]= [E]l,
[Y] =[EA]i,
[Z]= [E]total − ([X]+ [Y]),
}[X]= x[Y]
The significance of all symbols is: } = ki [S]k/l,
x= k3h/i,
= k2m/l
and h =1 +
[I] [S][I] [S] +c +e K4 K2K6 K2
i =1 +
[S] [I] [S][I] + + K2 K4 K2K6
k = 1 +b l =1 + + m=
[S] [I] [S][I] +d +f K1 K3 K1K5
[S] [I] [S][I] [I]2 [M] [S][M] [I][M] [S][I][M] + + + + + + + K1 K3 K1K5 K3K7 K8 K1K9 K3K10 K1K5K11
[I]2[M] K3K7K12
[M] [S][M] [I][M] [S][I][M] [I]2[M] +g +h +i +j K8 K1K9 K3K10 K1K5K11 K3K7K12
The rate of irreversible inhibition is d[Z]/dt =[X] Using conservation law d[X]/dt = −[X]/(1 +}/x) and upon integration ln([X]t /[X]0) = −t/(1 + }/x) /(1 +}/x) represents from Eq. (5) in the main text and after the rearrangement kh k2 3 m kik = k hl [S]i + 3 kik The rate of product formation is d[P]/dt = }[X]t = }[X]0e − t = }[E]total/(1+ }/ x)e- t.
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Integration gives Eq. (5) from the main text, where V0 =
k3[E]total[S]h . k hl [S]i + 3 kik
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[17] M.R. Pavlicˇ , Mechanism of methanesulfonylation of cholinesterase and accelaration by some ammonium compounds, Period. Biol. 86 (1984) 343 – 347. [18] M. Zorko, M.R. Pavlicˇ , Multiple binding of D-tubocurarine to acetylcholinesterase, Biochem. Pharmacol. 35 (1986) 2287 –2296. [19] Q. Gibbson, Rapid reaction methods in biochemistry, in: A. Neuberger, L.L.M. Van Deenen (Eds.), Modern Physical Methods in Biochemistry, Part B, Elsevier, Amsterdam, 1983, pp. 65 – 84. [20] H. Chaabihi, D. Fournier, Y. Fedon, J.P. Bossy, M. Ravallec, G. Devauchelle, M. Cerutti, Biochemical characterization of Drosophila melanogaster acetylcholinesterase expressed by recombinant baculoviruses, Biochem. Biophys. Res. Comm. 203 (1994) 734 – 742. [21] S. Estrada-Mondaca, D. Fournier, Drosophila acetylcholinesterase: effect of post-translational [correction of post-traductional] modifications on the production in the baculovirus system and substrate metabolization, Prot. Exp. Purif. 12 (1998) 166 – 172. [22] D. Levy, Y. Ashani, Synthesis and in vitro properties of a powerful quaternary methylphosphonate inhibitor of acetylcholinesterase. A new marker in blood – brain barrier research, Biochem. Pharmacol. 35 (1986) 143 –146. [23] L.M.C. Hall, P. Spierer, The Ace locus of Drosophila melanogaster: structural gene for acetylcholinesterase with unusual 5% leader, Eur. Mol. Biol. Org. J. 5 (1986) 2949 – 2954. [24] W.N. Aldridge, E. Reiner, Enzyme inhibitors as substrates. Interaction of esterases with esters of organophosphorus and carbamic acids, North Holland, Amsterdam, 1972, p. XV +328. [25] G.L. Ellman, K.D. Courtney, V. Andres, R.M. Feathersone, A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol. 7 (1961) 88 – 95. [26] W.W. Cleland, Determining the chemical mechanisms of enzyme-catalyzed reactions by kinetic studies, Adv. Enzymol. Relat. Areas Mol. Biol. 45 (1977) 273 – 387. [27] J. Stojan, Equations for progress curves of some kinetic models of enzyme-single substrate single slow binding modifier system, J. Enzyme Inhibit. 13 (1998) 161 – 176. [28] R.G. Duggleby, Regression analysis of non-linear Arrhenius plots: an empirical model and the computer program, Comput. Biol. Med. 14 (1984) 447 – 455. [29] J.F. Morrison, C.T. Walsh, The behavior and significance of slow-binding enzyme inhibitors, Adv. Enzymol. Relat. Areas Mol. Biol. 61 (1988) 201 – 301. [30] V. Marcel, S. Estrada-Mondaca, F. Magne, J. Stojan, A. Klae´ be´ , D. Fournier, Exploration of the Drosophila acetylcholinesterase substrate activation site using a reversible inhibitor (Triton X-100) and mutated enzymes, J. Biol. Chem. 275 (2000) 11603 – 11609. [31] S. Cha, A simple method for derivation of rate equations for enzyme-catalyzed reactions under the rapid equilibrium assumption or combined assumptions of equilibrium and steady state, J. Biol. Chem. 243 (1968) 820 –825.
www.elsevier.com/locate/chembiont
Acceleration of Drosophila melanogaster acetylcholinesterase methanesulfonylation: peripheral ligand D-tubocurarine enhances the affinity for small methanesulfonylfluoride Marko Golicˇnik a, Didier Fournier b, Jure Stojan a,* a
Institute of Biochemistry, Medical Faculty, Uni6ersity of Ljubljana, Vrazo6 trg 2, 1000 Ljubljana, Slo6enia b Laboratoire d’Entomologie et Groupe de Chimie Biologie, Uni6ersite` Paul Sabatier, 31062 Toulouse, France Received 14 May 2001; received in revised form 28 May 2001; accepted 3 November 2001
Abstract D-Tubocurarine, a reversible peripheral inhibitor of cholinesterases accelerates methanesulfonylation of Drosophila melanogaster wild type and W359L mutant. The kinetic evaluation of the process was performed in a step-by-step analysis. The second order overall sulfonylation rate constants, determined from classical residual activity measurements, were used in the subsequent analysis of progress curves. The latter were obtained by measuring the hydrolysis of acetylthiocholine in a complex reaction system of enzyme, substrate, irreversible and reversible inhibitor. The underlying kinetic mechanisms, from such a complex data, could only be untangled by targeted inspection and successive incorporation of reaction steps for which experimental evidence existed. The study showed that the peripheral ligand D-tubocurarine, by binding at the entrance into the active site of the two investigated enzymes (Golicˇnik et al., Biochemistry 40 (2001) 1214), enhances the affinity for small methanesulfonylfluoride, rather to speeding up the formation of a stable covalent enzyme-inhibitor complex. The specific arrangements at the rim of the active site of each individual
Abbre6iations: AChE, acetylcholinesterase; ATCh, acetylthiocholine; BChE, butyrylcholinesterase; ChE, cholinesterase; DmAChE, Drosophila melanogaster acetylcholinesterase; DTNB, 5,5%-dithio-bisnitrobenzoic acid; MSF, methanesulfonylfluoride; TC, D-tubocurarine. * Corresponding author. Tel.: +386-1-5437651; fax: +386-1-5437641. E-mail address: [email protected] (J. Stojan). 0009-2797/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 2 7 9 7 ( 0 1 ) 0 0 2 9 4 - 0
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enzyme dictate the actual events which can be detected by kinetic means. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Acetylcholinesterase; Kinetic models; curves
D-tubocurarine;
Methanesulfonylfluoride; Progress
1. Introduction The catalytic mechanism and inhibition of ChEs have been extensively investigated [1] during the past decades. Recently, the crystal structure determination of AChEs from vertebrates and from Drosophila melanogaster [2–5] revealed the active site, :20 A, deep inside the core of the molecule, at the bottom of a narrow gorge. It seems that such penetrated catalytic site is responsible for their peculiar kinetic behavior. All ChEs, either native or various specifically mutated enzymes, exhibit deviations from Michaelis–Menten hyperbolic kinetics. While moderate positive homotropic pseudo-cooperativity at intermediate substrate concentrations is present only in certain enzymes, the inhibition by excess substrate is seldom missing [6– 8]. Several kinetic models were proposed to describe non Michaelis– Menten behavior of ChEs [9,10], but they were able only to explain one deviation. Recently, a six parameter kinetic model valid for all ChEs was introduced [11]. It can describe both mentioned deviations and by an appropriate extension, also the data in the presence of various reversible or irreversible active site directed inhibitors [7,12– 14]. It was also found that D-tubocurarine (TC), an inhibitor, whose shape and size prevent the interaction with the active site at the bottom of the gorge, can enhance the substrate hydrolysis at high substrate concentrations in the case of Drosophila W359L AChE mutant [14]. This paradox has been explained as inhibition, less effective than the substrate inhibition itself. To obtain further information on the behavior of relatively narrow active site gorge in Drosophila AChE (50% less space as in Torpedo enzyme [5]) we investigated the methanesulfonylation of wild type DmAChE and the W359L mutant in the absence and presence of TC. Methanesulfonylfluoride (MSF), a poweful irreversible anticholinesterase agent, sulfonylates insect AChE with faster rates than vertebrate enzymes or BChEs [15–17]. It is also known that the action of this tiny sulfonate on vertebrate AChEs is accelerated by active site directed inhibitors like tetramethylammonium and decamethonium [16] but TC is an inhibitor of methanesulfonylation [18]. The fact, that TC can also activate W359L DmAChE, lead us to investigate its action on the methanesulfonylation of wild type DmAChE and the W359L mutant. We have performed the experiments, where the substrate hydrolysis was followed in the presence of two inhibitors, TC and MSF. Such experiments represent a very complex system, and the obtained data require sophisticated step-by-step kinetic analysis [19]. In the present paper, a two level description of the underlying events is given: (i) a logical explanation based on thorough inspection of raw experimental data and (ii) a profound successive kinetic analysis using modern mathematical modelling principles.
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2. Materials and methods
2.1. Chemicals Acetylthiocholine (ATCh) and 5,5%-dithio-bis-nitrobenzoic acid (DTNB) were purchased from Sigma Chemicals Co. (St. Louis, USA), D-tubocurarine chloride (TC) from Fluka Chemie AG (Buchs, Switzerland) and methanesulfonylfluoride (MSF) was a product of SERVA Biochemica (Heidelberg, Germany). MSF stock solution was prepared in spectroquality acetone. Other substances were reagent grade. All experiments were done at 25 °C in 25 mM sodium phosphate buffer with pH 7.0.
2.2. Source of enzymes Truncated cDNA encoding soluble AChE of D. Melanogaster was expressed with the baculovirus system [20]. Secreted enzymes were purified and stabilized with 1 mg/ml BSA according to Estrada-Mondaca and Fournier [21]. The titration of the enzyme’s active sites was carried out using 7-(methylethoxyphosphinyloxy)-1methylquinolinium iodide (MEPQ) synthesized as described by Levy and Ashani [22]. Residue numbering follows that of the precursor [23].
2.3. Kinetic experiments 2.3.1. Dilution experiments The time course of irreversible AChE inhibition by methanesulfonylfluoride was determined by measuring residual enzyme activity after various times of incubation in the absence and presence of TC, according to the dilution method of Aldridge [24]. The enzyme, in the presence of seven TC concentrations (0–500 mM), was incubated for 0.5– 20 min (eight time points on the average) with five different MSF concentrations (62.5– 750 mM). After a dilution of 300 times, the remaining enzyme activity was determined spectrophotometrically according to Ellman et al. [25]. Each measurement was repeated at least twice to obtain : 560 data points for the wild type enzyme and the W359L mutant, respectively. We added in the incubation mixture 5% of acetone. 2.3.2. Progress cur6e measurements In other different experiments, sulfonylation was followed on a stopped-flow apparatus in order to get important diagnostic information on the early stages of this process. Aliquots of two solutions, one containing only the enzyme and the other one ATCh, TC, DTNB, various concentrations of MSF and 6% of acetone were mixed together in the mixing chamber of the apparatus. The absorbance of the reaction mixture was recorded spectrophotometrically [25] at 412 nm. The measurements were performed in the presence of 1 mM substrate and with final MSF concentrations between 5.5 and 60 mM. The concentration of DTNB was 1 mM. In order to avoid possible product modulation, we stopped the measurement when : 60 mM concentration of the product was formed.
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Scheme 1.
2.4. Data analysis 2.4.1. Dilution experiments The sulfonylation of AChEs by MSF may be represented by Scheme 1 [16], where E is free enzyme, EMSF represents a reversible complex between the enzyme and MSF and E* is the inactivated enzyme. Ki is the equilibrium constant and k2 is the first order sulfonylation rate constant. It has been shown that Scheme 1 gives the following equation [16]: ln
[E]t k2·[MSF]·t =− [E]0 [MSF] +Ki
(1)
where [E]0 is the total enzyme concentration and [E]t is the remaining enzyme concentration at any time. Due to the relatively rapid sulfonylation of Drosophila AChE in the investigated MSF concentration range, sulfonylation appeared as a single step process. Since higher MSF concentrations are technically not applicable we used, for the analysis, the following simplified reaction scheme which permits the same flux through the step as the full mechanism [26] as in Scheme 2, where ks is the second order sulfonylation rate constant. In this case, Eq. (1) also simplifies: ln
[E]t = −ks·[MSF]·t [E]0
(2)
To describe the methanesulfonylation of DmAChE in the presence of TC, the appropriate steps should be added in the reaction scheme. According to previously collected kinetic data [14], the binding of one TC molecule to the wild type DmAChE can be detected, but two molecules of TC bind to the W359L mutant. Therefore, we constructed a general kinetic scheme for the methanesulfonylation of DmAChE in the presence of TC, which foresees a double successive TC binding as in Scheme 3 where I represents TC. According to this scheme, we derived the equation for residual activity, assuming instantaneous establishment of equilibria that involve the binding of TC and slow sulfonylation of all enzyme species. Under the conditions where [MSF], [I] [E] the following equation for residual activity was obtained: [E]t =[E]0·e − ks[MSF]st where
[I] [I]2 +q K3 K3K7 [I] [I]2 1+ + K3 K3K7
1+p
k=
(3)
(4)
Scheme 2.
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Scheme 3.
The kinetic parameters ks, p and q (Scheme 3) were determined by fitting Eq. (3) simultaneously to all experimental data obtained in dilution experiments, with each individual tested enzyme (:560 data points, see Section 2). K3 and K7 were set as previously evaluated [14] (for values see Table 1).
2.4.2. Progress cur6es measurements It should be emphasized that in these experiments the enzyme in the reaction vessel can react with the substrate (ATCh), TC and MSF. The kinetic representation of such a complex reaction system must include all these interactions, but it should also be in accordance with the data from the precedent dilution experiments. We included therefore, the interaction of MSF with all non-acylated enzyme species. We also included fast forming reversible MSF complexes with these species, since the rapid stopped-flow technique provided us the relevant information on these steps (Scheme 4).
Scheme 4.
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In this scheme, M denotes MSF, the symbol EA stands for acylated enzyme, while S, P1 and P2 are acetylthiocholine, thiocholine and acetate, respectively. The progress curve equation valid for this reaction scheme was derived essentially as described previously [27], again assuming that concentrations of MSF and TC were several grades of magnitude higher than the enzyme concentration (the derivation is given in Appendix A).
Table 1 Characteristic constants for the interactions of Drosophila wild type and W359L mutant acetylcholinesterases with ATCh, TC and MSF according to Scheme 4 Wild type
W359L
k Pi k P3 K P1 K P2 aP bP K P3 K P4 cP dP K P5 K P6 eP fP K P7
1.1×108/M/s 978/s 4.16 mM 24.1 mM 0.067 0.193 0.252 mM 46.7 mM 0.365 0.037 1.16 mM 1.27 mM 0 0.00461 –
8.23×107/M/s 5527/s 8.79 mM 2.29 mM 0.015 0.00040 165 mM 487 mM 0.302 0.884 – – – – 32.1 mM
ks pks qks gk2/K9 ik2/K11 k2 K8 K9 K10 K11 K12 gk2 hk2 ik2 jk2
13.2/M/s* 15.6/M/s* – 45.8/M/sa 49.8/M/sa ? ? ? ? ? – ? ? ? –
14.9/M/s* 14.7/M/s* 17.8/M/s* 7.5/M/s – ? ? 62.9 9 1.1 mM 32.4 9 25.5 mM – 17.9 9 6.0 mM 0.47 9 0.02/s 0.48 9 0.01/s 0.32 9 0.02/s
Superscript P denotes the previously determined constants (Ref. [14]); (?) not possible to determine (alone standing equilibrium constants were set to , the ratios in Eq. (5) were substituted by the relation in Eq. (6)); (–) unneeded in the model (discarded in the corresponding equations). * From dilution experiments (SDB1%). a Not possible to separate constants.
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[Pt ]=
V0(1 − e − t)
151
(5)
The expressions for V0 and are also given in Appendix A. The evaluation of ten missing kinetic parameters in Scheme 4 (K8, K9, K10, K11, K12, k2, gk2, hk2, ik2, jk2) was performed by fitting the progress curve equation (Eq. (5)) to all experimental curves, setting previously determined parameters as constants (Table 1). In order to do this we used the relation [26] ks =
k2 Ki
(6)
It follows from dilution experiments and Scheme 4 that ks = k2/K8, pks = hk2/K10 and qks =jk2/K12. So, instead of ten, we had to evaluate only seven missing parameters (K9, K11, k2, gk2, hk2, ik2, jk2) from progress curves data. All calculations were performed on IBM-PC compatible computer running LINUX, using C translation of non-linear regression program, originaly written by Duggleby [28].
3. Results Dilution experiments are the basic approach for the determination of acylation rate constants of irreversible cholinesterases’ inhibitors. Briefly, initial rates measured at various preincubation times yield pseudo first order rate constants which upon plotting vs. MSF concentration give second order sulfonylation rate constant. Subsequently, the effect of various TC concentrations on this second-order sulfonyl-
Fig. 1. Simulation of the dependence of second order sulfonylation rate constant on the concentration of D-tubocurarine for the wild type ( ) and the W359L mutant () of D. melanogaster AChEs. ksim represents the product between sulfonylation rate constant ks and s (see Eq. (4)). Concentrations on X-axis are linear from 0 to 20 mM, and logarithmic from 20 to 500 mM.
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Fig. 2. Progress curves for the hydrolysis of acetylthiocholine catalyzed by the wild type and the W359L mutant of D. melanogaster AChEs in the absence and presence of D-tubocurarine and methanesulfonylfluoride. Measurements were performed at 1 mM substrate concentration in the presence of 3% acetone. In panels A and C the concentrations of D-tubocurarine are 0, in panels B and D are 0.2 mM. Methanesulfonylfluoride concentrations are 0, 5.5, 11.1, 30 and 60 mM.
ation rate constant is inspected. We, however, performed a simultaneous analysis of all residual activity data using equation with three independent variables: time, [MSF] and [TC] (Eq. (3)). For clarity, only final dependence, the second order sulfonylation rate constants vs. TC concentration, is simulated in Fig. 1, according to the evaluated parameters ks, p and q. Although the curves for both studied enzymes describe acceleration of methanesufonylation, one can clearly distinguish different shapes of the two curves. For WT DmAChE, it shows a hyperbolic increase of second order overall sulfonylation rate constant (ks in Scheme 2) as a function of TC concentration. Such a pattern points to a simple Michaelis–Menten like saturation mechanism. It should be emphasized that the shape of hyperbolic curve provides the information for the high affinity binding of only one TC molecule (K3) and consequently, the analysis yields only the corresponding parameters (see Table 1, parameters denoted with ‘– ’ were omitted). In contrast, the acceleration of methanesulfonylation in the W359L mutant follows a sigmoidal curve, which can only be explained by multiple binding of TC (K3, K7). Therefore, we have included in the analysis the successive binding of two TC molecules (Scheme 3). The results of progress curve experiments with the wild type and the W359L mutant
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of DmAChE are presented in Fig. 2. A targeted inspection of the curves for the two enzymes reveals three very important differences in the effect of TC on the time course of product formation during the sulfonylation. (i) The initial slopes of the curves in panels A and B, representing WT DmAChE are MSF independent, but the progress curves for the W359L mutant (panels C and D) show clear MSF dependence of initial slopes. (ii) The next clearly visible characteristic of progress curves is the opposite effect of TC on the two enzymes, that can be recognized from the slope of the uppermost lines (the curves in the absence of MSF are linear). While TC inhibits WT DmAChE, it increases the initial rate of the W359L mutant. (iii) Finally, the sulfonylation is more accelerated by TC in the WT enzyme, but goes faster in the W359L mutant in the absence of TC. These can be seen by comparing the curvatures of the corresponding progress curves (faster sulfonylation, more concave progress curves) and is reflected by the values of the kinetic parameters in Table 1.
4. Discussion Methanesulfonylation and the influence of various anti-cholinesterase agents on this process have been extensively studied with various cholinesterases. Usually, the dilution method proposed by Aldridge [24] which excludes the interference of the substrate was used to determine the reversible Michaelis–Menten complex formation and irreversible sulfonylation rate constant. With vertebrate AChEs as well as with BChEs, the acceleration of methanesulfonylation by various active site-directed ligands is due to increased affinity of the enzyme for a small inhibitor rather to speeding up the formation of a stable covalent enzyme-inhibitor complex [16]. In Drosophila AChE, however, dilution experiments do not distinguish the two steps in the reaction. Our data agree completely with Krupka’s results [15] for AChE from the brain of the house fly: very low affinity of MSF for these enzymes and rather fast sulfonylation rate do not permit to use saturating MSF concentration. Surprisingly, TC turned to be an accelerator rather an inhibitor of methanesulfonylation in the WT enzyme and the biphasic activation is seen in the W359L mutant (Fig. 1). To be able to explain these findings, we measured the simultaneous influence of MSF and TC on the hydrolysis of ATCh by both enzymes. Indeed, these experiments also confirmed a very low affinity of the WT enzyme for MSF, characterized by non-dependent initial rates in Fig. 2, panels A, B [29]. On the other hand, the data for the W359L mutant show MSF dependent initial rates, allowing us to discriminate two steps in the process of sulfonylation: rapid initial complex formation is followed by slow irreversible inactivation. Of course, the enlargement of the kinetic model required for the analysis is such, that several sets of parameters apply unless some are fixed. This is reasonable, since Scheme 4 includes reaction intermediates which theoretically appear as the reaction system gets more and more complex. Therefore, in the analysis we fixed those parameters that we had determined in the analysis of dilution measurements but also in an independent study in the absence of MSF [14]. As mentioned in Section 2.4, there were seven parameters to be evaluated from progress curves. According to the conclusions based on the
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inspection of the data in Fig. 1 (Section 3), only six parameters for the WT enzyme and five for the W359L mutant remained to be determined. Unfortunately, our progress curves for the WT enzyme could only provide the information for two parameters and for the W359L mutant four of five. The experiments in the presence of the substrate do not discriminate k2 and K8 (Scheme 4), because the high substrate affinities of free enzymes prevent their accumulation (see the values of K1). The dissociation constants for the initial binding of MSF to the WT enzyme also cannot be evaluated as the initial rates are MSF independent. However, from the values of dissociation constants for the binding of MSF to the W359L mutant and the corresponding first order sulfonylation rate constants we can anticipate that K8 is of the same order as K9, K10 or K12. In this case it is evident that with dilution experiments it is technically impossible to evaluate K8. We can speculate that a similar situation happens when the substrate reacts with the enzyme: high affinity at the modulation site masks low affinity formation of the MichaelisMenten complex. Additionally, a continuous affinity gradient in the main catalytic pathway results kinetically in an overall second order acylation step (ki ) in Scheme 4. We have shown that by using rapid kinetic equipment and in the presence of TC it is possible to follow early stages in methanesulfonylation of W359L Drosophila AChE. The pattern of progress curves revealed the existence of an initial addition complex which gradually accumulates with the rising MSF concentrations or by the addition of reversible peripheral ligand TC. An appropriate analysis enabled us to evaluate dissociation constants for enzyme–MSF complexes but unfortunately, these study and all our previous attempts did not provide direct evidence for the existence of an analogous enzyme-substrate complex (ES). At the end, an important virtual discrepancy should be discussed. From our experimental data it is evident that methanesulfonylation is accelerated in both tested enzymes (Fig. 1), but the activity towards ATCh is only increased in the W359L mutant (uppermost lines in Fig. 2 and see also Ref. [14]). The explanation most probably lays in the size of the ligand entering the gorge of various DmAChEs. Small ligands are enhanced in their action under the influence of peripheral inhibitors and the ligands which exceed a certain size are hindered. The actual sign of the effect, however, depends upon specific arrangements at the rim of the active site of each individual enzyme. A similar conclusion can be drawn from the action of triton X-100, another peripheral inhibitor of WT Drosophila AChE: small carbamates and organophosphates are accelerated in their action but the large are inhibited [30].
Acknowledgements This work was supported by the Ministry of Science and Technology of the Republic of Slovenia, Grant No. P3-8720-0381, and by grants from DRET (94084), IFREMER and CNRS (GRD 1105 and ACS-SV3). The authors are indebted to Mrs Nevenka Klenovsˇek-S& pat for her valuable and skillful technical assistance.
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Appendix A. Derivation of Eq. (5) in the main text Scheme 4 can be written according to Cha [31]:
n
ZX X Y x
Assuming realistically, rapid equilibria in all reversible steps, mass conservation law and n, x it follows: [X]= [E]l,
[Y] =[EA]i,
[Z]= [E]total − ([X]+ [Y]),
}[X]= x[Y]
The significance of all symbols is: } = ki [S]k/l,
x= k3h/i,
= k2m/l
and h =1 +
[I] [S][I] [S] +c +e K4 K2K6 K2
i =1 +
[S] [I] [S][I] + + K2 K4 K2K6
k = 1 +b l =1 + + m=
[S] [I] [S][I] +d +f K1 K3 K1K5
[S] [I] [S][I] [I]2 [M] [S][M] [I][M] [S][I][M] + + + + + + + K1 K3 K1K5 K3K7 K8 K1K9 K3K10 K1K5K11
[I]2[M] K3K7K12
[M] [S][M] [I][M] [S][I][M] [I]2[M] +g +h +i +j K8 K1K9 K3K10 K1K5K11 K3K7K12
The rate of irreversible inhibition is d[Z]/dt =[X] Using conservation law d[X]/dt = −[X]/(1 +}/x) and upon integration ln([X]t /[X]0) = −t/(1 + }/x) /(1 +}/x) represents from Eq. (5) in the main text and after the rearrangement kh k2 3 m kik = k hl [S]i + 3 kik The rate of product formation is d[P]/dt = }[X]t = }[X]0e − t = }[E]total/(1+ }/ x)e- t.
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Integration gives Eq. (5) from the main text, where V0 =
k3[E]total[S]h . k hl [S]i + 3 kik
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