Báo cáo khoa học: Kinetics of inhibition of acetylcholinesterase in the presence of acetonitrile doc

Kinetics of inhibition of acetylcholinesterase in the presence of acetonitrile Markus Pietsch, Leonie Christian, Therese Inhester, Susanne Petzold and Michael Gutschow ă Pharmaceutical Chemistry I, Pharmaceutical Institute, University of Bonn, Germany Keywords acetylcholinesterase; enzyme kinetics; gallamine triethiodide; hyperbolic mixed-type inhibition; tacrine hydrochloride Correspondence M Pietsch, School of Chemistry & Physics, The University of Adelaide, Adelaide, SA 5005, Australia Fax: +61 8303 4358 Tel: +61 8303 5360 E-mail: markus.pietsch@adelaide.edu.au (Received 15 August 2008, revised 10 January 2009, accepted 11 February 2009) doi:10.1111/j.1742-4658.2009.06957.x The hydrolysis of acetylthiocholine by acetylcholinesterase from Electrophorus electricus was investigated in the presence of the inhibitors tacrine, gallamine and compound The interaction of the enzyme with the substrate and the inhibitors was characterized by the parameters KI, a¢, b or b, Km and Vmax, which were determined directly and simultaneously from nonlinear Michaelis–Menten plots Tacrine was shown to act as a mixedtype inhibitor with a strong noncompetitive component (a¢ % 1) and to completely block deacylation of the acyl-enzyme In contrast, acetylcholinesterase inhibition by gallamine followed the ‘steric blockade hypothesis’, i.e only substrate association to as well as substrate ⁄ product dissociation from the active site were reduced in the presence of the inhibitor The relative efficiency of the acetylcholinesterase–gallamine complex for the catalysis of substrate conversion was determined to be 1.7–25% of that of the free enzyme Substrate hydrolysis and the inhibition of acetylcholinesterase were also investigated in the presence of 6% acetonitrile, and a competitive pseudo-inhibition was observed for acetonitrile (KI = 0.25 m) The interaction of acetylcholinesterase with acetonitrile and tacrine or gallamine resulted in a seven- to 10-fold increase in the KI values, whereas the principal mode of inhibition was not affected by the organic solvent The determination of the inhibitory parameters of compound in the presence of acetonitrile revealed that the substance acts as a hyperbolic mixed-type inhibitor of acetylcholinesterase The complex formed by the enzyme and the inhibitor still catalysed product formation with 8.7–9.6% relative efficiency Acetylcholinesterase (AChE, EC 3.1.1.7) is a serine hydrolase [1], which belongs to the a ⁄ b hydrolase family [2,3] The enzyme hydrolyses a broad range of ester and amide substrates, showing the highest specificity for acetylselenocholine, acetylthiocholine (ATCh) and acetylcholine (ACh) [4] Substrate cleavage proceeds via a two-step mechanism: acylation of the enzyme, followed by deacylation involving a water molecule [5–7] This process is mediated by the catalytic triad Ser200–His440–Glu327 (Torpedo californica AChE, TcAChE, numbering [8]) located within the ˚ active site at the bottom of a 20 A deep gorge Substrate binding is facilitated by another component of the active site, the anionic site, which is characterized by several conserved aromatic residues, such as Trp84 and Phe330 These residues have been shown to interact with the quaternary ammonium groups of ACh or ATCh via cation–p interactions [7–12] Further stabilization of the quaternary moiety arises from an electrostatic interaction with the acidic side-chain of Glu199 [7,12] A second substrate-binding site, the peripheral anionic site (PAS), lies essentially on the Abbreviations ACh, acetylcholine; AChE, acetylcholinesterase; AD, Alzheimer’s disease; AP2238, 3-(4-{[benzyl(methyl)amino]methyl}phenyl)-6,7-dimethoxy2H-2-chromenone; ATCh, acetylthiocholine; Ab, amyloid-b; MeCN, acetonitrile; Nbs2, 5,5¢-dithiobis(2-nitrobenzoic acid); PAS, peripheral anionic site; Tc, Torpedo californica 2292 FEBS Journal 276 (2009) 2292–2307 ª 2009 The Authors Journal compilation ª 2009 FEBS M Pietsch et al Inhibition kinetics of acetylcholinesterase surface of AChE [8] and consists of five residues, Tyr70, Asp72, Tyr121, Trp279 and Tyr334, clustered around the entrance of the active site gorge [13–17] PAS binds ACh transiently as the first step in the catalytic pathway, enhancing the catalytic efficiency by trapping the substrate on its way to the active site, and allosterically modulates catalysis [7,12,18–22] The principal physiological function of AChE, mediated by the active site of the enzyme, is the rapid hydrolysis of the neurotransmitter ACh at cholinergic synapses and neuromuscular junctions, resulting in the termination of the nerve impulse In addition to this ‘classical’ function, several ‘nonclassical’ activities of AChE have been reported, which are associated with PAS [9,21,23,24] AChE is involved in neurite growth [25], haematopoiesis and osteogenesis [26], and acts as an adhesion protein in synaptic development and maintenance [9] AChE has also been shown to promote the pathophysiological assembly of the amyloid-b (Ab) peptide into amyloid fibrils in vitro [27,28] and in vivo [29,30], with complexes of AChE and Ab displaying an enhanced neurotoxicity in comparison with fibrils formed by Ab alone [31–33] AChE has been found to be associated with amyloid plaques and neurofibrillary tangles, two hallmarks of Alzheimer’s disease (AD), and may contribute to their development [34–36] A third characteristic symptom of AD is the decrease in cholinergic neurons, which causes a loss of cholinergic neurotransmission and may be responsible for the common signs of memory failure [37,38] This ‘cholinergic hypothesis’ provided the rationale for the current major therapeutic approach to AD: the inhibition of the catalytic function of AChE, thereby increasing the bioavailability of ACh at the synaptic cleft, resulting in an improvement in cholinergic neurotransmission and cognitive function [38–40] With regard to the involvement of PAS in the processes of AD, the use of PAS inhibitors and dual-site inhibitors of AChE allows for the inhibition of the catalytic activity of the enzyme and also lowers the incidence of Ab fibril assembly [33,41,42] Prototypes of AChE inhibitors known to bind at the active site, PAS or both sites simultaneously are tacrine, gallamine and donepezil (Fig 1), respectively The crystal structures of each complexed with AChE have been published [10,41,43] In a previous study, we described 7-benzyl5,6,7,8-tetrahydro-2-isopropylamino-4H-pyrido[4¢,3¢:4,5]thieno[2,3-d][1,3]thiazin-4-one (compound 1) (Fig 1), which inhibits AChE in the submicromolar range Kinetic analysis and structural similarities between donepezil, AP2238 (Fig 1) and compound suggest that these substances act as dual-site inhibitors of AChE and bind along the active site gorge [42–44] On the basis of these results, we performed a detailed kinetic study with the prototype inhibitors tacrine and gallamine, as well as compound The interaction of the inhibitors with AChE from Electrophorus electricus was characterized using the kinetic models of AChE inhibition, shown in Scheme [45,46] and Scheme [47], as well as the simplified model for hyperbolic mixed-type inhibitors (Scheme 2), i.e general modifiers [48–50] The analysis presented herein allowed for the simultaneous determination of the kinetic parameters KI, a¢, b or b, Km and Vmax directly from nonlinear Michaelis–Menten plots Recently, the interaction of gallamine and tacrine with AChE was found to be dependent on the presence of acetonitrile (MeCN) [51], a cosolvent frequently used in AChE inhibition assays [44,51–54] In our ongoing investigations, this finding N N NH2 O 3I O O O x HCl O N N O N Tacrine hydrochloride Donepezil Gallamine triethiodide N O O O N S S O AP2238 H N N O Fig Inhibitors of AChE FEBS Journal 276 (2009) 2292–2307 ª 2009 The Authors Journal compilation ª 2009 FEBS 2293 Inhibition kinetics of acetylcholinesterase S + kS E k–S + I + k–SI kS2 S + EI E + P2 H2O I kSI k–I k3 P1 + EA I + kI k2 ES M Pietsch et al ESI k–AI kAI ak2 bk3 P1 + EAI k–S2 EI + P2 H2O Scheme Kinetic model of AChE inhibition KS E I α'K s EI + E + P α'K I KI S kP + I + ES' + S ESI' β'kP EI + P Scheme Simplified kinetic model of the general modifier mechanism S + kS E ES k2 k–S + + E + P2 H2O I I kI k3 P1 + EA k–I EI k AI k–AI EAI bk EI + P2 H2O Scheme Kinetic model of AChE inhibition excluding the formation of ESI was analysed in detail by determining the effect of MeCN on the kinetic parameters of AChE inhibition Km ẳ kS ỵ k2 K   ẳ  S  k2 kS ỵ ỵ k2 k3 k3 Vmax ¼  Results and Discussion Characterization of AChE inhibition and estimation of the inhibitory parameters The inhibition of AChE from E electricus by tacrine, gallamine and compound was determined spectrophotometrically in a coupled assay with the substrate ATCh and 5,5¢-dithiobis(2-nitrobenzoic acid) (Nbs2) Inhibition studies were performed in the absence and presence of 6% v ⁄ v MeCN at various concentrations of both the substrate [S] and the inhibitor [I] For comparison, IC50 values were initially determined at a substrate concentration of 500 lm by plotting the rates versus [I] The inhibitory constants obtained for tacrine 2294 (IC50 = 0.047 ± 0.001 lm, no MeCN; IC50 = 0.34 ± 0.02 lm, 6% MeCN) and gallamine (IC50 = 1100 ± 60 lm, no MeCN; IC50 = 2930 ± 140 lm, 6% MeCN) were in good agreement with results from a previous study [51] In the case of compound 1, AChE inhibition was only determined in the presence of 6% MeCN because of a lack of solubility in the absence of an organic cosolvent As the enzyme was not completely inhibited at high concentrations of compound 1, residual activity at infinite concentration of the inhibitor (v[I] fi ¥) had to be considered Using Eqn (15) (see Experimental procedures), values of IC50 = 0.58 ± 0.02 lm and v[I] fi ¥ = 0.094 ± 0.004 (relative to the activity without inhibitor v0) were determined, which confirmed previously reported data [44] To characterize the inhibition of AChE, a kinetic model was considered (Scheme 1), which is analogous to that introduced by Barnett and Rosenberry [45] and Szegletes et al [46] In this model, the substrate S binds to the enzyme E to form an initial enzyme–substrate complex, also called Michaelis complex ES [55] This complex proceeds to an acylated enzyme intermediate EA, with the acylation rate constant k2, under simultaneous formation of the first product P1 The acyl-enzyme is then hydrolysed with the deacylation rate constant k3 to give the second product P2 and the free enzyme, which enters a new catalytic cycle If ATCh is used as substrate, thiocholine and acetate are formed as P1 and P2, respectively The Michaelis constant Km and the maximal velocity Vmax, which can be experimentally determined, are expressed by Eqns (1) and (2), respectively [56,57]: k2 ẵE0  ỵ k2 k3 1ị ð2Þ where [E]0 is the total enzyme concentration and KS is the dissociation constant of ES The parameter kcat is equal to the quotient Vmax ⁄ [E]0 [46] For the hydrolysis of ATCh by AChE from E electricus, values of the rate constants k2 (1.23 · 106 min)1) and k3 (9.3 · 105 min)1) have been obtained previously by direct measurements of the acetyl-enzyme As k2 is only about 1.3 times larger than k3, both constants are rate influencing [58] An inhibitor I can bind to each of the three enzyme species to form a binary enzyme–inhibitor complex EI, or the ternary complexes ESI and EAI with the FEBS Journal 276 (2009) 2292–2307 ª 2009 The Authors Journal compilation ª 2009 FEBS M Pietsch et al Inhibition kinetics of acetylcholinesterase enzyme–substrate complex and the acyl-enzyme, respectively [46] The EI complex is capable of binding S, and catalyses product formation via ESI and EAI, with the parameters a and b describing the factors by which k2 and k3 are altered As a general steady-state solution for the reaction rate v in Scheme (based on extension of the Michaelis–Menten expression) is too complex for useful comparison with experimental data [45,46,48,59], a virtual equilibrium has been assumed for all reversible reactions in Scheme (i.e k)S ) k2, k)S2 ) ak2, k)SI ) k2, k)SI ) ak2, k)AI ) k3, k)AI ) bk3) [45,46,59] The resulting expression for v is given by Eqn (3) with the dissociation constants KX expressed by the quotients k)X ⁄ kX v ¼ hyperbolic expression for v is, in general, more advantageous, as this method includes no transformation of the primary data, lower standard deviations and less bias in parameter estimates compared with algorithms using linearized plots [60] To apply such a nonlinear optimization, we simplified the kinetic model outlined in Scheme to that of the general modifier mechanism (Scheme 2) [48] In this model, ES and EA are not considered separately, but summed in a complex ES¢ that includes all enzyme–substrate intermediates In an analogous manner, ESI¢ represents the complexes of I with the substrate-bound enzyme and the acyl-enzyme [14] The dissociation constants of S from these binary and ternary complexes are KS = k)S ⁄ kS and a¢KS = k)S2 ⁄ kS2, respectively [49] Both ES and ESI Vmax ẵS 11 0 I I I B1ỵ K C B k B 1ỵK C  k B ỵ K CC SI C ỵ B B IC ỵ ẵS B AI CC Km B @ @ k2 ỵ k3 @ k2 ỵ k3 @ a I A a I A b I AA 1ỵ 1ỵ 1ỵ KSI KSI KAI A direct analysis of AChE inhibition using Eqn (3) is not possible, as contributions from the inhibition of both acylation and deacylation complicate the interpretation of the data In addition, estimates for a and b are not separately available Therefore, the parameters a and b were introduced to facilitate calculation At saturating concentrations of I, these parameters are expressed by Eqns (4) and (5), respectively [46]: a ¼ aKI aKS ¼ KSI KS2 4ị b ẳ ab k2 ỵ k3 ị ak2 ỵ bk3 5ị Under these conditions, i.e [I] Ơ, S exclusively binds to the EI complex and is converted to the products via ESI and EAI The reaction rate, v[I] fi ¥, at a given substrate concentration is defined by a combination of Eqns (35): vẵI!1 ẳ bVmax ẵS b Km ỵ ẵS a 6ị The kinetic parameters KI, a and b are usually determined by linearization of the Michaelis–Menten equation (Eqn 3) on the basis of the Lineweaver–Burk plot and replotting of the slopes and intercepts obtained (after normalization) [46,59] However, it was shown that an iterative nonlinear optimization based on the ð3Þ are capable of product formation (with P being both thiocholine and acetate [14]), governed by the catalytic constants kP and b¢kP, respectively The parameter b¢ reflects the efficiency of hydrolysis of ESI¢ compared with that of ES¢ This type of inhibition (b¢ > 0) is referred to as hyperbolic inhibition, as the shape of a reciprocal velocity ⁄ v versus [I] plot is hyperbolic In contrast, a value of b¢ = causes a linear dependence of ⁄ v on [I], and thus the inhibition is called linear [49,50] The dissociation constant of EI is KI = k)I ⁄ kI [49] and thus defined as in Scheme 1, whereas a¢KI reflects a composite of constants for inhibitor binding to the enzyme–substrate complex and the acyl-enzyme [14] The factor a¢ corresponds to the ratio of a¢KI and KI As the overall equilibrium constant for the formation of ESI¢ must be the same regardless of a path via ES¢ or EI, the same factor a¢ must be included in the model [49,61] Derivation of the general velocity equation for the system in Scheme can be performed assuming a rapid equilibrium or steady-state condition The first method gives a relatively simple expression, whereas the steady-state approach results in a very complex expression containing squared [S] and [I] terms However, the steady-state velocity equation simplifies to the same form as the rapid equilibrium velocity equation when pseudo-equilibrium conditions prevail (i.e k)S ) kP), as, in this case, the Michaelis constant Km = (k)S + kP) ⁄ kS substitutes for KS = k)S ⁄ kS in the velocity equation [49,61,62] In addition, FEBS Journal 276 (2009) 2292–2307 ª 2009 The Authors Journal compilation ª 2009 FEBS 2295 Inhibition kinetics of acetylcholinesterase M Pietsch et al Km has been found to be similar to KS for several substrates of AChE [63] For the purposes of the present study, a rapid equilibrium and an irreversible character of the catalytic step were assumed Under these conditions, the Michaelis–Menten equation for the general type of inhibition shown in Scheme is as follows: v ẳ V ẵS 1max I I B 1ỵ K C B ỵ a0 K C I C ỵ ẵS B IC à Km B @ @ b0 I A b0 I A 1ỵ 1ỵ a KI a KI ð7Þ At saturating concentrations of I, the products are exclusively formed from ESI¢ with a rate constant b¢kP Under these conditions, the rate v[I] fi ¥ can be expressed by Eqn (8) [44,49]: vẵI!1 ẳ b0 Vmax ẵS m ỵ ẵS a0 K 8ị As the rate v[I] ¥ must be the same, regardless of whether the model in Scheme or is applied, Eqns (6) and (8) can be set as equal Therefore, the parameters a and b in Eqn (6) can be expressed by means of a¢ and b¢ as follows: b = b¢ and a = b¢ ⁄ a¢ = b ⁄ a¢ In addition, the value KI is equally defined in both kinetic models (Schemes and 2); thus, all three parameters characterizing the inhibition according to the kinetic model in Scheme 1, i.e KI, a and b, can be determined on the basis of the simplified model depicted in Scheme This methodology was applied to the inhibition of AChE by gallamine (in the absence of MeCN) and compound (with 6% MeCN) The PAS inhibitor gallamine (without MeCN) has already been reported to follow the kinetic model in Scheme [46], whereas inhibition by compound was found not to be complete at saturating concentrations of I, i.e v[I] fi ¥ and therefore b are greater than zero For inhibitors attacking the active site of AChE and containing a positively charged quaternary nitrogen atom, it has been reported that they act not only by binding to the free enzyme at the same site as the substrate, but also by adding to the acyl-enzyme However, these compounds not inhibit through attachment to the Michaelis complex [47,55,64–66] An example of such an inhibitor is tacrine, which has been shown to occupy the anionic binding site of TcAChE by being sandwiched between the aromatic rings of Trp84 and Phe330, mainly through p–p interactions and cation–p interactions In addition, a direct hydrogen bond is formed between the acridinic protonated nitrogen of the inhibitor and the carbonyl oxygen of His440 [10] Crystallographic studies on AChE complexed with ACh and 2296 ATCh, as well as the nonhydrolysable substrate analogue 4-oxo-N,N,N-trimethylpentanaminium [7,12], revealed that these compounds also interact with Trp84 and Phe330 (TcAChE numbering [8]) Thus, it is unlikely that tacrine binds to the Michaelis complex, i.e no ternary complex ESI is formed However, in the crystal structure of AChE with tacrine, the immediate vicinity of the catalytic serine is not occupied by the inhibitor [10], and thus tacrine is probably able to bind to the EA complex [67] Under these conditions, the kinetic model in Scheme can be simplified to that shown in Scheme As I does not interact with ES to form ESI, the value of the dissociation constant KSI in Scheme becomes very large and the quotient KI ⁄ KSI is virtually zero Equation (4) reveals that the ratios KI ⁄ KSI and KS ⁄ KS2 are equal, and thus the dissociation constant KS2 must also become very large (i.e the formation of ESI does not occur via binding of S to EI) An identical model as depicted in Scheme has been used by Krupka and Laidler [47] to explain AChE inhibition caused by the interaction of I with E and EA The Michaelis–Menten equation for this type of inhibition is obtained by simplification of Eqn (3) with KSI fi Ơ: vẳ Vmax ẵS 11 0 1ỵ I   Ã    B B CC I k2 B KAI CC Km 1ỵ K ỵ ẵS B k3 @ k2 ỵk3 ỵ k2 ỵk3 @1ỵb I AA I KAI ð9Þ At saturating concentrations of I, the rate v[I] fi ¥ is equal to zero when calculated using Eqn (9) This means that the kinetic model in Scheme and Eqn (9) are only applicable if complete inhibition occurs In analogy with the kinetic model in Scheme 2, the dissociation constant KAI, obtained from Scheme 3, was termed a¢KI with a¢ corresponding to the ratio of a¢KI and KI Additional rearrangement of Eqn (9) results in the following expression for v: v ẳ Vmax ẵS  10ị  I ỵ bk3 B1 þ  k C B C  Ã  K ỵ k3 C B a I I B k2 C ỵ ẵS B Km ỵ K C I B C 1ỵb I B C @ A a KI Equation (10) was used to analyse AChE inhibition by tacrine in the absence and presence of 6% MeCN In the present study, we applied an iterative nonlinear optimization based on the hyperbolic Michaelis–Menten FEBS Journal 276 (2009) 2292–2307 ª 2009 The Authors Journal compilation ª 2009 FEBS M Pietsch et al Inhibition kinetics of acetylcholinesterase Eqn (7) (with b¢ = b) and Eqn (10) to calculate the parameters KI, a¢, b (Eqn 7) or b (Eqn 10), Km and Vmax simultaneously from plots of rate versus [S] in the presence of various inhibitor concentrations However, provisional estimates of the kinetic parameters were necessary prior to the computer-assisted iterative determination [60] To obtain such estimates for Km and Vmax, we determined these parameters separately for each set of data (tacrine without MeCN, tacrine with MeCN, gallamine without MeCN, gallamine with MeCN, and compound with MeCN) in the absence of inhibitor The data were analysed by a nonlinear regression according to Eqn (11), which represents a simplification of Eqn (3) with [I] = 0: v ¼ Vmax ẵS Km ỵ ẵS 11ị Using this method, the following values of Km and Vmax were calculated for the five sets of data: tacrine, no MeCN: Km = 101 ± 14 lm, Vmax = 110 ± 3%; tacrine, 6% MeCN: Km = 684 ± 41 lm, Vmax = 229 ± 5%; gallamine, no MeCN: Km = 135 ± 19 lm, Vmax = 116 ± 3%; gallamine, 6% MeCN: Km = 671 ± 20 lm, Vmax = 235 ± 3%; compound 1, 6% MeCN: Km = 606 ± 32 lm, Vmax = 229 ± 4% (The rate of the AChE-catalysed hydrolysis of 500 lm ATCh, corrected by the value of the nonenzymatic hydrolysis, was set to 100% in all experiments.) Provisional estimates for KI, a¢ and b in Eqn (7) (b¢ = b) were obtained by analysing the data of the AChE inhibition studies with the specific velocity plot developed by Baici [61] This method is advantageous over the commonly used Lineweaver–Burk plot or the similar Hanes–Woolf plot [49], as it always gives linear plots, independent of whether the inhibition is linear or hyperbolic The type of inhibition can be obtained by simple inspection of the specific velocity plot (Eqn 12), and linear replots permit the calculation of KI, a¢ and b [61] On the basis of Eqn (12), the quotient of the rate without inhibitor and the rate in the presence of inhibitor, v0 ⁄ vI, was plotted against r ⁄ (1 + r), with r being equal to [S] ⁄ Km (Doc S1, Fig S1A,B, see Supporting information):  v0 ¼ vI  Âà 1 Âà I À I 1ỵ K r a0 KI KI I þ 1þr b I b I 1þ 1þ a KI a KI ð12Þ Eqn (13), which is similar to the specific velocity plot (Doc S1, Fig S2A,B, see Supporting information):   bk3 I 1ỵ C B  k2  C B1 ỵ B ỵ k3  ÃC  Ã C B   a KI B I C r I v0 k C ỵ 1ỵ ẳB 1ỵ B vI KI C ỵ r KI b I C B 1ỵ C B a KI C B A @ ð13Þ Investigations of AChE inhibition by tacrine, in the absence and presence of 6% MeCN, on the basis of the modified specific velocity plot (Eqn 13, data not shown) indicated a mixed-type inhibition that tended to noncompetitive inhibition in both cases with KI = 0.038 lm, a¢ = 0.91 and KI = 0.25 lm, a¢ = 1.0, respectively The value b was determined to be equal to –0.004 for the enzyme–inhibitor interaction, both with and without MeCN As b < cannot be defined by the mechanism in Scheme 3, b was set to zero for the calculation of the kinetic parameters by Eqn (10) AChE inhibition by gallamine without MeCN and compound in the presence of 6% MeCN, analysed according to Eqn (12) (Fig S1A, see Supporting information), was found to follow a hyperbolic mixed-type inhibition with a¢ > and b > This is shown for gallamine by the common intersection point of the lines in the specific velocity plot at r ⁄ (1 + r) > 1; v0 ⁄ vI = (Fig S1A, see Supporting information), as well as by discrete intercepts of the replots (Fig S1B, see Supporting information) [61] On the basis of these replots, the parameters KI = 330 lm, a¢ = 5.7 and b = 0.31 were determined Using this method, KI = 0.52 lm, a¢ = 1.3 and b = 0.077 were calculated for the inhibition of AChE by compound (data not shown) In contrast with the study without MeCN, a plot of v0 ⁄ vI versus r ⁄ (1 + r) for AChE inhibition by gallamine in the presence of 6% MeCN showed an array of curves with a common intersection point close to r ⁄ (1 + r) = 1; v0 ⁄ vI = (Fig S2A, see Supporting information) A plot of int0 ⁄ (int0)1) versus ⁄ [I], where int0 is the intercept on the ordinate axis [r ⁄ (1 + r) = 0], and an initial analysis (Fig S2B, see Supporting information) gave an intercept equal to unity Such a behaviour indicates competitive inhibition [61], which is described by Eqn (14): v ¼ To obtain provisional estimates for KI, a¢ and b in Eqn (10), we developed a graphical method based on FEBS Journal 276 (2009) 2292–2307 ª 2009 The Authors Journal compilation ê 2009 FEBS Vmax ẵS   I ỵ ẵS Km ỵ K I 14ị 2297 Inhibition kinetics of acetylcholinesterase M Pietsch et al A 125 100 Rate (%) As an approximation, Eqn (14), a simplified form of the Michaelis–Menten Eqns (3) and (10) valid for competitive inhibitors, and an estimated value KI = 1130 lm, obtained on the basis of the modified specific velocity plot (Eqn 13; Doc S1, Fig S2B, see Supporting information), were used to quantify the interaction of AChE with gallamine in the presence of 6% MeCN 2298 50 25 Determination of the parameters of inhibition using the Michaelis–Menten equation 0 500 1000 1500 2000 2500 2000 2500 [ATCh] (µM) B 200 160 Rate (%) The final kinetic analysis of the inhibition by tacrine (Fig 2A,B), gallamine (Fig 3A,B) and compound (Fig 4) in the absence and presence of 6% MeCN was accomplished using Eqn (7) (b¢ = b), Eqn (10) or Eqn (14), as outlined above The rates of enzymecatalysed substrate cleavage were analysed as a function of both [S] and [I], and the parameters KI, a¢, b or b, Km and Vmax were calculated simultaneously (Table 1) Parameter estimates were taken from (modified) specific velocity plots and Michaelis–Menten plots in the absence of inhibitor (see above) All the values of Km and Vmax calculated independently by the nonlinear optimization of Eqns (7) and (10) (Table 1) were in good agreement with the parameter estimates obtained in the absence of the inhibitors (see above) In the case of gallamine investigated in the presence of MeCN, the calculation of the kinetic parameters was based on Eqn (14), as a competitive mode of inhibition was assumed from the modified specific velocity plot (Fig S2A,B, see Supporting information) The Km value of 778 lm obtained differed considerably from the parameter estimate of 671 lm In contrast, the calculated Vmax value of 245% was very similar to the parameter estimate of 235% This behaviour can be explained by the competitive mode of inhibition The substrate affinity of the enzyme will be reduced, i.e the apparent Km value will be increased, whereas the maximum velocity of product formation Vmax is not affected [49,50] With a given set of data for rates as a function of [S], the determination of Vmax and thus Km becomes less accurate when [S] is low relative to the apparent Km value [49] This occurred in our study for high concentrations of gallamine in the presence of 6% MeCN One possibility to avoid this accuracy problem would be to investigate the enzyme–inhibitor interaction in the presence of higher substrate concentrations However, in the case of AChE, it is known that substrate inhibition arises under such conditions, with PAS being involved in the mechanism [12,19,22,68–70] As gallamine binds to PAS [41], a more complex mode of inhibition might result [71] Therefore, we did not 75 120 80 40 0 500 1000 1500 [ATCh] (µM) Fig Inhibition of AChE by tacrine in the absence (A) and presence (B) of 6% MeCN Michaelis–Menten plots using mean values and standard deviations of rates from four separate experiments in 100 mM sodium phosphate, 100 mM NaCl, pH 7.3 with 350 lM Nbs2 and 0.033 mL)1 AChE (A) Concentrations of tacrine were as follows: open circles, [I] = 0; filled circles, [I] = 0.025 lM; open squares, [I] = 0.05 lM; filled squares, [I] = 0.1 lM; open triangles, [I] = 0.15 lM; filled triangles, [I] = 0.2 lM; open reversed triangles, [I] = 0.25 lM Nonlinear regression according to Eqn (10) gave KI = 0.027 ± 0.003 lM, a¢ = 1.4 ± 0.2, Km = 101 ± lM and Vmax = 110 ± 1% (B) Concentrations of tacrine were as follows: open circles, [I] = 0; filled circles, [I] = 0.125 lM; open squares, [I] = 0.25 lM; filled squares, [I] = 0.5 lM; open triangles, [I] = 0.75 lM; filled triangles, [I] = 1.0 lM; open reversed triangles, [I] = 1.25 lM Nonlinear regression according to Eqn (10) gave KI = 0.26 ± 0.02 lM, a¢ = 1.1 ± 0.2, Km = 691 ± 35 lM and Vmax = 229 ± 4% In (A) and (B), the b values were set to zero, as the starting values obtained from the modified specific velocity plots (data not shown) were b < use higher substrate concentrations to analyse AChE inhibition by gallamine in the presence of 6% MeCN, which might also have revealed a deviation from the apparent competitive inhibition Instead, we investigated the interaction of the inhibitor with AChE over a range of [S], where substrate inhibition was not observed [7,14] The maximum [S] value of 2250 lm corresponded to $ 16–22Km for experiments without FEBS Journal 276 (2009) 2292–2307 ª 2009 The Authors Journal compilation ª 2009 FEBS M Pietsch et al Inhibition kinetics of acetylcholinesterase 100 160 Rate (%) 200 Rate (%) A 125 75 50 25 80 40 0 500 1000 1500 2000 2500 [ATCh] (µM) 500 1000 1500 2000 2500 [ATCh] (µM) B 200 Fig Inhibition of AChE by compound in the presence of 6% MeCN Michaelis–Menten plot using mean values and standard deviations of rates from four separate experiments in 100 mM sodium phosphate, 100 mM NaCl, pH 7.3 with 350 lM Nbs2 and 0.033 mL)1 AChE Concentrations of compound were as follows: open circles, [I] = 0; filled circles, [I] = 1.5 lM; open squares, [I] = 3.0 lM; filled squares, [I] = 4.5 lM; open triangles, [I] = 6.0 lM; filled triangles, [I] = 7.5 lM Nonlinear regression according to Eqn (7) gave KI = 0.59 ± 0.05 lM, a¢ = 1.1 ± 0.1, b¢ = b = 0.096 ± 0.007, Km = 607 ± 25 lM and Vmax = 230 ± 3% A value of a = 0.087 was calculated as the quotient of b and a¢ 160 Rate (%) 120 120 80 40 0 500 1000 1500 [ATCh] (µM) 2000 2500 Fig Inhibition of AChE by gallamine in the absence (A) and presence (B) of 6% MeCN Michaelis–Menten plots using mean values and standard deviations of rates from four separate experiments in 100 mM sodium phosphate, 100 mM NaCl, pH 7.3 with 350 lM Nbs2 and 0.033 mL)1 AChE (A) Concentrations of gallamine were as follows: open circles, [I] = 0; filled circles, [I] = 500 lM; open squares, [I] = 1000 lM; filled squares, [I] = 2000 lM; open triangles, [I] = 3000 lM; filled triangles, [I] = 4000 lM; open reversed triangles, [I] = 5000 lM Nonlinear regression according to Eqn (7) gave KI = 270 ± 20 lM, a¢ = 15 ± 2, b¢ = b = 0.25 ± 0.03, Km = 135 ± lM and Vmax = 116 ± 1% A value of a = 0.017 was calculated as the quotient of b and a¢ (B) Concentrations of gallamine were as follows: open circles, [I] = 0; filled circles, [I] = 750 lM; open squares, [I] = 1500 lM; filled squares, [I] = 3000 lM; open triangles, [I] = 4500 lM; filled triangles, [I] = 6000 lM; open reversed triangles, [I] = 7500 lM Nonlinear regression according to Eqn (14) with Vmax being set to 235% gave KI = 2020 ± 50 lM and Km = 699 ± 10 lM MeCN and $3.2–3.7Km for experiments with MeCN (Table 1) To incorporate a more accurate Vmax value in the fitting process, this parameter was set to a value of 235%, obtained in the absence of gallamine (see above) An analysis of the data according to Eqn (14) using this set Vmax value gave Km = 699 lm (Table 1) This value is closer to the parameter estimate of 671 lm, as well as the Km values calculated for the inhibition of AChE by tacrine and compound in the presence of MeCN (Table 1) The KI value obtained using the predefined Vmax value in Eqn (14) was 2020 lm (Table 1), whereas an only slightly larger value of 2150 lm resulted when Vmax was determined independently The determination of the factors a¢, b and b (Table 1) was performed to obtain an insight into the mode of inhibition, and the KI values (Table 1) provided information on the inhibitory potency of the compounds As depicted in Schemes and 3, the factor a¢ defines whether the inhibitor binds to an enzyme–substrate species (ES and EA combined together as ES¢ in Scheme or EA in Scheme 3) with a greater affinity than to the free enzyme, or vice versa The preference of the inhibitor for binding to an enzyme–substrate species is reflected in values where a¢ < 1, which indicate mixed-type inhibition with a pronounced uncompetitive component A higher affinity of the inhibitor to the free enzyme, seen where a¢ > 1, corresponds to mixed-type inhibition with a more competitive character A pure noncompetitive mode of inhibition is characterized by a¢ = 1, i.e an equal affinity of the inhibitor to any form of the enzyme An investigation of AChE inhibition by tacrine in the absence of MeCN, according to the kinetic model in Scheme 3, gave values of KI = 0.027 lm and FEBS Journal 276 (2009) 2292–2307 ª 2009 The Authors Journal compilation ª 2009 FEBS 2299 Inhibition kinetics of acetylcholinesterase M Pietsch et al Table Inhibition of AChE from Electrophorus electricus Values with standard error were calculated using data (mean values) from four separate experiments, at five or six inhibitor concentrations and 10 or 12 substrate concentrations Analysis of the data was performed using Eqn (7) (gallamine triethiodide, no MeCN; compound 1, 6% v ⁄ v MeCN; b¢ = b), Eqn (10) (tacrine · HCl, no MeCN; tacrine · HCl, 6% v ⁄ v MeCN) or Eqn (14) (gallamine triethiodide, 6% v ⁄ v MeCN) Inhibitor MeCN (% v ⁄ v) Tacrine · HCl Tacrine · HCl Gallamine triethiodide Gallamine triethiodide Compound 6 0.027 0.26 270 2020 0.59 a¢a KI (lM) ± ± ± ± ± 0.003 0.02 20 50 0.05 b 1.4 ± 1.1 ± 15 ± NDe 1.1 ± 0.2 0.2 2d 0.1g b Km (lM) Vmax (%) 0b 0b NDc NDc NDc NDc 0.25 ± 0.03 NDc 0.096 ± 0.007 101 691 135 699 607 110 ± 229 ± 116 ± 235f 230 ± ± ± ± ± ± 35 10 25 a A value of 1.3 for k2 ⁄ k3 has been taken from the literature [58] to calculate a¢ for experiments with tacrine · HCl, no MeCN, and tacrine · HCl, 6% v ⁄ v MeCN b Starting value b < 0, thus b was set to zero c ND, nondeterminable d a = b ⁄ a¢ = 0.017 e b = in Scheme 3, thus a¢ is nondeterminable f Vmax was set to 235%, determined during provisional estimate investigations in the absence of gallamine triethiodide g a = b ⁄ a¢ = 0.087 a¢ = 1.4 (Table 1) The parameter b was necessarily set to zero for the nonlinear analysis (Table 1) and a catalytically inactive EAI (Scheme 3) could therefore be concluded Thus, the deacylation of the acyl-enzyme was completely blocked by the inhibitor A mixed-type inhibition, tending more to noncompetitive inhibition, was found with tacrine This was characterized by an a¢ value close to unity, and indicated that the affinity of tacrine towards AChE was only minimally affected by acylation of the active site serine Such a kinetic behaviour has been reported to occur only if b = and the acylation rate constant of substrate conversion k2 is equal to or larger than the deacylation rate constant k3 [47] Both requirements are fulfilled, as shown by the present study and as reported by Froede and Wilson [58], respectively These findings were in agreement with the results of Nochi et al [72] (obtained with ATCh and AChE from E electricus), who reported KI = 20.4 nm and KI* = a¢KI(1 + k3 ⁄ k2) = 38.3 nm for tacrine on the basis of the kinetic model in Scheme (with b = 0) [72,73] For comparison, we calculated a¢ = 1.1 using these values of KI and KI* This result also indicates a mixed-type inhibition with a pronounced noncompetitive component Inhibition of AChE by gallamine (in the absence of MeCN), analysed according to the kinetic model in Scheme 2, was characterized by values of KI = 270 lm, a¢ = 15 and b¢ = b = 0.25 A value of a = 0.017 was calculated as the quotient of b and a¢ (Table 1) The a¢ value obtained demonstrated the preference of gallamine to bind to the free enzyme rather than to the enzyme–substrate species ES and EA (combined in ESI¢, Scheme 2), which indicates a mixed-type inhibition with a pronounced competitive component Such a kinetic behaviour has recently been reported by Mooser and Sigman [74], who found pure competitive inhibition (KI = 140–320 lm) for the interaction of gallamine with AChE from E electricus 2300 The parameter b represents the substrate conversion catalysed by an inhibitor-bound enzyme species compared with that by the free enzyme (both Schemes and 2) Our study found a value of b = 0.25 for the interaction of AChE with gallamine (Table 1), which confirmed the parameter estimate based on the specific velocity plot (see above) Both b and the calculated value of a were in agreement with the data obtained by Szegletes et al [46] for the inhibition of AChE by gallamine with ATCh as substrate, who reported b = 0.44 and a = 0.019 Under the assumption of equilibrium conditions, a is represented by Eqn (4) Therefore, low values of this parameter require either that ESI (Scheme 1) is not formed (KS ⁄ KS2 and KI ⁄ KSI % 0) or that a % (Scheme 1) As shown in our study, a depends on both b and a¢, with the relatively large value of the latter parameter indicating a comparably low affinity of both the substrate and the inhibitor to form an enzyme–substrate–inhibitor species (Scheme 2) Thus, we hypothesize that the low a value results from a diminished formation of ESI rather than from inhibition of acylation (Scheme 1) Recently, nonequilibrium analysis of AChE inhibition by the PAS ligands propidium and gallamine resulted in the construction of the ‘steric blockade hypothesis’ (based on the model in Scheme 1) This hypothesis demonstrates that PAS ligands inhibit substrate hydrolysis without inducing conformational changes in the active site [46] Nonequilibrium conditions are characterized by k)S < k2 and k)S2 < ak2 [4,46], and thus a % kS2 ⁄ kS was concluded according to Szegletes et al [46] The ‘steric blockade hypothesis’ implies that a ligand bound to PAS slows down ligand entry into and exit from the active site of AChE (kS2 < kS and k)S2 < k)S) without affecting the thermodynamics of the binding of active site-directed ligands (KS2 = KS) It also stipulates that the PAS ligand has no effect on the rate constants of substrate acylation and deacylation FEBS Journal 276 (2009) 2292–2307 ª 2009 The Authors Journal compilation ª 2009 FEBS M Pietsch et al (a = b = 1), and that bound substrate does not alter the interaction of the PAS ligand with the enzyme (kI = kSI = kAI and k)I = k)SI = k)AI) Thus, only the ratio k)I ⁄ kI, i.e the KI value, is relevant [46] As an extension of the steric blockade model, it was proposed that bound PAS ligands also reduce the dissociation rate constants for product release from the active site, which becomes rate limiting at high [S] [19,46] On the basis of the ‘steric blockade hypothesis’, we concluded that the inhibition of AChE by gallamine (in the absence of MeCN) decreased the rate constants kS2 and k)S2 (Scheme 1) to values $ 1.7% of kS and k)S in our experiments This conclusion agrees with the result of a simulated gallamine inhibition of ATCh hydrolysis under nonequilibrium conditions [46], where kS2 and k)S2 were set to 1.5% of kS and k)S, respectively, to obtain optimal correlation between the calculated and experimentally determined parameters KI, a and b The last two parameters can also be used to characterize the relative efficiency of EI to catalyse substrate conversion: a is defined as the ratio of the second-order rate constant kcat ⁄ Km with saturating [I] to that in the absence of inhibitor, and b is the quotient of the first-order rate constants kcat for substrate conversion by EI (at saturating [I]) and E (Scheme 1) [46] According to Eqns (6) and (11), a and b represent the relative efficiency of EI (Scheme 1) if [S] < Km < and [S] ) Km, respectively Thus, the efficiency of the complex AChE–gallamine to hydrolyse ATCh is 1.7– 25% of that of free AChE Influence of MeCN on the inhibition of AChE The inhibition of AChE by tacrine, gallamine and compound was investigated in the presence of 6% v ⁄ v MeCN (corresponding to a concentration of 1.15 m), and the results are shown in Figs 2B,3B,4 and Table Even without the addition of inhibitors, our research showed that the presence of MeCN reduced the rate of enzyme-catalysed substrate conversion, which is in accordance with several literature reports on soluble and immobilized AChE from E electricus [75–80] At the highest substrate concentration used in our experiments, [S] = 2250 lm, the absolute enzyme activity without MeCN was 0.251 ± 0.052 min)1 (n = 8), whereas the addition of 6% MeCN resulted in a decrease in the rate to 0.089 ± 0.020 min)1 (n = 8), i.e to 36% (data not shown) As depicted in Table 1, MeCN also had an influence on the Km value of enzymatic substrate conversion, which increased from 101–135 to 607–699 lm when 6% MeCN was present in the assay A similar result was found in a Inhibition kinetics of acetylcholinesterase study by Ronzani [76], where Km values of 85 and 750 lm were determined for the AChE-catalysed conversion of ATCh in the absence and presence of 6.5% MeCN, respectively The latter Km value was calculated on the basis of a competitive mode of inhibition suggested for MeCN and KI = 0.16 m [75,76] For competitive inhibitors, KI can be calculated according to the equation KI = [I] ⁄ [(Km¢ ⁄ Km))1], where Km¢ is the Michaelis constant in the presence of a certain amount of inhibitor [75,76] Applying this equation to our experiments and using mean values of the data shown in Table (Km¢ = 666 lm at [MeCN] = 1.15 m, Km = 118 lm without MeCN), we calculated an equivalent KI value of 0.25 m for MeCN Considering the cosolvent MeCN as a competitive inhibitor, it might be included as a ‘second’ inhibitor in the fitting equations to analyse the influence of the ‘first’ inhibitor (i.e tacrine, gallamine or compound 1) Such attempts have, however, not been made in this study The inhibition experiments performed in the presence of 6% MeCN and tacrine or 6% MeCN and gallamine were analysed according to the kinetic model in Scheme 3, as ESI was assumed not to be formed in both cases (see above) Our investigations on the basis of Eqns (10) and (14) revealed increased KI values for the two inhibitors compared with the studies without MeCN For tacrine and gallamine, 9.6-fold and 7.5fold increases in the dissociation constant were observed, which resulted in KI = 0.26 lm and KI = 2020 lm, respectively (Table 1) The factors for the increase in KI are in good agreement with data from a previous study [76], where a sevenfold increase in KI was determined for the inhibition of AChE by neostigmine iodide in the presence of 6.5% MeCN and [S] = 20Km In addition, a considerable loss of inhibition of immobilized AChE by dichlorvos and paraoxon in the presence of increasing amounts of MeCN (0–15%) was reported [79] This loss was suggested to be caused by the denaturing effect of the organic solvent [79,81], which can also be considered as a pseudo-inhibition process [80] One reason for enzyme denaturation in the presence of water-miscible solvents, such as MeCN, might be the removal of essential water molecules from the enzyme, necessary for manifesting the catalytic activity [80,82] The inhibition of AChE by tacrine was characterized by values of a¢ = 1.1 and b = (Table 1), i.e tacrine (in the presence of 6% MeCN) acts as a mixed-type inhibitor with a strong noncompetitive component and completely blocks deacylation of EAI (Scheme 3) This behaviour is identical to that in the absence of MeCN In contrast with tacrine, the PAS ligand gallamine tends to inhibit AChE in a competitive manner During FEBS Journal 276 (2009) 2292–2307 ª 2009 The Authors Journal compilation ª 2009 FEBS 2301 Inhibition kinetics of acetylcholinesterase M Pietsch et al the course of the corresponding analysis, the parameter b was assumed to be unity (Table 1), i.e gallamine does not affect the deacylation of the acyl-enzyme Under these conditions, the parameter a¢ becomes irrelevant for the inhibition process and is therefore nondeterminable (Table 1) Comparison of AChE inhibition by gallamine in the absence and presence of MeCN revealed several similarities The inhibitor either acts with a pronounced competitive component or behaves in a purely competitive manner Binding of gallamine to PAS does not alter the deacylation rate constant of EAI (b = 1) when MeCN is present This has already been calculated for gallamine inhibition in the absence of MeCN under nonequilibrium conditions (see above [46]) On the basis of the conclusions drawn from this calculation, we propose that gallamine not only slows down substrate association with and substrate ⁄ product dissociation from the active site, as assumed in the absence of MeCN [19,46], but blocks these events if the organic solvent is present As a result of these findings, it can be concluded that the organic solvent has an influence on the inhibitory potency rather than on the mode of inhibition Such behaviour is found regardless of whether the inhibitor is active site directed, such as tacrine, or binds to PAS, such as gallamine The inhibition of AChE by compound was exclusively investigated in the presence of 6% MeCN because of solubility issues associated with compound without organic solvent An analysis of the enzyme–inhibitor interaction, according to Eqn (7), revealed a hyperbolic mixed-type inhibition characterized by KI = 0.59 lm, a¢ = 1.1 and b¢ = b = 0.096; a = 0.087 was calculated as the quotient of b and a¢ (Table 1) A value of a¢ % implies that compound exhibits the same dissociation constant towards AChE regardless of whether the free enzyme or an enzyme– substrate intermediate ES¢ (Scheme 2) is involved in the interaction [49] However, no statements can be made regarding the dissociation constants of ESI and EAI (Scheme 1), as a¢KI (Scheme 2) reflects a composite of these two constants [14] Nevertheless, the observed kinetic behaviour supports the proposed binding mode of compound to act as a dual-site inhibitor and bind along the active site gorge [44], as similarly concluded for heterobivalent tacrine inhibitors [52] The a and b values obtained indicate that the relative efficiency of EI to catalyse ATCh cleavage (at saturating [I]) ranges from 8.7% (if [S] < Km) to < 9.6% (if [S] ) Km) The residual activity of EI cannot be explained by the ‘steric blockade hypothesis’, as this hypothesis was constructed for inhibitors that exclusively interact with 2302 PAS In so doing, the inhibitor is proposed to simply act as a ‘permeable cork’ at the entrance of the active site gorge [46] In contrast, compound is proposed to bind along this gorge to both the active site at the bottom and PAS, which should prevent ligand access to the active site [83] Recently, an alternative route to and ⁄ or from the active site gorge that may be involved in substrate ⁄ product traffic was found on the basis of kinetic crystallography [84] In that study, opening of a hole adjacent to the choline-binding locus of the anionic site was observed to be particularly caused by rotation of Trp84 (TcAChE numbering [8]) Hints of a ‘back door’ exit were also obtained by mutagenesis experiments [85], molecular dynamics simulations [86] and from the absence of bulky leaving groups in crystal structures of TcAChE conjugated with more or less gorge-filling inhibitors covalently linked to Ser200 [83,87] In this context, the usage of inhibitors, such as compound 1, might be helpful to further elucidate alternative substrate access to the active site Conclusions Our investigations with the prototype AChE inhibitors tacrine and gallamine have shown that defined amounts of MeCN alter the KI value of the inhibitors (by a certain factor), but not their principal mode of inhibition This is a new finding which requires further investigation The presence of MeCN, however, repressed the catalytic activity of the ternary complex formed by AChE, ATCh and the PAS inhibitor gallamine On the basis of the ‘steric blockade hypothesis’ [46], where PAS inhibitors are proposed to act as a ‘permeable cork’ at the entrance of the active site gorge, we conclude that the ‘permeability’ of gallamine simply disappears in the presence of the organic solvent In other words, the PAS inhibitor blocks ligand access to the active site The kinetic analysis performed in this study was based on the simultaneous determination of the inhibitory parameters KI, a¢, b or b, as well as Km and Vmax, all obtained by a single calculation We also demonstrated two algorithms for the estimation of kinetic parameters to successfully perform kinetic analysis in such circumstances Finally, compound was demonstrated to be an effective dual-site inhibitor of AChE Experimental procedures Materials ATCh, Nbs2, gallamine triethiodide and tacrine hydrochloride were obtained from Sigma (Steinheim, Germany) AChE from E electricus and MeCN (HPLC grade) were FEBS Journal 276 (2009) 2292–2307 ª 2009 The Authors Journal compilation ª 2009 FEBS M Pietsch et al purchased from Fluka (Deisenhofen, Germany) MeCN was dried using phosphorus pentoxide, distilled and stored ˚ over molsieve A Compound was synthesized as described elsewhere [44] All measurements were performed on a Varian (Darmstadt, Germany) Cary 50 Bio UV ⁄ VIS spectrometer with a cell holder equipped with a Julabo (Seelbach, Germany) UC-5B constant temperature water bath Data were analysed using Grafit v5.0 (R J Leatherbarrow, Erithacus Software Ltd, Horley, Surrey, UK) AChE inhibition assay AChE activity was assayed spectrophotometrically at 25 °C according to the method of Ellman et al [88] in the absence or presence of 6% v ⁄ v MeCN Assay buffer was 100 mm sodium phosphate, 100 mm NaCl, pH 7.3 A stock solution of AChE (100 mL)1) in assay buffer was kept at °C A : 30 dilution of AChE stock was prepared in ice-cold assay buffer immediately before starting each measurement ATCh (0.5–45 mm) and Nbs2 (7 mm) were dissolved in assay buffer and kept at °C Stock solutions of tacrine, gallamine and compound were prepared in distilled water, assay buffer and MeCN, respectively, and kept at room temperature Inhibition of enzyme activity was determined with 12 (gallamine and tacrine: 25–2250 lm) or 10 (compound 1: 125–2250 lm) different substrate concentrations in the presence of six (gallamine: 500–5000 lm for measurements without MeCN, 750–7500 lm for measurements in the presence of 6% MeCN; tacrine: 0.025–0.25 lm for measurements without MeCN, 0.125–1.25 lm for measurements in the presence of 6% MeCN) or five (compound 1: 1.5– 7.5 lm) different inhibitor concentrations Progress curves were monitored at 412 nm over and characterized by a linear steady-state turnover of the substrate Inhibition studies in the absence or presence of 6% MeCN were performed in a volume of mL containing 350 lm Nbs2, 0.033 mL)1 AChE and different substrate and inhibitor concentrations The enzymatic substrate conversion was initiated by adding 50 lL of ATCh solution after incubating the enzyme–inhibitor mixture for 15 at 25 °C Uninhibited enzyme activity was determined by adding the corresponding solvent instead of the inhibitor solution The rates of AChE-catalysed ATCh cleavage were corrected by those of the nonenzymatic hydrolysis of ATCh obtained in the absence of enzyme and inhibitor The rate of the AChEcatalysed hydrolysis of 500 lm ATCh was determined in duplicate without inhibitor in each experiment and, after correction by the value of the nonenzymatic hydrolysis, was set to 100% Mean values of percentage rates obtained in four separate experiments were used for all calculations IC50 values were determined by plotting the rates v obtained at [ATCh] = 500 lm against the inhibitor concentrations [I] In the case of compound 1, this plot did not become asymptotic to the abscissa Therefore, residual activity at infinite concentration of the inhibitor v[I] fi ¥ was Inhibition kinetics of acetylcholinesterase included in the calculation of the inhibition constant [44] using Eqn (15): ðv0 À v Â Ã Þ I !1   ỵ v v ẳ  I !1 I ỵ IC50 15ị where v0 is the velocity in the absence of the inhibitor and IC50 is the concentration of the inhibitor which reduces the velocity of the enzyme-catalysed reaction to a value halfway between v0 and v[I] fi ¥ To determine IC50 values for gallamine and tacrine (in the presence and absence of 6% MeCN), v[I] fi ¥ was set to zero in Eqn (15) Acknowledgements This work was supported by grants from the Research Training Group 804 ‘Analysis of cellular functions by combinatorial chemistry and biochemistry’ (to L.C and M.G.) M.P gratefully acknowledges financial support from Professor Andrew Abell (School of Chemistry and Physics, The University of Adelaide, Adelaide, Australia), ARC grant DP0771901 and a fellowship within the ‘Postdoc-Programm’ of the German Academic Exchange Service (DAAD) The authors wish to thank Megan Garvey for critical reading of the manuscript References Soreq H & Seidman S (2001) Acetylcholinesterase – new roles for an old actor Nat Rev Neurosci 2, 294– 302 Cygler M, Schrag JD, Sussman JL, Harel M, Silman I, Gentry MK & Doctor BP (1993) Relationship between sequence conservation and three-dimensional structure in a large family of esterases, lipases, and related proteins Protein Sci 2, 366–382 Nardini M & Dijkstra BW (1999) a ⁄ b Hydrolase fold enzymes: the family keeps growing Curr Opin Struct Biol 9, 732–737 Quinn DM (1987) Acetylcholinesterase: enzyme structure, reaction dynamics, and virtual transition states Chem Rev 87, 955–979 Wilson IB & Cabib E (1956) Acetylcholinesterase: enthalpies and entropies of activation J Am Chem Soc 78, 202–207 Silman I, Millard CB, Ordentlich A, Greenblatt HM, Harel M, Barak D, Schafferman A & Sussman JL (1999) A preliminary comparison of structural models for catalytic intermediates of acetylcholinesterase Chem Biol Interact 119–120, 43–52 ´ Bourne Y, Radic Z, Sulzenbacher G, Kim E, Taylor P & Marchot P (2006) Substrate and product trafficking through the active center gorge of acetylcholinesterase FEBS Journal 276 (2009) 2292–2307 ª 2009 The Authors Journal compilation ª 2009 FEBS 2303 Inhibition kinetics of acetylcholinesterase 10 11 12 13 14 15 16 17 18 19 M Pietsch et al analyzed by crystallography and equilibrium binding J Biol Chem 281, 29256–29267 Sussman JL, Harel M, Frolow F, Oefner C, Goldman A, Toker L & Silman I (1991) Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein Science 253, 872–879 Silman I & Sussman JL (2005) Acetylcholinesterase: ‘classical’ and ‘non-classical’ functions and pharmacology Curr Opin Pharmacol 5, 293–302 Harel M, Schalk I, Ehret-Sabatier L, Bouet F, Goeldner M, Hirth C, Axelsen PH, Silman I & Sussman JL (1993) Quaternary ligand binding to aromatic residues in the active site gorge of acetylcholinesterase Proc Natl Acad Sci USA 90, 9031–9035 Ordentlich A, Kronman C, Flashner Y, Leitner M, Segall Y, Ariel N, Cohen S, Velan B & Shafferman A (1993) Dissection of the human acetylcholinesterase active center determinants of substrate specificity J Biol Chem 268, 17083–17095 Colletier JP, Fournier D, Greenblatt HM, Stojan J, Sussman JL, Zaccai G, Silman I & Weik M (2006) Structural insights into substrate traffic and inhibition in acetylcholinesterase EMBO J 25, 2746–2756 Shafferman A, Kronman C, Flashner Y, Leitner M, Grosfeld H, Ordentlich A, Gozes Y, Cohen S, Ariel A, Barak D et al (1992) Mutagenesis of human acetylcholinesterase Identification of residues involved in catalytic activity and in polypeptide folding J Biol Chem 267, 17640–17648 ´ Radic Z, Pickering NA, Vellom DC, Camp S & Taylor P (1993) Three distinct domains in the cholinesterase molecule confer selectivity for acetyl- and butyrylcholinesterase inhibitors Biochemistry 32, 12074– 12084 ´ Radic Z, Duran R, Vellom DC, Li Y, Cervenansky C & Taylor P (1994) Site of fasciculin interaction with acetylcholinesterase J Biol Chem 269, 11233–11239 Barak D, Kronman C, Ordentlich A, Ariel N, Bromberg A, Marcus D, Lazar A, Velan B & Shafferman A (1994) Acetylcholinesterase peripheral anionic site degeneracy conferred by amino acid arrays sharing a common core J Biol Chem 269, 6296–6305 Ordentlich A, Barak D, Kronman C, Ariel N, Segall Y, Velan B & Shafferman A (1995) Contribution of aromatic moieties of tyrosine 133 and of the anionic subsite tryptophan 86 to catalytic efficiency and allosteric modulation of acetylcholinesterase J Biol Chem 270, 2082–2091 ´ Hosea NA, Radic Z, Tsigelny I, Berman HA, Quinn DM & Taylor P (1996) Aspartate 74 as a primary determinant in acetylcholinesterase governing specificity to cationic organophosphonates Biochemistry 35, 10995– 11004 Szegletes T, Mallender WD, Thomas PJ & Rosenberry TL (1999) Substrate binding to the peripheral site of 2304 20 21 22 23 24 25 26 27 28 29 30 31 32 acetylcholinesterase initiates enzymatic catalysis Substrate inhibition arises as a secondary effect Biochemistry 38, 122–133 Mallender WD, Szegletes T & Rosenberry TL (2000) Acetylcholine binds to Asp74 at the peripheral site of human acetylcholinesterase as the first step in the catalytic pathway Biochemistry 39, 7753–7763 Johnson G & Moore SW (2006) The peripheral anionic site of acetylcholinesterase: structure, functions and potential role in rational drug design Curr Pharm Des 12, 217–225 Stojan J, Brochier L, Alies C, Colletier JP & Fournier D (2004) Inhibition of Drosophila melanogaster acetylcholinesterase by high concentrations of substrate Eur J Biochem 271, 1364–1371 ´ Massoulie J (2002) The origin of the molecular diversity and functional anchoring of cholinesterases Neurosignals 11, 130–143 Munoz-Muriedas J, Lopez JM, Orozco M & Luque FJ ˜ (2004) Molecular modelling approaches to the design of acetylcholinesterase inhibitors: new challenges for the treatment of Alzheimer’s disease Curr Pharm Des 10, 3131–3140 Layer PG, Weikert T & Alber R (1993) Cholinesterases regulate neurite growth of chick nerve cells in vitro by means of a non-enzymatic mechanism Cell Tissue Res 273, 219–226 Grisaru D, Sternfeld M, Eldor A, Glick D & Soreq H (1999) Structural roles of acetylcholinesterase variants in biology and pathology Eur J Biochem 264, 672–686 Bartolini M, Bertucci C, Cavrini V & Andrisano V (2003) Beta-amyloid aggregation induced by human acetylcholinesterase: inhibition studies Biochem Pharmacol 65, 407–416 Alvarez A, Opazo C, Alarcon R, Garrido J & Inestrosa NC (1997) Acetylcholinesterase promotes the aggregation of amyloid-b-peptide fragments by forming a complex with the growing fibrils J Mol Biol 272, 348–361 Rees T, Hammond PI, Soreq H, Younkin S & Brimijoin S (2003) Acetylcholinesterase promotes beta-amyloid plaques in cerebral cortex Neurobiol Aging 24, 777–787 ´ Chacon MA, Reyes AE & Inestrosa NC (2003) Acetylcholinesterase induces neuronal cell loss, astrocyte hypertrophy and behavioral deficits in mammalian hippocampus J Neurochem 87, 195–204 Alvarez A, Alarcon R, Opazo C, Campos EO, Munoz FJ, Calderon FH, Dajas F, Gentry MK, Doctor BP, De Mello FG et al (1998) Stable complexes involving acetylcholinesterase and amyloid-b peptide change the biochemical properties of the enzyme and increase the neurotoxicity of Alzheimer’s fibrils J Neurosci 18, 3213–3223 ´ Reyes AE, Chacon MA, Dinamarca MC, Cerpa W, Morgan C & Inestrosa NC (2004) Acetylcholinesterase–Ab FEBS Journal 276 (2009) 2292–2307 ª 2009 The Authors Journal compilation ª 2009 FEBS M Pietsch et al 33 34 35 36 37 38 39 40 41 42 43 44 45 46 complexes are more toxic than Ab fibrils in rat hippocampus Am J Pathol 164, 2163–2174 Inestrosa NC, Dinamarca MC & Alvarez A (2008) Amyloid–cholinesterase interactions Implications for Alzheimer’s disease FEBS J 275, 625–632 Carson KA, Geula C & Mesulam MM (1991) Electron microscopic localization of cholinesterase activity in Alzheimer brain tissue Brain Res 540, 204–208 Wright CI, Geula C & Mesulam MM (1993) Protease inhibitors and indoleamines selectively inhibit cholinesterases in the histopathologic structures of Alzheimer disease Proc Natl Acad Sci USA 90, 683–686 Inestrosa NC & Alarcon R (1998) Molecular interactions of acetylcholinesterase with senile plaques J Physiol (Paris) 92, 341–344 Recanatini M & Valenti P (2004) Acetylcholinesterase inhibitors as a starting point towards improved Alzheimer’s disease therapeutics Curr Pharm Des 10, 3157– 3166 Musia A, Bajda M & Malawska B (2007) Recent developments in cholinesterase inhibitors for Alzheimer’s disease treatment Curr Med Chem 14, 2654–2679 Benzi G & Moretti A (1998) Is there a rationale for the use of acetylcholinesterase inhibitors in the therapy of Alzheimer’s disease? Eur J Pharmacol 346, 1–13 Colombres M, Sagal JP & Inestrosa NC (2004) An overview of the current and novel drugs for Alzheimer’s disease with particular reference to anti-cholinesterase compounds Curr Pharm Des 10, 3121–3130 ´ Bourne Y, Taylor P, Radic Z & Marchot P (2003) Structural insights into ligand interactions at the acetylcholinesterase peripheral anionic site EMBO J 22, 1–12 Piazzi L, Rampa A, Bisi A, Gobbi S, Belluti F, Cavalli A, Bartolini M, Andrisano V, Valenti P & Recanatini M (2003) 3-(4-{[Benzyl(methyl)amino]methyl}phenyl)6,7-dimethoxy-2H-2-chromenone (AP2238) inhibits both acetylcholinesterase and acetylcholinesterase-induced b-amyloid aggregation: a dual function lead for Alzheimer’s disease therapy J Med Chem 46, 2279– 2282 Kryger G, Silman I & Sussman JL (1999) Structure of acetylcholinesterase complexed with E2020 (Aricept): implications for the design of new anti-Alzheimer drugs Struct Fold Des 7, 297–307 Pietsch M & Gutschow M (2005) Synthesis of tricyclic ă 1,3-oxazin-4-ones and kinetic analysis of cholesterol esterase and acetylcholinesterase inhibition J Med Chem 48, 8270–8288 Barnett P & Rosenberry TL (1977) Catalysis by acetylcholinesterase Acceleration of the hydrolysis of neutral acetic acid esters by certain aromatic cations J Biol Chem 252, 7200–7206 Szegletes T, Mallender WD & Rosenberry TL (1998) Nonequilibrium analysis alters the mechanistic interpre- Inhibition kinetics of acetylcholinesterase 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 tation of inhibition of acetylcholinesterase by peripheral site ligands Biochemistry 37, 4206–4216 Krupka RM & Laidler KJ (1961) Molecular mechanisms for hydrolytic enzyme action III A general mechanism for the inhibition of acetylcholinesterase J Am Chem Soc 83, 1454–1458 Botts J & Morales M (1953) Analytical description of the effects of modifiers and of enzyme multivalency upon the steady state catalyzed reaction rate Trans Faraday Soc 49, 696–707 Segel IH (1993) Enzyme Kinetics Wiley and Sons, New York, NY Cornish-Bowden A (2004) Fundamentals of Enzyme Kinetics, 3rd edn Portland Press, London Elsinghorst PW, Cieslik JS, Mohr K, Trankle C & ă Gutschow M (2007) First gallaminetacrine hybrid: ă design and characterization at cholinesterases and M2 muscarinic receptor J Med Chem 50, 5685–5695 ´ Elsinghorst PW, Gonzalez Tanarro CM & Gutschow M ă (2006) Novel heterobivalent tacrine derivatives as cholinesterase inhibitors with notable selectivity toward butyrylcholinesterase J Med Chem 49, 7540–7544 Lin G, Lai CY & Liao WC (1999) Molecular recognition by acetylcholinesterase at the peripheral anionic site: structure–activity relationships for inhibitions by aryl carbamates Bioorg Med Chem 7, 2683–2689 Darvesh S, Darvesh KV, McDonald RS, Mataija D, Walsh R, Mothana S, Lockridge O & Martin E (2008) Carbamates with differential mechanism of inhibition toward acetylcholinesterase and butyrylcholinesterase J Med Chem 51, 4200–4212 Krupka RM & Laidler KJ (1961) Molecular mechanisms for hydrolytic enzyme action I Apparent non-competitive inhibition, with special reference to acetylcholinesterase J Am Chem Soc 83, 1445–1447 Hillman GR & Mautner HG (1970) Hydrolysis of electronically and sterically defined substrates of acetylcholinesterase Biochemistry 9, 2633–2638 Wilson IB & Alexander J (1962) Acetylcholinesterase: reversible inhibitors, substrate inhibition J Biol Chem 237, 1323–1326 Froede HC & Wilson IB (1984) Direct determination of acetyl–enzyme intermediate in the acetylcholinesterasecatalyzed hydrolysis of acetylcholine and acetylthiocholine J Biol Chem 259, 11010–11013 Eastman J, Wilson EJ, Cervenansky C & Rosenberry ˜ TL (1995) Fasciculin binds to the peripheral site on acetylcholinesterase and inhibits substrate hydrolysis by slowing a step involving proton transfer during enzyme acylation J Biol Chem 270, 19694–19701 Markus M, Hess B, Ottaway JH & Cornish-Bowden A (1976) The analysis of kinetic data in biochemistry A critical evaluation of methods FEBS Lett 63, 225–230 Baici A (1981) The specific velocity plot A graphical method for determining inhibition parameters for both FEBS Journal 276 (2009) 2292–2307 ª 2009 The Authors Journal compilation ª 2009 FEBS 2305 Inhibition kinetics of acetylcholinesterase 62 63 64 65 66 67 68 69 70 71 72 73 74 75 M Pietsch et al linear and hyperbolic enzyme inhibitors Eur J Biochem 119, 9–14 Garrido MN, Lisa TA & Domenech CE (1988) Pseudomonas aeruginosa acid phosphatase contains an anionic site with a trimethyl subsite Kinetic evidences obtained with alkylammonium ions Mol Cell Biochem 84, 41–49 Hasan FB, Cohen SG & Cohen JB (1980) Hydrolysis by acetylcholinesterase Apparent molal volumes and trimethyl and methyl subsites J Biol Chem 255, 3898– 3904 Krupka RM & Laidler KJ (1961) Molecular mechanisms for hydrolytic enzyme action II Inhibition of acetylcholinesterase by excess substrate J Am Chem Soc 83, 1448–1454 Krupka RM (1964) Acetylcholinesterase: trimethylammonium-ion inhibition of deacetylation Biochemistry 3, 1749–1754 Krupka RM (1965) Acetylcholinesterase: structural requirements for blocking deacetylation Biochemistry 4, 429–435 Fallarero A, Oinonen P, Gupta S, Blom P, Galkin A, Mohan CG & Vuorela PM (2008) Inhibition of acetylcholinesterase by coumarins: the case of coumarin 106 Pharmacol Res 58, 215–221 ´ Radic Z, Reiner E & Taylor P (1991) Role of the peripheral anionic site on acetylcholinesterase: inhibition by substrates and coumarin derivatives Mol Pharmacol 39, 98–104 ´ Simon S & Massoulie J (1997) Cloning and expression of acetylcholinesterase from Electrophorus Splicing pattern of the 3¢ exons in vivo and in transfected mammalian cells J Biol Chem 272, 33045–33055 Johnson JL, Cusack B, Hughes TF, McCullough EH, Fauq A, Romanovskis P, Spatola AF & Rosenberry TL (2003) Inhibitors tethered near the acetylcholinesterase active site serve as molecular rulers of the peripheral and acylation sites J Biol Chem 278, 38948–38955 Johnson JL, Cusack B, Davies MP, Fauq A & Rosenberry TL (2003) Unmasking tandem site interaction in human acetylcholinesterase Substrate activation with a cationic acetanilide substrate Biochemistry 42, 5438– 5452 Nochi S, Asakawa N & Sato T (1995) Kinetic study on the inhibition of acetylcholinesterase by 1-benzyl-4[(5,6-dimethoxy-1-indanon)-2-yl]methylpiperidine hydrochloride (E2020) Biol Pharm Bull 18, 1145–1147 Froede HC, Wilson IB & Kaufman H (1986) Acetylcholinesterase: theory of noncompetitive inhibition Arch Biochem Biophys 247, 420–423 Mooser G & Sigman DS (1974) Ligand binding properties of acetylcholinesterase determined with fluorescent probes Biochemistry 13, 2299–2307 Ronzani N (1993) Effet de solvents sur l’acetylcholinesterase Tetrahedron Lett 34, 3867–3870 2306 76 Ronzani N (1994) Effet de solvents sur l’acetylcholines´ ´ terase I Mesure de l’activite anticholinesterasique de composes hydrophobes Analusis 22, 249–256 77 Ingkaninan K, de Best CM, van der Heijden R, Hofte AJP, Karabatak B, Irth H, Tjaden UR, van der Greef J & Verpoorte R (2000) High-performance liquid chromatography with on-line coupled UV, mass spectrometric and biochemical detection for identification of acetylcholinesterase inhibitors from natural products J Chromatogr A 872, 61–73 78 Rhee IK, Appels N, Luijendijk H, Irth H & Verpoorte R (2003) Determining acetylcholinesterase inhibitory activity in plant extracts using a fluorimetric flow assay Phytochem Anal 14, 145–149 79 Dondoi MP, Bucur B, Danet AF, Toader CN, Barthelmebs L & Marty JL (2006) Organophosphorus insecticides extraction and heterogeneous oxidation on column for analysis with an acetylcholinesterase (AChE) biosensor Anal Chim Acta 578, 162–169 80 Turdean GL & Turdean MS (2008) Synergetic effect of organic solvents and paraoxon on the immobilized acetylcholinesterase Pestic Biochem Physiol 90, 73–81 ` 81 Fremaux I, Mazeres S, Brisson-Lougarre A, Arnaud M, Ladurantie C & Fournier D (2002) Improvement of Drosophila acetylcholinesterase stability by elimination of a free cysteine BMC Biochem 3, 21 82 Mionetto N, Marty JL & Karube I (1994) Acetylcholinesterase in organic solvents for the detection of pesticides: biosensor application Biosens Bioelectron 9, 463–470 83 Bartolucci C, Perola E, Cellai L, Brufani M & Lamba D (1999) ‘Back door’ opening implied by the crystal structure of a carbamoylated acetylcholinesterase Biochemistry 38, 5714–5719 84 Colletier JP, Royant A, Specht A, Sanson B, Nachon F, Masson P, Zaccai G, Sussman JL, Goeldner M, Silman I et al (2007) Use of a ‘caged’ analogue to study the traffic of choline within acetylcholinesterase by kinetic crystallography Acta Crystallogr D Biol Crystallogr 63, 1115–1128 85 Nachon F, Stojan J & Fournier D (2008) Insights into substrate and product traffic in the Drosophila melanogaster acetylcholinesterase active site gorge by enlarging a back channel FEBS J 275, 2659–2664 86 Gilson MK, Straatsma TP, McCammon JA, Ripoll DR, Faerman CH, Axelsen PH, Silman I & Sussman JL (1994) Open ‘back door’ in a molecular dynamics simulation of acetylcholinesterase Science 263, 1276– 1278 87 Bartolucci C, Siotto M, Ghidini E, Amari G, Bolzoni PT, Racchi M, Viletti G, Delcanale M & Lamba D (2006) Structural determinants of Torpedo californica acetylcholinesterase inhibition by the novel and orally active carbamate based anti-Alzheimer drug ganstigmine (CHF-2819) J Med Chem 49, 5051–5058 FEBS Journal 276 (2009) 2292–2307 ª 2009 The Authors Journal compilation ª 2009 FEBS M Pietsch et al 88 Ellman GL, Courtney KD, Andres V & Featherstone RM (1961) A new rapid colorimetric determination of acetylcholinesterase activity Biochem Pharmacol 7, 88–95 Supporting information The following supplementary material is available: Doc S1 A complete list of equations, including those that are not part of the manuscript, together with corresponding annotations Fig S1 (A) Specific velocity plot for the inhibition of AChE by gallamine in the absence of MeCN (B) Plot of int0 ⁄ (int0)1) and int1 ⁄ (int1)1) versus the reciprocal concentrations of gallamine Inhibition kinetics of acetylcholinesterase Fig S2 (A) Modified specific velocity plot for the inhibition of AChE by gallamine in the presence of 6% MeCN (B) Plot of int0 ⁄ (int0)1) versus the reciprocal concentrations of gallamine This supplementary material can be found in the online version of this article Please note: Wiley-Blackwell is not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 276 (2009) 2292–2307 ª 2009 The Authors Journal compilation ª 2009 FEBS 2307 ... the inhibition of AChE by gallamine in the absence of MeCN (B) Plot of int0 ⁄ (int0)1) and int1 ⁄ (int1)1) versus the reciprocal concentrations of gallamine Inhibition kinetics of acetylcholinesterase. .. AChE–gallamine to hydrolyse ATCh is 1.7– 25% of that of free AChE In? ??uence of MeCN on the inhibition of AChE The inhibition of AChE by tacrine, gallamine and compound was investigated in the presence of. .. involvement of PAS in the processes of AD, the use of PAS inhibitors and dual-site inhibitors of AChE allows for the inhibition of the catalytic activity of the enzyme and also lowers the incidence of