Tài liệu Báo cáo khoa học: Kinetic analysis of effector modulation of butyrylcholinesterase-catalysed hydrolysis of acetanilides and homologous esters pdf

Kinetic analysis of effector modulation of butyrylcholinesterase-catalysed hydrolysis of acetanilides and homologous esters ´ ` Patrick Masson1, Marie-Therese Froment1, Emilie Gillon1, Florian Nachon1, Oksana Lockridge2 and Lawrence M Schopfer2 ´ ´ ´ ´ Unite d’Enzymologie, Departement de Toxicologie, Centre de Recherches du Service de Sante des Armees, La Tronche Cedex, France University of Nebraska Medical Center, Eppley Institute, Omaha, NE, USA Keywords aryl acylamidase; benzalkonium; butyrylcholinesterase; serotonin; tyramine Correspondence ´ P Masson, Unite d’Enzymologie, ´ Departement de Toxicologie, Centre de ´ Recherches du Service de Sante des ´ Armees, BP 87, 38702 La Tronche Cedex, France Fax: +33 76 63 69 62 Tel: +33 76 63 69 59 E-mail: pmasson@unmc.edu (Received 30 December 2007, revised 27 February 2008, accepted 17 March 2008) doi:10.1111/j.1742-4658.2008.06409.x The effects of tyramine, serotonin and benzalkonium on the esterase and aryl acylamidase activities of wild-type human butyrylcholinesterase and its peripheral anionic site mutant, D70G, were investigated The kinetic study was carried out under steady-state conditions with neutral and positively charged aryl acylamides [o-nitrophenylacetanilide, o-nitrotrifluorophenylacetanilide and m-(acetamido) N,N,N-trimethylanilinium] and homologous esters (o-nitrophenyl acetate and acetylthiocholine) Tyramine was an activator of hydrolysis for neutral substrates and an inhibitor of hydrolysis for positively charged substrates The affinity of D70G for tyramine was lower than that of the wild-type enzyme Tyramine activation of hydrolysis for neutral substrates by D70G was linear Tyramine was found to be a pure competitive inhibitor of hydrolysis for positively charged substrates with both wild-type butyrylcholinesterase and D70G Serotonin inhibited both esterase and aryl acylamidase activities for both positively charged and neutral substrates Inhibition of wild-type butyrylcholinesterase was hyperbolic (i.e partial) with neutral substrates and linear with positively charged substrates Inhibition of D70G was linear with all substrates A comparison of the effects of tyramine and serotonin on D70G versus the wild-type enzyme indicated that: (a) the peripheral anionic site is involved in the nonlinear activation and inhibition of the wild-type enzyme; and (b) in the presence of charged substrates, the ligand does not bind to the peripheral anionic site, so that ligand effects are linear, reflecting their sole interaction with the active site binding locus Benzalkonium acted as an activator at low concentrations with neutral substrates High concentrations of benzalkonium caused parabolic inhibition of the activity with neutral substrates for both wild-type butyrylcholinesterase and D70G, suggesting multiple binding sites Benzalkonium caused linear, noncompetitive inhibition of the positively charged aryl acetanilide m-(acetamido) N,N,N-trimethylanilinium for D70G, and an unusual mixed-type inhibition ⁄ activation (a > b > 1) for wild-type butyrylcholinesterase with this substrate No fundamental difference was observed between the effects of ligands on the butyrylcholinesterase-catalysed hydrolysis of esters and amides Thus, Abbreviations AAA, aryl acylamidase; ASCh, acetylthiocholine; ATMA, m-(acetamido) N,N,N-trimethylanilinium; BuChE, butyrylcholinesterase; DFP, diisopropylfluorophosphate; NATAc, N-acetylanthranilic acid; Nbs2, 5,5¢-dithiobis(2-nitrobenzoic acid); o-NA, o-nitroaniline; o-NAC, o-nitroacetanilide; o-NP, o-nitrophenol; o-NPA, o-nitrophenylacetate; o-NTFNAC, o-nitrotrifluoroacetanilide; o-NTMNPA, o-N-trimethylnitrophenylaniline; PAS, peripheral anionic site FEBS Journal 275 (2008) 2617–2631 ª 2008 The Authors Journal compilation ª 2008 FEBS 2617 Modulation of butyrylcholinesterase catalytic activitiy P Masson et al butyrylcholinesterase uses the same machinery, i.e the catalytic triad S198 ⁄ H448 ⁄ E325, for the hydrolysis of both types of substrate The differences in response to ligand binding depend on whether the substrates are neutral or positively charged, i.e the differences depend on the function of the peripheral site in wild-type butyrylcholinesterase, or the absence of its function in the D70G mutant The complex inhibition ⁄ activation effects of effectors, depending on the integrity of the peripheral anionic site, reflect the allosteric ‘cross-talk’ between the peripheral anionic site and the catalytic centre Cholinesterases are structurally related hydrolases [1] Acetylcholinesterase (EC 3.1.1.7) plays a key role in the cholinergic system in terminating the action of acetylcholine, but no clear physiological function has yet been assigned to butyrylcholinesterase (BuChE; EC 3.1.1.8) [2] BuChE may have physiological functions related to its esterase activity In particular, it has been proposed that BuChE may play a role in fatty acid [3] and lipoprotein [4] metabolism Studies with knock-out mice for acetylcholinesterase indicate that BuChE can act in the central nervous system as a surrogate acetylcholine-hydrolysing enzyme [5] Both cholinesterases also display noncholinergic activities Cholinesterase isoforms may have nonenzymatic roles in axonal outgrowth, synaptogenesis, cell adhesion, neuronal migration and developmental neurotoxicity to organophosphates [6–8] Certain nonenzymatic functions of acetylcholinesterase have been found to depend on the peripheral anionic site (PAS) [9]; others appear to be related to a peptide derived from the enzyme C-terminus [10] However, the physiological relevance of these activities is still unclear [2,11] One noncholinergic activity displayed by cholinesterases is aryl acylamidase (AAA; EC 3.5.1.13) activity [12,13] Indeed, there is some evidence that the AAA activity of BuChE plays a role in early brain development [14] and in the formation of amyloid plaques in Alzheimer’s disease [2,15] Human plasma BuChE is of toxicological and pharmacological importance, because it scavenges and detoxifies numerous carboxyl ester drugs and prodrugs [16–18], and carbamyl and phosphoryl esters, including nerve agents [19] Numerous widely used chemicals are aryl acylamides (drugs: acetaminophen, phenacetin, flutamide, isocarboxazid, lidocaine, butanilicaine; pesticide: acephate; herbicides and fungicides: acetochlor, propanil and butachlor) The AAA activity of BuChE in plasma and tissues could participate in the metabolism of these aryl acylamide drugs and xenobiotics However, the potential detoxification role of the AAA activity of BuChE needs to be addressed 2618 Known AAAs are serine hydrolases that catalyse the deacylation of N-acyl arylamines [20,21] Several AAAs have been identified in mammalian tissues [22,23] Certain acryl acylamidases are identical to carboxylesterases [24] Albumin also displays an AAA activity [25,26] A correspondence between certain molecular forms of AAAs and cholinesterases has been demonstrated in different organs [22] Deacetylation of retinal melatonin into 5-methoxytryptamine is catalysed by an eye AAA [27] However, no clear physiological function has yet been ascribed to most mammalian AAAs At a minimum, AAAs are toxicologically relevant because they deacylate arylamide xenobiotics [20,21,28] The crystal structures of acetylcholinesterase and BuChE reveal that these enzymes have a common architecture, with only one catalytic triad located at the bottom of a deep gorge [29] However, it has been suggested that esterase and amidase active centres are nonidentical, although they are overlapping [29–33] Contrary to this proposal, recent kinetic studies and structure–activity relationships have clearly indicated that BuChE utilizes the same catalytic site to hydrolyse anilides and esters [24,25] It has been reported that the AAA activity of BuChE, as well as that of acetylcholinesterase, can be either activated or inhibited by various ligands These ligands include: (a) biogenic amines: serotonin (5-hydroxytryptamine), tryptamine and related molecules [22,32,36–39]; (b) kynuramine [22] and tyramine [22,36,37,39]; (c) procainamide [39]; (d) anti-Alzheimer drugs: (+)huperzine A, donepezil, galantamine and tacrine [32,40,41]; and (e) a cationic detergent that is an acetylcholine (nicotinic) agonist: benzalkonium [42] Although most ligands were found to be reversible inhibitors of the BuChE-catalysed hydrolysis of o-nitroacetanilide (o-NAC), tyramine was found to be an activator However, kinetic analysis of these inhibiting or activating effects was either incomplete [22,36–39] or debatable [33,42] In particular, reported results were interpreted in terms of an AAA site distinct from FEBS Journal 275 (2008) 2617–2631 ª 2008 The Authors Journal compilation ª 2008 FEBS P Masson et al the ester site Moreover, certain of these studies were performed using partially purified enzymes from sera [22,36,42], biological fluids [37,43] or commercial preparations [32,42] that very probably contained serum albumin as a contaminant Human serum albumin has been found to display intrinsic AAA activity [25,26] Thus, in order to provide a complete analysis and to clarify debated issues, we investigated the effects of tyramine, serotonin and benzalkonium on the BuChEcatalysed hydrolysis of neutral and charged aryl acetyl amides [o-NAC, o-nitrotrifluoroacetanilide (o-NTFNAC) and m-(acetamido) N,N,N-trimethylanilinium (ATMA)] and acetyl esters [o-nitrophenylacetate (o-NPA) and acetylthiocholine (ASCh)] under steadystate conditions All of these substrates give the same acyl enzyme intermediate Effects on wild-type human BuChE and its PAS mutant D70G were compared Because the presence of contaminating proteins displaying AAA activity, e.g albumin, in the BuChE preparation could have biased the results, experiments were carried out on highly purified recombinant enzymes free of albumin and any other AAAs It was found that there was no fundamental difference in the mechanisms of inhibition and activation for either the AAA or esterase activities by these ligands In addition, differences between the behaviour of the wild-type enzyme and D70G were found to reflect alterations in the binding of positively charged substrates ⁄ ligands on PAS, regardless of the type of substrate (acetyl amide or acetyl ester) Results and Discussion Action of reversible effectors on AAA activity of BuChE The investigation of the effects of the ligands (tyramine, serotonin and benzalkonium) on the AAA and esterase activities of BuChE was performed in parallel on wild-type enzyme and the D70G mutant The substrates were neutral and positively charged acetanilides (o-NAC and ATMA) and esters (o-NPA and ASCh) The hydrolysis of neutral substrates by BuChE, in the absence of effectors, obeys the Michaelis–Menten model (Scheme 1, boxed mechanism in Scheme 2) that is described by Eqn (1): Scheme General scheme for hydrolysis of neutral substrates by BuChE Modulation of butyrylcholinesterase catalytic activitiy Scheme General scheme for hydrolysis of positively charged substrates by BuChE vẳ with kcat ẵEẵS Km ỵ ẵS 1ị Ks k3 Ks ẳ k2 ỵ k3 ị ẵ1 ỵ k2 =k3 ị 2ị k2 k3 k2 ỵ k3 ị 3ị kcat =Km ẳ k2 =Ks Km ẳ 4ị kcat ẳ The hydrolysis of positively charged substrates by BuChE, in the absence of effectors, shows either activation or inhibition by excess substrate The BuChE-catalysed hydrolysis of positively charged substrates is conveniently described by Scheme [44] In Scheme 2, the enzyme–substrate complex SpE corresponds to S bound on PAS Once the first substrate molecule has bound to the catalytic binding site (ES), a second substrate molecule can bind to PAS to form the ternary complex SpES The kinetics of this scheme are described by Eqn (5):    kcat ẵE ỵ bẵS=Kss 5ị vẳ ỵ Km =ẵS ỵ ẵS=Kss where Kss is the dissociation constant of complexes SpE and SpES (Kss > Km) The parameter b reflects the efficiency with which SpES forms products When b > 1, there is substrate activation; when b < 1, there is substrate inhibition; when b = 1, the enzyme kinetics obey the simple Michaelis–Menten model (Eqn 1) BuChE shows substrate activation with ATMA (b = 1.53, Kss = 0.7 mm [35]) and ASCh (b = 2.7, Kss = 0.6 [45]) The ligands act as either inhibitors or activators depending on the nature of the substrate: neutral or charged Homologous pairs of substrates (e.g acetyl anilide ⁄ acetyl ester) show the same type of inhibition The binding constants for D70G were generally higher than those for wild-type BuChE, indicating that PAS is involved in some of these effects Additional complexities are seen with benzalkonium The results are summarized in Tables and The following is an analysis of these effects FEBS Journal 275 (2008) 2617–2631 ª 2008 The Authors Journal compilation ª 2008 FEBS 2619 Modulation of butyrylcholinesterase catalytic activitiy P Masson et al Table Effect of ligands (tyramine, serotonin and benzalkonium) on esterase and AAA activities of BuChE with the neutral substrates oNPA versus o-NAC Values are means ± standard error from three to five independent determinations H, hyperbolic; L, linear; P, parabolic These terms refer to the appearance of the Dixon plots Hyperbolic curves appear when there is partial inhibition or when there is activation Parabolic curves indicate multiple ligand binding A, activation; C, competitive; I, inhibition ND, not determined Tyramine Serotonin Benzalkonium Substrate o-NPA o-NAC o-NPA o-NAC o-NPA o-NAC Wild-type: I or A type Ka (mM) Ki (mM) a b b⁄a D70G: I or A type Ka (mM) Ki (mM) a b HA ± 0.3 – ND (< 1) 2.8 ND (> 3) LA 9.1 ± 1.9 – ND (< 1) >1 HA 0.8 ± 0.2 – 0.4 5.5 14 ± LA NDa – ND (> 1) >1 HCI – 1.7 ± 0.5 1.9 ± 1.2 0.6 ± 0.3 0.3 ± 0.3 LCI – 12.5 ± 0 LCI – 7.7 ± 0.2 – ) – LCI – 7.9 ± 0.4 0 PCI –b 0.18 ± 0.02 – – – HA + PCI NDc 0.2 ± 0.08 – – HA + PCI 0.03 ± 0.01 0.37 ± 0.03 – – – HA + PCId 0.015 ± 0.015 (> 0.3)d – – a No binding up to mM tyramine; weak activation beyond mM b No activation even at low [benzalkonium] c Weak activation at low [benzalkonium] at low [o-NPA] d Competitive inhibition occurs beyond 0.3 mM benzalkonium at the lowest [o-NAC] Table Effect of ligands (tyramine, serotonin and benzalkonium) on esterase and AAA activities of BuChE with the positively charged substrates ASCh versus ATMA Values are the means ± standard error from three to five independent determinations H, hyperbolic; L, linear These terms refer to the appearance of the Dixon plots Hyperbolic curves appear when there is partial inhibition or when there is activation A, activation; C, competitive; I, inhibition; M, mixed; N, noncompetitive; U, uncompetitive Tyramine Serotonin Benzalkonium Substrate ASCh ATMA ASCh ATMA ASCha ATMA Wild-type: I or A type Ka (mM) Ki (mM) a b D70G I or A type Ka (mM) Ki (mM) a b LCI – 0.78 ± 0.07 0 LCI – 9.65 ± 0.7 0 LCI – 2.4 ± 0.9 0 LCI – 6.15 ± 1.8 0 LCI – 0.53 ± 0.19 0 LCI – 2.74 ± 0.35 0 LUI – 0.09 ± 0.03 0 LNI – 0.27 ± 0.03 0 LMIb – 1.03 lMb 4.52b 0b NDa – NDa – – LMIc NDd 0.05 lMd 5.7d 3.1 LNI – 13 ± lM 0 a Benzalkonium chloride precipitated with Nbs2 under our assay conditions b At high substrate concentration [42] c Under experimental conditions, but theory predicts HA at high substrate concentration because a > b > d Inhibition at substrate concentration lower than [S]cross ([S]cross = 0.66 mM) and activation at [S] > [S]cross Effects of tyramine Tyramine was found to be an activator of both D70Gand wild-type BuChE-catalysed hydrolysis for neutral substrates (b > 1, a > 0) (Table 1), but was an inhibitor of hydrolysis for charged substrates (a = 0) (Table 2) The affinity of wild-type BuChE for tyramine was higher than that of D70G with both acetyl anilides and acetyl esters Our results with o-NPA and o-NAC confirm the reports that tyramine is an activator for the hydrolysis 2620 of o-NAC by wild-type BuChE [22,36,37,39] The activating effect of tyramine yields the expected hyperbolic Dixon and Cornish–Bowden plots (Fig 1A,B) for hydrolysis of both o-NPA and o-NAC This nonessential activation can be mathematically treated in a manner similar to partial mixed-type inhibition (see Experimental procedures, Scheme 3) Similar activating effects on the BuChE-catalysed hydrolysis of o-NPA have been reported for the positively charged ligands dibucaine [45], amiloride [46] and tetraalkylammonium compounds [47] This activation was interpreted in FEBS Journal 275 (2008) 2617–2631 ª 2008 The Authors Journal compilation ª 2008 FEBS P Masson et al Modulation of butyrylcholinesterase catalytic activitiy A 16 60 S = o-NPA 10 [S]/n 1n(1/ΔA λ 410nm),min 12 40 30 10 0 [Tyramine] mM B [Tyramine] mM 600 S = o-NAC 500 1/n(1/ΔAλ 430nm),min 20 Fig Activating effect of tyramine on wild-type BuChE-catalysed hydrolysis of o-NPA and o-NAC in 0.1 M phosphate buffer at 25 °C (A) o-NPA (d, 0.1 mM; s, 0.2 mM; , 0.4 mM; h, 0.6 mM): left panel, Dixon plots of v)1 versus [tyramine]; right panel, Cornish–Bowden plots of [S] ⁄ v versus [tyramine] Nonlinear Dixon plots are expected for activation (B) o-NAC (d, mM; s, mM; , 3.5 mM; h, mM); Dixon plots of v)1 versus [tyramine] S = o-NPA 14 50 400 300 200 100 0 [Tyramine] mM terms of binding of the positively charged ligands to PAS This would form a ternary complex, LPASES, that linearly accelerates catalysis (bkcat with b > 1) The degree of activation in the presence of tyramine was higher for the hydrolysis of o-NAC than for the hydrolysis of o-NPA This was determined from the nonactivated and asymptotic limits in the nonlinear hyperbolic acceleration plots (Fig 1A,B), which provided estimates of b For o-NPA hydrolysis b = 2.8, and for o-NAC hydrolysis b = 5.5 For o-NAC, because the hydrolysis kinetics were performed under first-order conditions, the b ⁄ a ratio was determined using Eqn (14) (see Experimental procedures): b ⁄ a = 14 ± and a = 0.4 (Table 1) For o-NPA, experiments were performed at [S] close to Km, so that Eqn (13) (see Experimental procedures), which describes velocity, gives inaccurate values for a (a < 1) and therefore b ⁄ a > The difference in the extent of activation can be explained by differences in the rate-limiting steps Because the rate-limiting step for the hydrolysis of o-NAC is acylation [35], it follows that the activating effects of tyramine take place at the level of acylation For the hydrolysis of o-NPA, both acylation and deacylation are partly rate limiting [35] If, by analogy with its effect on o-NAC, the activating effects of tyramine reflect the acceleration of acylation, the activation of hydrolysis of o-NPA should become limited by the deacylation rate This predicts that activation should result in a modest increase in activity Because the difference between the rates for acylation and deacylation must be greater for o-NAC, the activation would be expected to be greater, matching the observations obtained (Table 1) The binding of tyramine to D70G, in the presence of o-NPA, is weaker (9.1-fold) than binding to the wild-type enzyme, and induces an activating effect on o-NPA hydrolysis (Table 1) However, it was not possible to determine the a and b parameters by nonlinear fitting of Eqn (13) The activating effect on o-NAC hydrolysis is apparent only for tyramine concentrations greater than mm It is so small that it cannot be quantified It is clear, however, that there is a reduction in affinity with D70G for this ligand This supports the hypothesis that PAS plays a role in binding of this ligand to BuChE It was found that tyramine inhibited the turnover of the positively charged substrates ASCh and ATMA in a linear competitive manner (Table 2) The affinity of tyramine for wild-type BuChE, in this inhibitory capacity (0.78–2.4 mm), is essentially the same as its affinity for the wild-type enzyme in its activating capacity for neutral substrates (0.8–1.0 mm) The same is true for the binding of tyramine to D70G, although the affinity of D70G for tyramine is weaker than the affinity of the wild-type enzyme From the data in Tables and 2, it can reasonably be stated that, for each enzyme form, Ki = Ka This strongly suggests that FEBS Journal 275 (2008) 2617–2631 ª 2008 The Authors Journal compilation ª 2008 FEBS 2621 Modulation of butyrylcholinesterase catalytic activitiy P Masson et al both the competitive inhibition of hydrolysis of positively charged substrates and activation of hydrolysis of neutral substrates result from tyramine binding to PAS Such qualitatively opposite effects can be tentatively interpreted in terms of allosteric inhibition ⁄ activation: the binding of tyramine to PAS induces a conformational change that affects the formation of the productive enzyme–substrate complex It should be remembered that PAS and the binding locus (W82) of the active site are connected through an W loop [29,44] For positively charged substrates, the conformational change prevents the productive binding of substrate, probably by disrupting the W82–p-cation interaction; in contrast, for neutral substrates, the conformational change optimizes the enzyme–substrate orientation in the active site pocket for acylation Effects of serotonin It was found that serotonin inhibited both esterase and AAA activities of BuChE (Tables and 2), in contrast with previous reports [22] The inhibition of wild-type BuChE was partially (hyperbolic) competitive with the neutral substrate o-NPA (Fig 2A) It was linearly competitive with o-NAC (Fig 2B) and with the positively charged substrates ASCh and ATMA (data not shown) The inhibition of D70G was linear with all four substrates The affinity of D70G for serotonin was generally lower than that of the wild-type enzyme: 7.3-fold in the presence of o-NPA, 5.2-fold with ASCh, A 1/n (1/ΔAλ 410mn), The hydrolysis of both o-NPA and o-NAC by wildtype BuChE displayed complexities in the presence of benzalkonium The hydrolysis of o-NAC was activated at low benzalkonium concentration, and then inhibited as the benzalkonium concentration increased (Fig 3; Table 1) There was no activation phase for the hydrolysis of o-NPA Inhibition was parabolic and partial This biphasic behaviour suggests at least two binding sites for benzalkonium Activation of the BuChE-catalysed hydrolysis of o-NAC by low concentrations of benzalkonium has been reported previously [42] With o-NPA and o-NAC, benzalkonium shows parabolic competitive inhibition Parabolic inhibition suggests that the binding of more than one benzalkonium contributes to the inhibition (Fig 4) The multiplicity of cation binding sites was revealed with phenoxazine ⁄ phenothiazine dyes for wild-type BuChE [48], 40 [S]/n 30 20 10 10 12 14 16 [Serotonin] mM 10 12 14 16 [Serotonin] mM 10000 4000 3500 S = o-NAC 8000 3000 2500 [S]/n 1/n (ΔAλ 430nm), Effects of benzalkonium on o-NPA and o-NAC hydrolysis 50 S = o-NPA B three-fold with ATMA and unaffected with o-NAC The actual binding site of serotonin cannot be inferred from these results Although serotonin can bind to the active site binding locus (W82), binding to PAS cannot be ruled out If serotonin binds only to W82, the affinity differences between wild-type BuChE and D70G mutant for this ligand could reflect differences in the conformational plasticity of the active site gorge of these enzymes 2000 1500 1000 6000 4000 2000 500 -10 2622 -5 10 [Serotonin] mM 15 0 [Serotonin] mM 10 12 Fig Inhibitory effect of serotonin on wildtype BuChE-catalysed hydrolysis of o-NPA and o-NAC in 0.1 M phosphate buffer at 25 °C (A) o-NPA (d, 0.1 mM; s, 0.2 mM; , 0.4 mM; h, 0.6 mM; , 0.8 mM): left panel, Dixon plots of v)1 versus [serotonin]; right panel, Cornish–Bowden plots of [S] ⁄ v versus [serotonin] Nonlinear plots indicate partial inhibition (B) o-NAC (d, mM; s, mM; , 10 mM): left panel, Dixon plots of v)1 versus [serotonin]; right panel, Cornish– Bowden plots of [S] ⁄ v versus [serotonin] Converging Dixon plots and parallel Cornish–Bowden plots indicate competitive inhibition FEBS Journal 275 (2008) 2617–2631 ª 2008 The Authors Journal compilation ª 2008 FEBS P Masson et al Modulation of butyrylcholinesterase catalytic activitiy 0.010 ΔA430nm/min 0.008 0.006 0.004 0.002 S = o-NAC 0.000 0.0 0.2 0.1 0.3 0.5 0.4 [benzalkonium] mM Fig Concentration-dependent activation and inhibition of BuChEcatalysed hydrolysis of o-NAC (bottom curves, mM; top curves, mM) by benzalkonium Full lines, wild-type enzyme; broken lines, D70G mutant With D70G, benzalkonium showed a clear hyperbolic activation of o-NAC hydrolysis at low concentrations, and parabolic inhibition at high concentrations (Fig 3; Table 1) There was slight activation of o-NPA hydrolysis at low [S] and low benzalkonium concentration (data not shown) Under these conditions, the hydrolysis kinetics are first order (cf Eqn 12) with kcat ⁄ Km = k2 ⁄ Ks At low [S], the ‘buffer’ contribution of deacylation does not take place, and activation reflects an effect on acylation (k2) The fact that D70G is slightly activated at low benzalkonium concentrations, whereas the wild-type enzyme is not, indicates that b ⁄ a ‡ for D70G, whereas b ⁄ a < for the wildtype enzyme This subtle difference in behaviour between the two enzyme forms reflects the higher conformational plasticity of the active site gorge of D70G compared with that of the wild-type enzyme for acylation with neutral ester Effects of benzalkonium on ATMA hydrolysis 1200 1/n (1/ΔAλ430nm), 1000 S = o-NAC 800 600 400 200 0 0.1 0.2 0.3 0.4 0.5 [benzalkonium] mM Fig Dixon plot of the inhibitory portion of the effect of benzalkonium on the wild-type BuChE-catalysed hydrolysis of o-NAC The plot shows only benzalkonium concentrations greater than 0.1 mM The substrate concentration was mM o-NAC Nonlinearity indicates multiple binding and with propidium for a mutant (A277W ⁄ G283D) having PAS similar to that of acetylcholinesterase [49] The fact that benzalkonium acts as an apparent activator at low concentrations with o-NAC and not with o-NPA suggests that the activation of wild-type BuChE occurs at the level of the acylation step, similar to the mechanism suggested for tyramine Activation is observable with o-NAC because acylation is rate limiting (kcat = k2), whereas it is ‘buffered’ with o-NPA because acylation and deacylation are both partly rate limiting [35] Hydrolysis of ATMA by wild-type BuChE in the presence of increasing concentrations of benzalkonium gave unusual Lineweaver–Burk plots (Fig 5A) in which the lines intersected in the upper right quadrant at ⁄ [S]cross $ ± 0.5 mm)1 This is consistent with benzalkonium being an inhibitor at low substrate concentration and an activator as the substrate concentration is increased The highest ATMA concentration (0.5 mm) was below Kss = 0.70 mm [35], so that activation by excess substrate did not take place This pattern of inhibition has been reported previously for decamethonium inhibition of the hydrolysis of 7-acetoxy-4-methylcoumarin by acetylcholinesterase [50] The inhibition of wild-type BuChE hydrolysis of ATMA by benzalkonium can be described by Scheme (see Experimental procedures) For this scheme, the Lineweaver–Burk plot is given by Eqn (6) When a > b, a > and b > 1, the Lineweaver–Burk lines intersect in the first quadrant at ⁄ [S]cross     aKm ẵL ỵ Ki 1 ẵL ỵ aKi ẳ ỵ v Vmax bẵL ỵ aKi ẵS Vmax bẵL ỵ aKi 6ị with the coordinates of the intersecting point: b1 Km a bị a1 ẳ Vmax a bị 1=ẵScross ẳ 1=Vmax;cross 7ị 8ị This very rare situation in which ligand L is an inhibitor at low [S] and an activator at high [S], FEBS Journal 275 (2008) 2617–2631 ª 2008 The Authors Journal compilation ª 2008 FEBS 2623 Modulation of butyrylcholinesterase catalytic activitiy A aKi Vmax 1 bVmax ẳ ỵ Dslope Km a bị ẵL Km a bị 7000 0.005 6000 5000 0.00 0.00 0.001 4000 –10 10 20 1/[benzalkonium] àM 3000 2000 Dintercept ẳ wild type 1000 10 1/[ATMA] mM ð11Þ aKi Vmax bVmax ẳ ỵ Dintercept b 1ị ½LŠ ðb À 1Þ ð12Þ 5000 4000 1/v (1/ΔAλ290 nm), ẵL ỵ aKi ị Vmax bẵL ỵ aKi Þ Vmax and B ð10Þ when [L] fi ¥, the intercept on the Dslope)1 axis in the re-plot is bVmax ⁄ Km(a – b), the intercept on the [L])1 axis is – b ⁄ aKi and the slope is aKiVmax ⁄ Km (a ) b) Dintercept is the difference between the intercept of the Lineweaver–Burk plot at ligand concentration [L] and the intercept without ligand (Eqn 11): 0.004 1/Δ slope 1/v (1/ΔAλ290 nm), P Masson et al 3000 2000 1000 0.0 D70G 0.2 0.4 0.6 0.8 1.0 1/[ATMA] mM Fig Lineweaver–Burk plots for the inhibition of BuChE-catalysed hydrolysis of ATMA (0.1–0.5 mM) by different concentrations of benzalkonium in 0.1 M phosphate buffer at 25 °C (A) Wildtype BuChE:d, no benzalkonium;s, 0.05 lM; , 0.1 lM; h, 0.2 lM; Ô, 0.3 lM Inset: re-plot of Dslope)1 of the Lineweaver– Burk plot as a function of the reciprocal of the benzalkonium concentration (B) Mutant D70G:d, no benzalkonium; s, lM; , lM; h, lM beyond [S]cross, is symmetrical to system C5 of partial and mixed inhibition as described by Segel [51] The values for a and b can be determined from the re-plots of ⁄ Dslope versus ⁄ [L] and ⁄ Dintercept versus ⁄ [L] [51]; Dslope of the Lineweaver–Burk plot is the difference between the slope at ligand concentration [L] and the slope without ligand (Eqn 9): Dslope ¼ and 2624 aKm ẵL ỵ Ki ị Km Vmax bẵL þ aKi Þ Vmax ð9Þ Parameter b = 3.1 was determined from the intercept on the Dintercept)1 axis re-plot, i.e bVmax ⁄ (b ) 1) Then, parameter a was determined from the intercept on the Dslope)1 axis in the re-plot of Dslope)1 versus [L])1, and Ki was determined from the intercept on the [L])1 axis of the Dslope)1 re-plot (Fig 5A, inset) This gave a = 5.7 and Ki = 0.05 lm The high value of a reflects the decreased affinity of benzalkonium for the enzyme–substrate complex This result is consistent with the proposal that the binding site for benzalkonium is either at PAS or in the active site gorge close to PAS The binding of benzalkonium would then have to induce a conformational change at the active site that is responsible for the increase in kcat at high [S] beyond [S]cross Thus, the activating effect of benzalkonium produces an effect similar to the activation by excess substrate that has been found to be dependent on the integrity of PAS [44,49,52,53] Because the rate-limiting step for the hydrolysis of ATMA is acylation (k2 > k3) [35], it is probable that activation reflects an increase in the acylation rate This is similar to the activating effect of tyramine binding on the hydrolysis of o-NAC and o-NPA The observation that benzalkonium is an inhibitor of BuChE-catalysed hydrolysis of ATMA at low [S] and an activator at high [S] suggests that the conformational change induced by the occupancy of PAS is different at low and high [S] This difference could be the result of the binding of a second substrate molecule in the gorge at high concentration, which causes a different (activating) conformational change in the active site Because the rate-limiting step of BuChE-catalysed hydrolysis of ATMA is acylation, the second substrate molecule must bind in the active site gorge of the enzyme already complexed with the first substrate molecule In acetylcholinesterase, binding of an additional FEBS Journal 275 (2008) 2617–2631 ª 2008 The Authors Journal compilation ª 2008 FEBS P Masson et al substrate molecule to PAS and in the gorge has been shown to inhibit enzyme activity by preventing the exit of reaction products [54,55] In contrast, the active site ˚ gorge of BuChE is about 200 A3 larger than that of the acetylcholinesterase gorge, and therefore may easily accommodate several ligands ⁄ substrates [29,56] without inhibition of substrate and product traffic The inhibition of D70G by benzalkonium appears to be purely noncompetitive, i.e the lines in the Lineweaver–Burk plot cross on the y-axis (Fig 5B) with Ki = 13 ± lm Thus, the affinity of D70G for benzalkonium is at least 260-fold weaker than that of the wild-type enzyme This suggests that PAS is the binding site for benzalkonium, and supports the proposal that the complexity encountered with the wild-type enzyme reflects the binding of benzalkonium to PAS Effects of benzalkonium on ASCh hydrolysis Under our experimental conditions, a study of the inhibition of ASCh hydrolysis was not possible because 5,5¢-dithiobis(2-nitrobenzoic acid) (Nbs2) precipitated with benzalkonium However, the inhibition of BuChE-catalysed hydrolysis of ASCh by benzalkonium has been reported [42] It is unclear how these authors avoided the precipitation problem In that study, the inhibition of human BuChE was found to be of the partial mixed type Unfortunately, the experiments were performed at high substrate concentration, in the concentration range corresponding to substrate activation (cf Experimental procedures, Scheme 2, Eqn 10) Thus, the reported Ki value (1.03 lm) [42] probably reflects the inhibition of substrate activation That is, benzalkonium is probably competing with the formation of both SpE and the productive ternary complex SpES (cf Scheme 2) Under these conditions, the formation of SpE and SpES is governed by a high Kss (Kss = 0.6 mm), which is about 10 times higher than Km The Ki value of 1.03 lm reported by these authors is 20 times higher than our Ki value for the inhibition of the BuChE-catalysed hydrolysis of ATMA (at low concentrations of ATMA) This difference supports our interpretation that these authors were observing effects related to the substrate activation portion of the mechanism and not to the primary hydrolytic steps Interaction of propanil with BuChE Propanil (3¢,4¢-dichloroacetanilide) was not hydrolysed by wild-type BuChE under our experimental conditions, i.e [E] > [S] Yet, propanil binds to BuChE and linearly inhibits the hydrolysis of o-NTFNAC and Modulation of butyrylcholinesterase catalytic activitiy ASCh over a large substrate concentration range Inhibition constants were determined from Dixon plots and Cornish–Bowden plots (data not shown) Propanil is a pure competitive inhibitor (a = 0) of the BuChEcatalysed hydrolysis of both substrates: Ki = 0.49 ± 0.05 mm with ASCh, and Ki = 0.74 ± 0.58 mm with o-NTFNAC Thus, propanil interferes with the formation of ES, but not with SpE or SpES In the BuChE–ASCh complex, the choline head group strongly interacts with W82 [29] The fact that propanil is a competitive inhibitor suggests that it also binds to the p-cation binding site W82 These results imply that other acetyl anilide substrates (i.e o-NAC, o-NTFNAC, ATMA) may bind to W82 in the active centre This would place the substrate in the BuChE– acetanilide substrate complexes into a favourable position for the use of the catalytic triad Ser198 ⁄ H438 ⁄ E325 to make products The resistance of propanil to hydrolysis by BuChE probably results from electronic effects contributed by the polar chlorine atoms in the aromatic ring that could hamper the rotational flexibility of the amide bond [57] This could impair appropriate orientation of the carbonyl oxygen in the oxyanion hole Reaction of N-acetylanthranilic acid (NATAc) with BuChE Hydrolysis of the negatively charged acetanilide NATAc by BuChE was attempted We chose this substrate because it is homologous to aspirin (N-acetylsalicylic acid), a negatively charged acetyl ester that is a BuChE substrate [58] We found that BuChE, even at high concentration ([E] = 0.03 lm), does not hydrolyse NATAc ([S]max = 0.5 mm) Moreover, NATAc up to mm did not inhibit the BuChE-catalysed hydrolysis of ASCh (0.035–1 mm) or o-NTFNAC (2–3 mm) Therefore, it does not appear to bind Competition of NATAc with the three selected ligands was not investigated Active structure site responsible for AAA activity It has recently been suggested that Ser224 is the nucleophile involved in the hydrolysis of aryl acylamides by BuChE [33] However, several lines of structural evidence clearly rule out this hypothesis First, kinetic analysis of organophosphate inhibition of the ester and AAA activities of BuChE indicates that there is a single nucleophilic serine, Ser198, for both activities [35] Second, studies on mutant forms, e.g silent allozyme and S198C ⁄ D mutants of BuChE, support the kinetic findings with wild-type BuChE and rule out the FEBS Journal 275 (2008) 2617–2631 ª 2008 The Authors Journal compilation ª 2008 FEBS 2625 Modulation of butyrylcholinesterase catalytic activitiy P Masson et al A Ser198 Ser224 B Ser198 2.8 His438 2.7 wat 2.7 Ser224 2.9 2.5 Glu325 wat Fig (A) Overall view of the three-dimensional structure of human BuChE The solvent-accessible surface is represented by a mesh Helices are represented as coils and b-sheets by arrows Ser224 is represented by cyan balls and Ser198 is represented by green balls with their respective Oc in red (B) Hydrogen bond network associated with Ser224 and Ser198 Participating residues are represented as sticks and water molecules as balls Hydrogen bond distances are given for the catalytic triad residues Ser198 ⁄ His438 ⁄ Glu325 (green) and Ser224 (cyan) hypothesis that a nucleophile other than Ser198 is responsible for the AAA activity [35] Third, inspection of the three-dimensional structure of human BuChE shows that Ser224 is deeply buried inside the protein, ˚ with Oc pointing away from the surface, about 6–7 A from the bulk solvent [29] (Fig 6A) Therefore, no access for substrate to Ser224 is possible The latter problem was acknowledged by the authors of the Ser224 proposal However, it was argued that the binding of ligands such as benzalkonium may induce a conformational change that activates a Ser224 ⁄ His438 ⁄ E197 triad [33] Our present results 2626 show that the effect of benzalkonium on the AAA activity of BuChE can be interpreted without postulating the unmasking of an alternative nucleophile A conformational change of the enzyme that would give accessibility to Ser224 is unlikely, because it would require a large movement of the main chain and subsequent disorganization of the central b-sheet Moreover, catalysis relies on optimal angles and distances between the nucleophile and the base in order to allow the formation of short, strong hydrogen bonds The observed spatial position of Ser224 and His438 does not allow the formation of such a short, strong hydrogen bond For example, the distance between Ne of ˚ His438 and Oc of Ser224 is 4.8 A In addition, Ser224 is strongly locked in a dense hydrogen bond network that is essential for the integrity of the active site This dense hydrogen bond network prevents any conformational change of this residue (Fig 6B) Ser224 notably makes a strong hydrogen bond with Glu325 ˚ (d $ 2.5 A) Moreover, Ser224 is also hydrogen bonded to two key water molecules that are strictly conserved in all crystal structures of BChE in complexes with charged or uncharged ligands that have been solved to date Ligands invariably fill the pocket near Trp82 without triggering any alteration of this hydrogen bond network Finally, His438 is totally restrained because of a stabilizing interaction with Phe398 In contrast with the observations reported for acetylcholinesterases [59–61], no mobility of the catalytic histidine of human BChE has been observed in crystal structures A change in the position of this catalytic histidine would be necessary for the formation of a Ser224 ⁄ His438 ⁄ E197 triad Conclusions Despite the complexity of interactions between BuChE, tyramine, serotonin and benzalkonium, no fundamental differences were found between the effects of these compounds on the AAA and esterase activities of human BuChE The concentrations of tyramine and serotonin that activate or inhibit the AAA activity of BuChE (and also its esterase activity) are several orders of magnitude higher than the concentrations of these compounds that can be encountered in vivo under physiological conditions or even during pathological processes The concentration of serotonin in human plasma of normal subjects is nm [62]; it is increased several fold as a consequence of migraine headache, schizophrenia, hypertension or carcinoid syndrome The concentration of tyramine in the plasma of normal subjects is about nm [63]; it is increased as a FEBS Journal 275 (2008) 2617–2631 ª 2008 The Authors Journal compilation ª 2008 FEBS P Masson et al consequence of cirrhosis, and on consumption of food containing tyramine Tyramine poisoning causes headache, hypertensive crisis and adverse interactions with antidepressive drugs (cf ‘cheese syndrome’), but, even in these extreme situations, tyramine concentrations never reach the millimolar range In addition, BuChE does not hydrolyse melatonin (P Masson & M T Froment, unpublished results), a neutral aryl acylamide, that is a serotonin metabolite [64] The nocturnal peak concentration of melatonin in blood is 0.3 nm [64] Thus, owing to the low affinity of BuChE for serotonin and neutral aryl acylamides, it is not surprising that this enzyme does not contribute to the hydrolysis of the neurohormone Therefore, the physiological, pharmacological and toxicological relevance of tyramine and serotonin effects on BuChE activity is questionable In this respect, it is unlikely that the AAA activity of BuChE could be a link between serotonergic and cholinergic systems, as has been suggested [38] Exogenous aryl acylamide compounds, drugs and xenobiotics are mostly metabolized in the liver and other organs by amidase-carboxylesterases [20,21] There is no carboxylesterase in human plasma [60,65,66] The only AAA activities present in human plasma are those of albumin and BuChE Both activities are very slow with acetanilides (o-NAC, o-NTFNAC) [26,35] In addition, BuChE does not hydrolyse m-nitroacetanilide [35], the fungicide propanil (m,p-dichlorocacetanilide) (present study) or drugs such as acetaminophen (p-hydroxyacetanilide) and phenacetin (p-ethoxyacetanilide) (P Masson & M T Froment, unpublished results) Although BuChE may interact with high concentrations of these compounds, e.g the inhibition of AAA activity by propanil occurs at a Ki value of about 0.6 mm, such concentrations are far higher than would be expected to occur in blood during treatments or intoxications, even in the most severe cases Therefore, it is unlikely that BuChE plays a significant role in the metabolism of endogenous or exogenous aryl acylamides Experimental procedures Chemicals o-NAC was obtained from Merck (Limonest, France) and benzalkonium chloride was obtained from Interchim (Montlucon, France) Tyramine, serotonin (5-hydroxytryp¸ tamine), o-NPA, o-nitrophenol (o-NP) and o-nitroaniline (o-NA) were purchased from Sigma Chemical France (Saint Quentin Fallavier, France) NATAc and diisopropylfluorophosphate (DFP) were obtained from Acros Organics Modulation of butyrylcholinesterase catalytic activitiy France (Noisy-le-Grand, France) ATMA was a gift from T L Rosenberry (Mayo Clinic, Jacksonville, FL, USA) o-NTFNAC was a gift from S Darvesh (Dalhousie University, Halifax, Canada) Propanil (3¢,4¢-dichloroacetanilide) was obtained from CIL-Cluzeau (Sainte-Foy-La-Grande, France) Other chemicals were of biochemical grade Enzymes Wild-type recombinant human BuChE and the mutant enzyme D70G were expressed in CHO-K1 cells (ATCC, N°CCL 61, Rockville, MD, USA) Enzymes were highly purified by anion exchange and affinity chromatography in 20 mm potassium phosphate pH 7.0 containing mm EDTA, as described previously [44,67] The absence of contaminating AAAs in purified enzymes, i.e cell carboxylesterases and albumin, was controlled by nondenaturing PAGE and activity staining [26,68] Kinetics of substrate hydrolysis in the absence of ligand Kinetic assays with neutral and positively and negatively charged acetanilides and acetyl esters were carried out at 25 °C in 0.1 m phosphate buffer pH 7.0, except for o-NTFNAC for which the buffer was 60 mm Tris ⁄ HCl pH 8.0 The measured rates were corrected for spontaneous hydrolysis of the substrates BuChE activity on a negatively charged substrate, NATAc, was studied under the same conditions Acetanilides Kinetic analysis of BuChE AAA activity was carried out with a positively charged acetanilide (ATMA), a negatively charged acetanilide (NATAc) and neutral acetanilides (o-NAC and o-NTFNAC) The stock solution of ATMA was in phosphate buffer, that of o-NAC was in methanol, and those of o-NTFNAC and NATAc were in 50% acetonitrile Assays with ATMA were carried out according to Johnson et al [69] The substrate concentration ranged from 0.025 to mm The hydrolysis of ATMA was recorded for 30 at 290 nm (eo)NTMNPA = 1850 m)1Ỉcm)1), and rate measurements were performed on the steady-state phase [26] Assays with NATAc were carried out according to Kolkenbrock et al [70] The hydrolysis of NATAc was recorded for 30 at 293 and 325 nm Assays with o-NAC were carried out according to the method of Hoagland and Graf [71] The final methanol concentration in the assays was 5% The substrate concentration in the assays was in the range 0.05– mm Assays with o-NTFNAC were carried out according to Darvesh et al [34] The final concentration of acetonitrile in the assays was 3.5% The hydrolysis of o-NAC was recorded for 45 at 430 nm for o-NA (eo-NA = 4000 m)1Ỉcm)1) FEBS Journal 275 (2008) 2617–2631 ª 2008 The Authors Journal compilation ª 2008 FEBS 2627 Modulation of butyrylcholinesterase catalytic activitiy P Masson et al The hydrolysis of o-NTFNAC was recorded at 430 nm (e = 3954 m)1Ỉcm)1) for Acetyl esters Kinetic analysis of the BuChE-catalysed hydrolysis of o-NPA was followed by monitoring the rate of appearance of o-NP at 410 nm (eo-NP = 3190 m)1Ỉcm)1) The stock solution of o-NPA was in methanol The substrate concentration in the assays ranged from 0.05 to 3.5 mm The final methanol concentration in each assay was 5.3% [45] Hydrolysis of ASCh was followed at 420 nm using the method of Ellman et al [72] with 0.5 mm Nbs2 as the chromogenic reagent The substrate concentration in the assays ranged from 0.05 to 3.5 mm Kinetics under steady-state conditions tration of tyramine in the assays varied from 0.1 to 15 mm, that of serotonin from 0.05 to 15 mm, and that of benzalkonium from 0.05 lm to 0.5 mm The type of inhibition or activation was determined by visual inspection of velocity versus substrate concentration plots (at various ligand concentrations), Lineweaver–Burk plots, and a combination of Dixon and Cornish–Bowden plots [74] For benzalkonium, interactions between BuChE (E), substrate (S) and ligand (L) showed complications, but can be depicted by Scheme at low [L] The ligand L, acting either as an inhibitor or an activator, can bind to the free enzyme (E) or the enzyme–substrate complex (ES) and modify Km and kcat K is either an inhibition constant (Ki) or an activation constant (Ka) Assuming rapid equilibrium, the velocity equation for Scheme is described by Eqn (13): v¼ Under our experimental conditions, the BuChE-catalysed hydrolysis of o-NAP can be investigated up to saturation However, because of the lower solubility of o-NAC and o-NTFNAC, studies with these substrates were carried out at concentrations much lower than Km (cf [35]) The steady-state catalytic parameters Km, kcat, Kss and b were determined by nonlinear computed fitting of Eqns (1,5) using the sigma plot program (Jandel Science, San Raphael, CA, USA) The active site concentration [E] of highly purified enzyme preparations was determined according to [73] with DFP as the titrant The active site concentration of wild-type BuChE was 0.16 ± 0.01 lm, and that of the D70G mutant was 0.196 ± 0.02 lm Effects of ligands on steady-state kinetics The effects of the ligands (L: tyramine, serotonin and benzalkonium) on the AAA and esterase activities of wild-type BuChE and its D70G mutant were investigated Assays were performed in 0.1 m phosphate buffer pH 7.0 at 25 °C The effect of each ligand on related pairs of substrates (neutral substrates o-NAC and o-NPA, and positively charged substrates ATMA and ASCh) was compared To avoid complications caused by substrate activation with ATMA and ASCh, assays were performed at low and intermediate substrate concentrations ([S] < Kss) The concen- kcat ẵEẵS aKm fẵL ỵ Kị=bẵL ỵ aKịg ỵ ẵSfẵL ỵ aKị=bẵL ỵ aKịg 13ị At low substrate concentrations, [S] > Km: vẳ kcat f1 ỵ bẵLị=aKịg ẵEẵS Km f1 ỵ ẵL=Kịg 14ị If a = 0, inhibition is competitive; if a = 1, inhibition is noncompetitive; if a > 1, inhibition is mixed If the ternary complex ESL is nonproductive (b = 0), there is linear mixed inhibition If ESL is productive with b > 1, there is activation Activation and inhibition of mixed type are symmetric If ESL makes product with < b < and b ⁄ a < 1, there is nonlinear hyperbolic inhibition In hyperbolic activation, < a < and b ⁄ a > More complex situations that can be encountered, e.g multiple binding, are examined in the Results and Discussion section It should be noted that only the b ⁄ a ratio can be determined from Eqn (14) (cf Table 1; activation of BuChE-catalysed hydrolysis of o-NAC by tyramine) Kinetic constants were determined by: (a) linearizing the Dixon ⁄ Cornish–Bowden plots [74], followed by fitting the line with Sigma Plot 4; (b) re-plotting the slopes from Lineweaver–Burk plots according to Segel [51], followed by fitting the resulting line with Sigma Plot 4; or (c) fitting the data directly to the rate equations that describe Scheme GOSA-fit, a simulated annealing based fitting software (BioLog, Toulouse, France; http://www.bio-log.biz) was used for the fitting of nonlinear inhibition ⁄ activation kinetics only Effect of propanil Scheme General scheme for nonessential activation or partial and mixed-type inhibition of a Michaelian enzyme 2628 Propanil was tested as a possible acetanilide substrate of BuChE It was assayed with wild-type enzyme in the same manner as o-NAC Absorbance measurements were carried out at 430 nm for The effect of propanil (seven FEBS Journal 275 (2008) 2617–2631 ª 2008 The Authors Journal compilation ª 2008 FEBS P Masson et al concentrations ranging from 0.05 to 0.5 mm) on the BuChE-catalysed hydrolysis of o-NTFNAC and ASCh was examined at four concentrations of o-NTFNAC (0.1, 0.3, 0.5 and mm) and seven concentrations of ASCh (0.025, 0.05, 0.075, 0.1, 0.25, 0.5 and mm) Acknowledgements The authors thank Professor T Rosenberry (Mayo Clinic, Jacksonville, FL, USA) for the gift of ATMA and Dr S Darvesh (Dalhousie University, Halifax, Canada) for the gift of o-NTFNAC The authors are grateful to Ellen Duysen and Hasmik Grigoryan (UNMC, Eppley Institute, Omaha, NE, USA) for their ´ ´ interest in this study This work was supported by Dele´ ´ gation Generale pour l’Armement/Programme d’Etudes Amont (DGA/PEA) No 01 08 07 ⁄ 03 CO 010-05 to PM Modulation of butyrylcholinesterase catalytic activitiy 10 11 12 13 14 15 References ´ Massoulie J, Pezzementi L, Bon S, Krejci E & Vallette FM (1993) Molecular and cellular biology of cholinesterases Prog Neurobiol 41, 31–91 Darvesh S, Hopkins DA & Geula C (2003) Neurobiology of 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FEBS 2621 Modulation of butyrylcholinesterase catalytic activitiy P Masson et al both the competitive inhibition of hydrolysis of positively charged substrates and activation of hydrolysis of neutral