Eur. J. Biochem. 269, 1154–1161 (2002) (cid:211) FEBS 2002
Concentration-dependent reversible activation-inhibition of human butyrylcholinesterase by tetraethylammonium ion
Jure Stojan1, Marko Golicˇ nik1, Marie-The´ re` se Froment2, Francois Estour2 and Patrick Masson2 1Institute of Biochemistry, Medical Faculty, University of Ljubljana, Slovenia; 2Centre de Recherches du Service de Sante´ des Arme´es, Unite´ d’Enzymologie, La Tronche, France
pH 7.0 in the presence of tetraethylammonium (TEA). It appears that human enzymes with more intact structure of the PAS show more prominent activation phenomenon. The following explanation has been put forward: TEA competes with the substrate at the peripheral site thus inhibiting the substrate hydrolysis at the CS. As the inhibition by TEA is less effective than the substrate inhibition itself, it mimics activation. At the concentrations around 40 mM, well within the range of TEA competition at both substrate binding sites, it lowers the activity of all tested enzymes.
cholinesterases;
tetraalkylammonium com-
Keywords: pounds; kinetics; reaction mechanism.
Tetraalkylammonium (TAA) salts are well known reversible inhibitors of cholinesterases. However, at concentrations around 10 mM, they have been found to activate the hydrolysis of positively charged substrates, catalyzed by wild-type human butyrylcholinesterase (EC 3.1.1.8) [Erdoes, E.G., Foldes, F.F., Zsigmond, E.K., Baart, N. & Zwartz, J.A. (1958) Science 128, 92]. The present study was under- taken to determine whether the peripheral anionic site (PAS) of human BuChE (Y332, D70) and/or the catalytic substrate binding site (CS) (W82, A328) are involved in this phenom- enon. For this purpose, the kinetics of butyrylthiocholine (BTC) hydrolysis by wild-type human BuChE, by selected mutants and by horse BuChE was carried out at 25 (cid:176)C and
importance:
Acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholi- nesterase (BuChE; EC 3.1.1.8) are closely related serine hydrolases [1]. No clear physiological function has yet been assigned to BuChE; it appears to play a role in neurogenesis and neural disorders [2] and it is of pharmacological and toxicological it hydrolyses numerous ester like AChE is inhibited by containing drugs [3–5] and, similar compounds. Thus, an understanding of BuChE catalysis and inhibition mechanisms is of paramount importance, especially for the research of new treatments against organophosphate and carbamate poisoning [6], i.e. for the design of new reactivators of phosphylated choli- nesterases and of mutated enzymes capable of hydrolyzing organophosphates or carbamates [7].
and it is slightly inhibited by excess BTC [8,9]. In contrast, AChE shows only negative pseudo-cooperativity at high acetylthiocholine (ATC) concentrations [10,11]. Further- more, some cationic ligands, such as TAA salts, choline, and also uncharged trialkylammonium compounds act as acti- vators or inhibitors, depending on both the concentration of the ligand and the substrate [12], the solvent and the presence of cosolvent [13,14]. The goal of this work was to locate the site of interaction between BuChE and tetra- alkylammonium (TAA) salts responsible for activation and to reach a mechanistic explanation of the phenomenon. In particular, tetraethylammonium (TEA) at the concentra- tions above 40 mM, reversibly inhibits the wild-type human BuChE, but at the concentrations around 10 mM it accelerates BuChE catalyzed hydrolysis of positively charged substrates.
BuChE catalysis of charged substrates and inhibition by charged ligands are complex reactions. In particular, they show homotropic and heterotropic pseudo-cooperative effects. At intermediate substrate concentrations BuChE hydrolyses its optimal substrate BTC with rates exceeding those expected by simple Michaelis–Menten dependence
The active site serine, S198 in human BuChE, is located at the bottom of a 20-A˚ deep cleft [15,16]. Ligands can bind on two distinct sites: a peripheral anionic site (PAS) located at the mouth of the active site cleft, regarded as the substrate/ ligand recognition site, and the (cid:212)anionic(cid:213) subsite of the CS [1,15]. Residues D70 (D72, Torpedo AChE numbering) and Y332 (Y334) are the key elements of the PAS in human BuChE [9,17]. For positively charged substrates, the CS is W82 (W84) where the binding occurs through p–cation interactions [7,9,16]. Residue A328 (F330), which is also a part of this hydrophobic subsite, was found to be involved in substrate/inhibitor binding, too [18]. To determine the site involved in the effect of TAA salts, we carried out the steady-state and progress curve analysis of BTC hydrolysis by recombinant wild-type human BuChE, by four selected mutants (Y332A/D70G, Y332D/D70Y, W82A, A328Y) and by commercial horse serum BuChE in the presence of TEA. Additionally, we tested the hydrolytic activity toward BTC of the mixture between horse enzyme and W82A
Correspondence to J. Stojan, Institute of Biochemistry, Medical Faculty, Vrazov trg 2, 1000 Ljubljana, Slovenia. Fax: + 386 1 5437641, Tel.: + 386 1 5437649, E-mail: stojan@ibmi.mf.uni-lj.si Abbreviations: AChE, acetylcholinesterase; BuChE, butyrylcholine- sterase; CS, catalytic site; PAS, peripheral anionic site; TAA, tetra- alkylamonium; TEA, tetraethylammonium; BTC, butyrylthiocholine; DTNB, dithiobisnitrobenzoic acid; ATC, acetylthiocholine. Note: a coordinate file of the homology built model of human wild- type butyrylcholin-esterase with docked TEA can be downloaded from http://www2.mf.uni-lj.si/(cid:24)stojan/stojan.html (Received 27 September 2001, revised 17 December 2001, accepted 19 December 2001)
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to explain kinetically such observations, we analyzed the data according to the six parameter model (Scheme 1) introduced previously [21].
recombinant human enzyme in order to see whether such a low activity mutant still can tie up substrate by binding it with high affinity.
ak3 SEA (cid:135) P1 (cid:255)!
SE (cid:135) P2
M A T E R I A L S A N D M E T H O D S
Chemicals and equipment
bki S (cid:135) SE (cid:255)! "# K1 S (cid:135)
"# K2 S (cid:135)
ki
S (cid:135) E (cid:255)!
k3 EA (cid:135) P1 (cid:255)!
E (cid:135) P2
Scheme 1.
Butyrylthiocholine and buffer components of biochemical grade were purchased from Sigma Chemical Co. (St Louis, MO, USA). Tetraethylammonium chloride was obtained from Fluka (Buchs, Switzerland), chlorpyrifos-oxon (CPO) was from Dow Chemical Co. (Indianapolis, IN, USA) and diisopropylfluorophosphate (DFP) was from Acrosorganics France (Noisy-le-Grand, France).
In this scheme, E is the free enzyme, EA the acylated enzyme, while SE and SEA represent the complexes with the substrate molecule bound at the modulation site. The products P1 and P2 are thiocholine and butyrate, respect- ively. K1 and K2 are the equilibrium constants for the substrate binding to the nonproductive site, while ki and k3 are the rate constants. a and b are the partitioning ratios.
Mixed equilibrium and steady-state assumptions [22] in
Classical kinetic experiments were performed on a Beckman DU-7500 diode array spectrophotometer. Rapid kinetic measurements were curried out on a Hi-Tech (Salisbury, UK) PQ/SF-53 stopped-flow apparatus connec- ted to a SU-40 spectrophotometer and Apple E-II micro- computer, equipped with high speed AD converter.
the derivation give the following rate equation:
(cid:17)
Enzyme sources
(cid:133)1(cid:134)
v0 (cid:136)
(cid:1)
k3
(cid:17)
(cid:16)
K2
K1
(cid:135)
(cid:1)(cid:255)1 (cid:135) a(cid:137)S(cid:138) (cid:1)
(cid:137)S(cid:138) 1 (cid:135) (cid:137)S(cid:138) K2
(cid:255)1 (cid:135) b(cid:137)S(cid:138)
(cid:16) E0k3(cid:137)S(cid:138) 1 (cid:135) a (cid:137)S(cid:138) K2 (cid:255)1 (cid:135) a(cid:137)S(cid:138) ki
K1
The corresponding kinetic parameters were evaluated by fitting this equation to the initial rate data obtained in the experiments using recombinant wild-type and the four mutated human enzymes as well as the horse enzyme.
Recombinant wild-type and mutant human BuChEs. Two amino-acid residues (D70 and Y332) in the PAS and two (W82 and A328) in the CS, known to play a role in the binding of positively charged ligands and in inhibition control of BuChE, were selected. The BuChE gene was mutated to make the single mutants W82A and A328Y and the double mutants Y332A/D70G and Y332D/D70Y. Wild-type and mutant enzymes were expressed in stably transfected CHO cells as previously described [9].
For the analysis of the experiments in the presence of TEA we made an extension of the model to allow the competition between TEA and BTC at both substrate binding sites and consequently also the occupation of the two sites by two TEA molecules (Scheme 2).
I (cid:135) SE (cid:135) S (cid:255)!
ak3 SE (cid:135) P2
"# K1 S (cid:135)
I (cid:135) E (cid:135) S (cid:255)!
k3 E (cid:135) P2
Horse Serum BuChE. This was purchased from Worth- ington. It was chosen because the major difference between human and horse BuChEs at the cleft entrance is an additional negative charge in the loop opposite to the omega loop. As the two enzymes have 90% identical amino-acid residues [19], we may see the horse enzyme, in terms of peripheral site differences, as a human A277V/G283D/ P285L triple mutant (W279, D283, I287 homologous, in Torpedo AChE).
Kinetic experiments and data analysis
K7 SEI ¢ "# S (cid:135) K5 EI ¢ (cid:135) I "#
bki SEA (cid:135) P1 (cid:255)! "# K2 S (cid:135) ki EA (cid:135) P1 (cid:255)! (cid:135) I "# K4
(cid:135) I "# K3
K6 IEI ¢
dki I (cid:135) IE (cid:135) S (cid:255)!
ck3 IEA (cid:135) P1 (cid:255)!
IE (cid:135) P2
Scheme 2.
In this scheme, I stands for TEA and c and d are again the corresponding partitioning ratios.
An analogous derivation as described for Scheme 1 leads
to the following rate equation:
(cid:17)
v0 (cid:136)
(cid:1)
k3
(cid:17)
(cid:135)c (cid:137)I(cid:138) K4
K1
K2
(cid:135) (cid:137)S(cid:138)(cid:137)I(cid:138) K1 K7
(cid:135) (cid:137)I(cid:138)2 K3K6
Hydrolysis of BTC was measured by Ellman’s method in 0.1 M potassium phosphate buffer, pH 7.0 at 25 (cid:176)C [20]. The substrate concentration ranges depended on the human enzyme mutants: 0.6 lM to 90 mM for the wild-type, 0.015– 100 mM for double mutants, 3–100 mM for W82A mutant and 0.015–3 mM for A328Y mutant; the substrate concen- trations used with the commercial horse serum BuChE were between 0.05 and 10 mM. The concentration of enzyme active sites E0, was determined by the method of residual activity using CPO and/or DFP as the titrating reagents. Inhibition experiments were carried out at TEA concentra- tions from 0 to 100 mM.
(cid:135)
(cid:135) (cid:137)I(cid:138) K5 (cid:1)
(cid:16) (cid:137)S(cid:138) 1 (cid:135) (cid:137)S(cid:138) K2
(cid:135) (cid:137)I(cid:138) K4
(cid:16) E0k3(cid:137)S(cid:138) 1 (cid:135) a(cid:137)S(cid:138) (cid:135) c (cid:137)I(cid:138) K2 K4 (cid:1)(cid:255)1(cid:135) (cid:137)S(cid:138) (cid:255)1(cid:135)a(cid:137)S(cid:138) (cid:255)1(cid:135)b(cid:137)S(cid:138) ki
(cid:135) (cid:137)I(cid:138) K3 (cid:135)d (cid:137)I(cid:138) K3
K1
(cid:133)2(cid:134)
Initial rate data in the absence of TEA showed, in most enzymes, deviations from Michaelis–Menten kinetics: at intermediate substrate concentrations an apparent activa- tion is seen, while inhibition is detectable at the substrate concentrations approaching maximum solubility. In order
Final evaluation of kinetic constants relevant for each individual enzyme was carried out by fitting this equation
1156 J. Stojan et al. (Eur. J. Biochem. 269)
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same amount of buffer. The mixture was prepared by mixing together the aliquots without adding buffer. In this way the mixture contained the same concentrations of the two enzymes as the solution of each individual enzyme. The activities of the three solutions were now tested at various substrate concentrations in the range from 5 lM to 75 mM. The concentration of W82A was 16 lM and that of the horse enzyme was 10 nM. The experimental conditions were the same as in classical initial rate measurements (pH 7.0 and 25 (cid:176)C).
simultaneously to the data in the absence and presence of TEA. We started with fixed values of parameters obtained from the analysis without the inhibitor, to determine rough estimates of TEA binding parameters. Eventually, all parameters were released to achieve the best accordance between theoretical curves and the data. It should be stressed that some parameters in the reaction Scheme are closely related to certain parts of data. For instance, the parameter a set to zero, would denote complete blocking of deacylation. Solubility maximum of the substrate, however, only allows to statistically anticipate the real value unless the clear plateau is reached [23].
We analyzed the data for W82A by fitting a system of stiff differential equations, that described the six-parameter model in Fig. 1 under combined steady-state and equilib- rium assumptions (cf. [24]) to the data of all experimental progress curves simultaneously. Initial rates were obtained as numerical derivatives at zero time of each progress curve. The same procedure was used to evaluate data obtained with commercial horse BuChE. The initial rates at various substrate concentrations for the mixture of the two enzymes (W82A mutant and horse serum BuChE) were determined analytically by fitting the equation for single exponential curve to each individual progress curve and than taking derivatives at time zero.
Model building
The initial rate data for the W82A mutant differed substantially from the data for other enzymes. It appeared that the hydrolysis of BTC by this enzyme obeyed Michaelis–Menten kinetics. In order to investigate the kinetics of this mutant more closely, we measured the hydrolysis of BTC catalyzed by the W82A mutant, by the horse enzyme and by the mixture of the two enzymes on a stopped-flow apparatus. Aliquots of two solutions, one containing the enzyme and the other the substrate and DTNB were mixed together in the mixing chamber of the apparatus. The absorbance of the reaction mixture was recorded spectrophotometrically [20] at various concentra- tions of the substrate in the presence of 0.66 mM DTNB. In order to avoid possible product modulation, we stopped the measurement when approximately 60 lM concentration of the product was formed. The stock solutions of the two enzymes were prepared by dilution of the aliquots with the
Modelling was performed with WHATIF [25], starting with the homology built model of human BuChE (CODE P06276) from Swiss-Model, an automated protein modeling
Fig. 1. pS curves for the hydrolysis of butyryl- thiocholine catalyzed by the wild-type, by var- ious human butyrylcholinesterase mutants and by horse butyrylcholinesterase in 0.1 M phos- phate buffer at pH 7.0 and 25 (cid:176)C.
Activation inhibition of human butyrylcholinesterase (Eur. J. Biochem. 269) 1157
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server [26], on an IBM compatible PC running under LINUX. Further refinement and the molecular dynamics were carried out using the macromolecular simulation program CHARMM [27] on a cluster of four PCs. Topology and force field parameters for TEA from CHARMM distribution c27n1 were used. Energy minimizations were performed with a constant dielectric constant (e (cid:136) 1). Electrostatic force was treated without cutoffs and van der Waals forces were calculated with the shift method with a cutoff of 10 A˚ . All lysines and arginines were protonated and aspartic and glutamic acids were deprotonated. Histidines were neutral with a hydrogen on N d1.
in all selected enzymes except in the W82A mutant, that apparently obeys Michaelis–Menten kinetics. Additionally, to obtain comparable activities, the concentration of this mutant had to be raised almost hundred times in comparison to the wild-type enzyme and the A328Y mutant and was still 10 times higher than the concentrations of the double mutants. Experimental data in all diagrams cannot predict the extent of inhibition at saturating substrate concentrations and in the case of W82A enzyme even the plateau/optimum is not reached. On the other hand, the theoretical curves for other enzymes, that were obtained by putting kinetic parameters from Table 1 into the Eqn 1, are in very good agreement with the data and they stipulate complete substrate inhibition. In other words, the fitting converged with the parameter a set to zero. It should be recalled that the data in the absence and presence of TEA were used for the determination of the kinetic parameters listed in Table 1.
(a) activation at
The corrections of the starting structure were performed in iterative steps as follows: the protein molecule was put in the cube of water molecules (9091), subjected to 150 relaxation steps (50 steps of steepest descent optimization, 50 steps of optimization by adopted basis Newton–Raphson method, 50 steps of steepest descent lattice optimization) and followed by 10 picoseconds constant pressure and temperature (CPT) dynamic simulation (300 K, 1 bar, time step of 1 fs) invoking the Ewald summation for calculating the electrostatic interactions. The last frame was devoided of all water molecules but those in the coat of 2.9 A˚ around the protein, relaxed with 100 optimization steps and checked by the CHECK module in WHATIF. The unrealistic protein portions were exchanged by the DGFIX command or by using the SCAN LOOP command in SPDBVIEWER [26]. After some 20 steps the check score improved substantially, so a continuous simulation run was performed for 300 ps. The final frame was used in a subsequent simulation involving TEA. Docking was performed by superimposing TEA to the trimethylamino group of docked acetycholine from Protein Data Bank entry 2ACE [15]. In order to remove overlapping between existing water molecules and newly introduced TEA we performed 150 relaxation steps (see previously) with fixed protein and TEA, followed by further 150 steps without any constrains. Finally, the dynamics simulation as described was run for 180 ps.
R E S U L T S
Initial rate data for the hydrolysis of BTC by five (wild-type and four mutants) recombinant human BuChEs and horse BuChE are presented in Fig. 1. The pS diagrams show that activation at intermediate substrate concentrations is present
Figure 2 shows the dependence of activity on the TEA concentration of all enzymes at different substrate concen- trations. From the panels in this figure we can see some low TEA important characteristics: concentrations is clearly visible in the wild-type enzyme, in the A328Y mutant and in the (cid:212)compensatory(cid:213) mutant (Y332D/D70Y). It can only be perceived in Y332A/D70G mutant but is absent in the W82A and in horse enzyme. (b) In the wild-type enzyme the activation is the most prom- inent at intermediate substrate concentrations. (c) Increas- ing inhibition at higher TEA concentrations is seen in all enzymes and the curves in the presence of the lowest substrate concentration approach to zero. This is the most evident in the wild-type and A328Y enzymes. The linear decrease in double mutants also indicates such a tendency. (d) Interestingly, TEA shows no activation of commercial wild-type horse BuChE at any concentration. Moreover, inhibition by TEA is very effective and the rate of hydrolysis clearly approaches zero even at the highest substrate concentration. (e) Inhibition by TEA is the most prominent in the A328Y mutant of human BuChE. It occurs at much lower TEA concentrations as in other enzymes, but activation is also present. Unlike in the wild-type enzyme, activation in the A328Y mutant appears stronger at higher substrate concentrations. (f) Regarding TEA inhibition, the W82A mutant is a special case: the inhibition emerges only at higher substrate concentrations indicating that either the interaction of TEA with the free enzyme is very weak or an
Table 1. Characteristic constants for the interactions of various human butyrylcholinesterases and horse butyrylcholinesterse with butyrylthiocholine and tetraethylammonium according to Scheme 2. Values in parenthesis are for the Michaelis–Menten mechanism (see Discussion).
)1Æs)1)
Y332D/D70Y (220 nM) Y332A/D70G (245 nM) W82A (2.4 lM) A328Y (39 nM) Horse BuChE (10 nM) Wild-type (39.5 nM)
3.45 (cid:139) 0.46 · 106 467 (cid:139) 26 46.9 (cid:139) 7.7 77.2 (cid:139) 12.5 6.88 (cid:139) 0.97 · 105 113 (cid:139) 2 292 (cid:139) 136 88.6 (cid:139) 4.2 7.82 (cid:139) 0.57 · 105 132 (cid:139) 7 60.9 (cid:139) 9.9 85.3 (cid:139) 9.3 0 0 0 2.08 (cid:139) 0.12 · 107 8.51 (cid:139) 0.2 · 106 3800 (cid:139) 1700 25.5 (cid:139) 2.9 1.03 (cid:139) 0.92 0.117 (cid:139) 0.059 (1.44) 88.2 (cid:139) 1.3 (0079) 18.0 (cid:139) 0.6 17.0 (cid:139) 3.4 0.27 (cid:139) 0.05 0.0242 (cid:139) 0.0032 0.0119 (cid:139) 0.0022 0.0114 (cid:139) 0.0017 1282 (cid:139) 87 100 (cid:139) 9.9 38.8 (cid:139) 9.3 0.376 (cid:139) 0.053 0.134 (cid:139) 0.032 7.19 (cid:139) 2.8 0.325 (cid:139) 0.029 39.0 (cid:139) 8.9 0 – – – 0.028 (cid:139) 0.005 8.58 (cid:139) 3.91 177 (cid:139) 82.9 0.393 (cid:139) 0.096 0.926 (cid:139) 0.395 2.97 (cid:139) 1.15 0.440 (cid:139) 0.056 23.8 (cid:139) 2.2 129.4 (cid:139) 38.6 0.257 (cid:139) 0.118 0.915 (cid:139) 0.236 59.8 (cid:139) 27.0 0.166 (cid:139) 0.015 13.1 (cid:139) 3.4 397 (cid:139) 7.5 0.407 (cid:139) 0.030 0.511 (cid:139) 0.049 296 (cid:139) 93 – (340) 5.7 (cid:139) 0.4 – – – 0.093 (cid:139) 0.013 1.75 (cid:139) 0.22 93.7 (cid:139) 12.7 ki (M k3 (s)1) K1 (lM) K2 (mM) a b K3 (mM) K4 (mM) c d K6 (mM)
1158 J. Stojan et al. (Eur. J. Biochem. 269)
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Fig. 2. Dependence of the activity of various human BuChEs and horse BuChE on the con- centration of TEA at various butyrylthiocholine concentrations. BTC concentrations for human enzymes are 15 lM, 25 lM, 50 lM, 100 lM, 1 mM, 2 mM, 3 mM, from the lowest to the highest curve. For horse enzyme BTC concentrations are: 50 lM, 200 lM, 1 mM and 2 mM.
independent binding of TEA and substrate at low concen- trations occurs on different sites.
seen that at low substrate concentrations the mixture of the enzymes is less active than the horse enzyme alone. Additionally, the theoretical curves for W82A mutant agree very good with the experimental progress curves. It should be stressed, however, that we could only achieve such an agreement with six-parameter model according to Scheme 1 and not with simple Michaelis–Menten reaction mechanism.
In order to find out the reason for the very low activity of the W82A mutant, we tested the activity of the enzyme mixture: W82A human BuChE and horse BuChE. Figure 3 shows the progress curves obtained in this experiment and the pS diagram of calculated initial rates. It can be clearly
Fig. 3. Progress curves for the hydrolysis of butyrylthiocholine catalyzed by the horse butyrylcholinesterase, by the W82A mutant of human butyrylcholinesterase and by the enzyme mixture. Measurements were performed at substrate concentrations ranging from 5 lM to 75 mM. Lower right panel shows the depend- ence of the initial rates in the form of pS curves.
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possible perturbations at the PAS are kinetically invisible. Slow acylation, again, should be the consequence of changed architecture in the catalytic site which firstly, cannot help to accommodate the substrate in forming Michaelis–Menten complex and secondly, enhance the stability of the acylated enzyme.
Molecular dynamics calculations on the wild-type human BuChE in water reveal after 180 ps very interesting TEA positioning. From the starting site in the vicinity of W82 indole ring it moved upward the cleft and accommodated just below Y332 and D70, the major constituents of the PAS in human BuChE (Fig. 4). It seems that the A328 plays a role in this rapid movement (compare K3 values). In A328Y mutant and in vertebrate AChEs, the homologous F330 or Y330 would prevent such positioning of TEA.
Fig. 4. Stereo view of important active site residues in superimposed structures of Torpedo acetylcholinesterase (2ACE) and human butyryl- cholinesterase. Docked as a tetrahedral adduct is butyrylthiocholine. The starting position of tetraethylammonium is position superimposed on the substrate trimethyl group. An intermediate position of tetraethylammonium and final position (uppermost) after 180 picoseconds molecular dynamics are also seen. Note the overlaping of Torpedo AChE residue F330 and tetraethylammonium in the final position. Corresponding A328 in butyrylcholinesterase does not prevent the final orientation of tetraethylammonium. Labelling and numbering are according to human butyrylcholinesterase.
D I S C U S S I O N
The kinetic behavior of ChEs shows deviations from the Michaelis–Menten model. Although it has long been believed that the only deviation in vertebrate AChEs is inhibition by excess substrate and that BuChEs are analo- gically activated, a more detailed investigations on insect AChEs, nematode enzymes and also BuChEs from various sources reveal both phenomena [28].
In order to find out whether the extremely poor activity of the W82A mutant is due to low affinity for the substrate and/or to the slow acylation-deacylation, we mixed W82A mutant with horse serum BuChE and tested the hydrolytic activity of the mixture at low BTC concentrations. The aim was to perform the experiments where the concentrations of the substrate and the W82A mutant were similar, while the concentration of the horse enzyme was at least thousand times lower. Under such conditions it might be incorrectly assumed that the low activity enzyme in such large concentration must tie up substrate by binding it to a number of sites with varying affinity. Of course, only a single specific interaction is possible when the enzyme is a reaction partner in stoichiometric amount to the substrate. We can conclude therefore that the lower activity of the mixture, compared to horse enzyme alone, is a consequence of good affinity of W82A for BTC (17 lM) and rather ineffective catalysis.
the binding of
Recent studies on various mutated enzymes showed, that an appropriate mutation can mask one or the other deviation, but can also introduce it, if missing (cf. [10,23,29]). Exactly this can be seen from our experiments in Fig. 1. While the pS curve for the wild-type enzyme shows clearly deviations at intermediate and very high substrate concentrations none of them is evident in the W82A mutant. However, the progress curves for W82A in Fig. 3, which include the information at very low substrate concentrations (the plateaus confirm complete hydrolysis), can only be explained by introducing an additional deviation from Michaelian kinetics into the reaction mech- anism (see parameters in Table 1). Moreover, we could also speculate that unless prevented by the solubility maximum, inhibition too might occur. Consequently, in W82A mutant a plateau/optimum shift towards higher substrate concen- trations appeared to take place. The explanation for such a shift may be the very low turnover of this mutant (kcat (cid:136) 100 min)1, [30]). The probability for the substrate to encounter the acylation site correctly is so low, that the
Our experiment is the first kinetic evidence that in spite of high substrate affinity the activity of a cholinesterase may be very low. It is well known that transition state analogues are extremely good inhibitors. As BTC is the substrate, a substantional shift of the pS curve towards high concentra- tions suggests the inability to reach transition state rather to stabilize in it. It is well founded therefore to corroborate this finding with apparent activating deviation from Michaelis– Menten kinetics, which has also been reported for several other cholinesterases. It was suggested that deviations from Michaelis–Menten kinetics reflect the substrate molecule to the PAS [9,10,31,32]. In enzymes, showing apparent activation, the substrate affinity for the PAS appears to be relatively high, but overall catalytic power of such enzymes is low [23]. It seems that inhibition at
1160 J. Stojan et al. (Eur. J. Biochem. 269)
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substrate concentrations in the range of high affinity binding constant, mimics apparent activation.
with additional, more or less realistic assumptions. Our kinetic model needs no additional assumptions and a unique set of six parameters can be evaluated if the two deviations can be inspected.
In conclusion, the major arguing point in the interpret- ation of the results obtained by this model is a great difference between the binding of the substrate to the modifier site of free and acylated enzyme (K1 vs. K2). We have designed the experiment with the mixture of a normal and a low activity enzyme to confirm at least one high affinity substrate binding site in the enzyme showing two deviations from Michaelian kinetics (see K1 for W82A). We agree, that the model does not predict the exact spot and orientation in this binding but clearly explains the observed deviation at that substrate concentration as homotropic inhibition rather as activation. The concentration dependent activation-inhibition pattern by TMA and other quaternary and tertiary ammonium compounds [12] strongly supports this interpretation.
A C K N O W L E D G E M E N T S
All this is supported by the inhibitory pattern of TEA on various enzymes. The activation of some enzymes by TEA in low concentrations might be the consequence of the competition between the substrate and TEA at the PAS. In comparison to the substrate, TEA inhibits the substrate hydrolysis less effectively (d > b, see Table 1). This might be true for all enzymes showing activation by TEA, especially because in different enzymes it (cid:212)appears(cid:213) at different substrate concentrations (compare the wild-type and the A328Y). At the highest TEA concentrations it competes with the substrate at both sites, thus, inhibiting the enzyme. The question rises, why some enzymes do not show activation by TEA, but show substrate affinity at the PAS. Two explanations come to mind. The first one would be that the same substrate orientation at the PAS, in various enzymes, cannot affect the events at the acylation site. As in the W82A mutant, the missing bulky indole ring at the bottom of the cleft allows multiple substrate orientations at the acylation site, thus preventing the influence of the ligand from the PAS. In this enzyme the weak inhibition at the highest TEA concentrations corroborates the explanation. The second plausible possibility would be different orien- tation of the substrate at the PAS. It might be the case in the Y332A/D70G double mutant and in the horse enzyme. In addition to an extra negative charge at the mouth of the cleft in horse enzyme (D283, identical in Torpedo), the substrate affinity at the PAS of both these enzymes appears lower as in other tested enzymes.
We thank Dr Oksana Lockridge (Eppley Institute, University of Nebraska, Omaha, USA) for generously providing us with human butyrylcholinesterase mutants. This work was partially supported by the Ministry of Science and Technology of the Republic of Slovenia, Grant No. P3-8720-0381 to J. S. and by DGA/DSP/STTC, grant no. 97/08 and 99 CO 029 to P. M.
R E F E R E N C E S
1. Massoulie´ , J., Pezzementi, L., Bon, S., Krejci, E. & Vallette, F.M. (1993) Molecular and cellular biology of cholinesterases. Prog. Neurobiol. 41, 31–91.
2. Mack, A. & Robitzki, A. (2000) The key role butyrylcholinesterase during neurogenesis and neural disorders. Prog. Neurobiol. 60, 607–628. 3. Lockridge, O. (1992) In Pharmacogenetics of Drug Metabolism (Kalow, W., ed.), pp. 15–50. Pergamon Press, New York.
The important role of PAS residues is further supported by docking and dynamics simulations of TEA in the cleft of the wild-type human BuChE. Similar to the simulations on human AChE [33], a gradual movement is seen of the TEA molecule from its starting position at p-electron interactions with W82 indole ring, upwards to the vicinity of the two PAS constituting residues, Y332 and D70. Although longer simulation run might reveal yet another position, such a movement indicates that at low concentrations TEA might preferentially occupy PAS, thus, preventing substrate to bind and to inhibit its own metabolization.
4. Cashman, J.R., Perotti, B.Y.T., Berkman, C.E. & Lin, J. (1996) Pharmacokinetics and molecular detoxication. Health Persp. 104, 23–40.
5. Mattes, C.E., Belendiuk, G.W., Lynch, T.J., Brady, R.O. & Dretchen, K.L. (1998) Butyrylcholinesterase: an enzyme antidote for cocaine intoxication. Addict. Biol. 3, 171–178.
6. Ballantyne, B. & Marss, T.C. (1992) Clinical and Experimental Toxicology of Organophosphates and Carbamates. Butterworth- Heinemann, Oxford.
7. Broomfield, C.A., Lockridge, O. & Millard, C.B. (1999) Protein engineering of a human enzyme that hydrolyses V and G nerve agents: design, construction and characterization. Chem. Biol. Interact. 119/120, 413–418.
8. Masson, P., Froment, M.Th, Bartels, C.F. & Lockridge, O. (1996) Asp 70 in the peripheral anionic site of human butyryl- cholinesterase. Eur. J. Biochem. 235, 36–48.
9. Masson, P., Legrand, P., Bartels, C.F., Froment, M.Th, Schopfer, C.M. & Lockridge, O. (1997) Role of aspartate 70 and trypto- phane 82 in binding of succinyldithiocholine to human butyryl- cholinesterase. Biochemistry 36, 2266–2277.
10. Radi\¢C.Z., Pickering, N.A., Vellom, D.C., Champ, S. & Taylor, P. (1993) Three distinct domains in the cholinesterase molecule confer selectivity for acetyl- and butyrylcholinesterase inhibitors. Biochemistry 32, 12074–12084.
Finally, we would like to discuss the significance of kinetic parameters that we evaluated with our six parameter model. One could argue at this point that the model can very exactly reproduce the experimental data but does not reflect the realistic events during the catalytic process and thus the constants and their values are meaningless. Three points should be emphasized in this connection. Firstly, the kinetic model is one possible reduction of the traditional reaction scheme generally valid for all cholinesterases. It assumes that Michaelis–Menten complex is not accumula- ting, but it does not deny it. Such an approach is well justified in the kinetic analysis and the simplification is introduced according to well known principles [34]. More- over, some parameters can easily be interpreted with the classical terms of Michalis–Menten kinetics. For instance, k3 in the model represents kcat and ki is in fact kcat/Km. Secondly, the six parameters are sufficient but also neces- sary to reproduce two deviations from Michaelian kinetics, for which more and more evidence exists, that they are a rule rather an exception with cholinesterases. It should be very clear, that many realistic models with more than six parameters can equally precise reproduce the data, but only
11. Ordentlich, A., Barak, D., Kronman, C., Ariel, N., Segal, B., Velan, B. & Shafferman, A. (1995) Contribution of aromatic
Activation inhibition of human butyrylcholinesterase (Eur. J. Biochem. 269) 1161
(cid:211) FEBS 2002
tion or combined assumptions of equlibrium and steady state. J. Biol. Chem. 243, 820–825. moities of tyrosine 133 and of the anionic subsite tryptophane 86 to catalytic e(cid:129)ciency and allosteric modulation of acetyl- cholinesterase. J. Biol. Chem. 270, 2082–2091.
23. Golinik, M., Fournier, D. & Stojan, J. (2001) Interaction of Drosophila acetylcholinesterases with D-tubocurarine: an explan- ation of the activation by an inhibitor. Biochemistry 40, 1214– 1219. 12. Erdoes, E.G., Foldes, F.F., Zsigmond, E.K., Baart, N. & Zwartz, J.A. (1958) Acceleration of plasma colinesterase activity by qua- ternary ammonium salts. Science 128, 92.
24. Stojan, J. (1997) Analysis of progress curves in an acetyl- integration treatment. cholinesterase reaction: a numerical J. Chem. Inf. Comput. Sci. 37, 1025–1027. 25. Vriend, G. (1990) WHAT IF: a molecular modeling and drug design program. J. Mol. Graph. 8, 52–56. 13. Cle´ ry, C., Heiber-Langen, I., Channac, L., David, L., Balny, C. & Masson, P. (1995) Substrate dependence of amiloride- and soman-induced conformation changes of butyrylcholinesterase as evidence by high-pressure perturbation. Biochim. Biophys. Acta 1250, 19–28.
26. Guex, N. & Peitsch, M.C. (1997) SWISS-MODEL and the Swiss- PdbViewer: an environment for comparative protein modeling. Electrophoresis 18, 2714–2723. 14. Levitsky, V., Xie, W., Froment, M.Th, Lockridge, O. & Masson, P. (1999) Polyol-induced activation by excess substrate of the D70G butyrylcholinesterase mutant. Biochim. Biophys. Acta 1429, 422– 430.
27. Brooks, B.R., Bruccoleri, R.E., Olafson, B.D., States, D.J., Swaminathan, S. & Karplus, M. (1983) CHARMM: a program for macromolecular energy minimization and dynamic calcula- tions. Comput. Chem. 4, 187–217. 15. Sussman, J.L., Harel, M., Frolow, F., Oefner, C., Goldman, A., Toker, L. & Silman, I. (1991) Atomic structure of acetylcholine- sterase from Torpedo californica: a prototypic acetylcholine- binding protein. Science 253, 872–878.
28. Marcel, V., Palacios, L.G., Pertuy, C., Masson. P. & Fournier, D. (1998) Two invertabrate acetylcholinesterases show activation followed by inhibition with substrate concentration. Biochem. J. 329, 329–334. 16. Harel, M., Sussman, J.L., Krejci, E., Bon, S., Chanal, P., Massoulie´ , J. & Silman, (1992) Conversion of acetyl- I. cholinesterase to butyrylcholinesterase: modeling and mutagen- esis. Proc. Natl Acad. Sci. USA 89, 10827–10831.
29. Marcel, V., Estrada-Mondaca, S., Magne´ , F., Stojan, J., Klae´ be´ , A. & Fournier, D. (2000) Exploration of the Drosophila acet- ylcholinesterase substrate activation site using a reversible inhibitor (Triton X-100) and mutated enzymes. J. Biol. Chem. 275, 11603–11609. 17. Nachon, F., Ehret-Sabatier, L., Loew, D., Colas, C. & van Dorsselaer and Goldner, M. (1998) Trp82 and Tyr332 are involved in two quaternary ammonium binding domains of human butyrylcholinesterase as revealed by photoa(cid:129)nity labeling with [3H]DDF. Biochemistry 37, 10507–10513.
30. Masson, P., Xie, W., Froment, M.-T. & Lockridge, O. (2000) Effects of mutations of active site residues and amino acids interacting with omega loop on substrate activation of butyr- ylcholinesterase. Biochim. Biophys. Acta 1564, 166–176. 31. Szegletes, T., Mallender, W.D. & Rosenberry, T.L. 18. Saxena, A., Redman, A.M.G., Quian, N., Lockridge, O. & Doc- tor, B.P. (1997) Differences in active site gorge dimensions of cholinesterases revealed by binding of inhibitors to human butyrylcholinesterase. Biochemistry 36, 14642–14651.
(1998) Nonequilibrium analysis alters the mechanistic interpretation of inhibition of acetylcholinesterase by peripheral site ligand. Bio- chemistry 37, 4206–4216. 19. Wierdl, M., Morton, C.L., Dunks, M.K. & Potter, P.M. (2000) Isolation and characterization of a cDNA encoding a horse liver butyrylcholinesterase: evidence for CPT-11 drug activation. Bio- chem. Pharmacol. 59, 773–781.
32. Mallender, W.D., Szegletes, T. & Rosenberry, T.L. (2000) Acet- ylcholine binds to Asp74 at the peripheral site of human acet- ylcholinesterase as the first step in the catalytic pathway. Biochemistry 39, 7753–7763. 20. Ellman, G.L., Courtney, K.D., Andres, V. & Feathersone, R.M. (1961) A new and rapid colorimetric determination of acet- ylcholinesterase activity. Biochem. Pharmacol. 7, 88–95.
33. Van Belle, D., De Maria, L., Iurcu, G. & Wodak, S.J. (2000) Pathways of ligand clearance in acetylcholinesterase by multiple copy sampling. J. Mol. Biol. 298, 705–726. 21. Stojan, J., Marcel, V., Estrada-Mondaca, S., Klaebe, A., Masson, P. & Fournier, D. (1998) A putative kinetic model for substrate metabolization by Drosophila acetylcholinesterase. FEBS Lett. 440, 85–88.
34. Cleland, W.W. (1977) Determining the chemical mechanisms of enzyme-catalyzed reactions by kinetic studies. Adv. Enzymol. 45, 273–387. 22. Cha, S. (1968) A simple method for derivation of rate equation for enzyme-catalyzed reactions under the rapid equlibrium assump-
