©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Fasciculin 2 Binds to the Peripheral Site on Acetylcholinesterase and Inhibits Substrate Hydrolysis by Slowing a Step Involving Proton Transfer during Enzyme Acylation (*)

(Received for publication, January 26, 1995; and in revised form, May 18, 1995)

Jean Eastman Erica J. Wilson Carlos Cerveñansky (1) Terrone L. Rosenberry (§)

From the Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44120-4965 and the Instituto de Investigaciones Biologicas, Clemente Estable, Montevideo, Uruguay 11600

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
APPENDIX
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The acetylcholinesterase active site consists of a gorge 20 Å deep that is lined with aromatic residues. A serine residue near the base of the gorge defines an acylation site where an acyl enzyme intermediate is formed during the hydrolysis of ester substrates. Residues near the entrance to the gorge comprise a peripheral site where inhibitors like propidium and fasciculin 2, a snake neurotoxin, bind and interfere with catalysis. We report here the association and dissociation rate constants for fasciculin 2 interaction with the human enzyme in the presence of ligands that bind to either the peripheral site or the acylation site. These kinetic data confirmed that propidium is strictly competitive with fasciculin 2 for binding to the peripheral site. In contrast, edrophonium, N-methylacridinium, and butyrylthiocholine bound to the acylation site and formed ternary complexes with the fasciculin 2-bound enzyme in which their affinities were reduced by about an order of magnitude from their affinities in the free enzyme. Steady state analysis of the inhibition of substrate hydrolysis by fasciculin 2 revealed that the ternary complexes had residual activity. For acetylthiocholine and phenyl acetate, saturating amounts of the toxin reduced the first-order rate constant k to 0.5-2% and the second-order rate constant k/K to 0.2-2% of their values with the uninhibited enzyme. To address whether fasciculin 2 inhibition primarily involved steric blockade of the active site or conformational interaction with the acylation site, deuterium oxide isotope effects on these kinetic parameters were measured. The isotope effect on k/K increased for both substrates when fasciculin 2 was bound to the enzyme, indicating that fasciculin 2 acts predominantly by altering the conformation of the active site in the ternary complex so that steps involving proton transfer during enzyme acylation are slowed.


INTRODUCTION

Acetylcholinesterase (EC 3.1.1.7, AChE) (^1)hydrolyzes its physiological substrate acetylcholine at a very high catalytic rate (Rosenberry, 1975a, 1979). The unique features of AChE structure that determine its catalytic power have been pursued for many years, and the recent determination of the three-dimensional structure of torpedo AChE (Sussman et al., 1991) offers opportunities for new insights. A few key features of the AChE structure are diagrammed in Fig. 1. The active site is a narrow gorge lined with aromatic residues that is about 20 Å deep and penetrates nearly to the center of the 70-kDa catalytic subunit. At the base of the gorge is the Ser residue (torpedo sequence numbering) that is acylated and deacylated during substrate turnover and His and Glu residues that together with Ser participate in a catalytic triad similar to those found in other serine proteases and esterases (Glu-His-Ser). Because acetylcholine occupies this site as acyl transfer is initiated, we shall refer to it as the acylation site. Certain cationic inhibitors bind selectively to this site. For example, crystal structure analysis shows that edrophonium is bound with its hydroxyl group making hydrogen bonds to both the N atom of His and the O atom of Ser (Sussman et al., 1992). Our earlier kinetic studies on eel AChE showed that the active site has a high net negative charge that can electrostatically attract cationic substrates and inhibitors (Nolte et al., 1980), and molecular modeling calculations (Ripoll et al., 1993) from the three-dimensional structure suggest that a number of negatively charged residues in or near the active site gorge can provide such electrostatic attraction. The three-dimensional structure confirms several features of the active site that were inferred from kinetic investigations of AChE catalysis. Early kinetic studies emphasized that the enzyme active site is composed of several ``subsites.'' Anionic and esteratic subsites were proposed to accommodate the two ends of the acetylcholine molecule (Nachmansohn and Wilson, 1951). Model building of acetylcholine in the AChE active site (Sussman et al., 1991) reveals the substrate trimethylammonium group in the anionic subsite to be adjacent to Trp, while in the esteratic subsite the substrate carbonyl carbon is positioned to make a tetrahedral bond with the O of Ser and the acetoxy methyl group appears clamped in an ``acyl pocket'' formed by Phe and Phe. Site-directed mutagenesis has confirmed the importance of these 2 residues in the acyl pocket (Vellom et al., 1993; Ordentlich et al., 1993).


Figure 1: Schematic diagram of the sites for ligand binding in AChE.



The peripheral site was first defined by cationic inhibitors like propidium that do not compete with edrophonium in binding to the active site (Taylor and Lappi, 1975). Studies involving affinity labeling (Weise et al., 1990; Schalk et al., 1992) and site-specific mutants (Radic et al., 1993; Barak et al., 1994) indicate that Trp on the rim of the active site gorge is a key component of the peripheral site and that residues from at least two other polypeptide loops at the gorge rim also contribute. Bisquaternary ligands like decamethonium or BW284C51 compete with both propidium and edrophonium for enzyme binding, and site-specific mutants indicate that these ligands bridge the 12-15 Å distance between the acylation and peripheral sites. Recently the fasciculins, a family of very similar snake venom neurotoxins from mambas (genus Dendroaspis), have emerged as new probes of the AChE active site. These 61-amino-acid polypeptides have three-dimensional structures that are very similar to those of the short alpha-neurotoxins with four disulfide bonds that bind to nicotinic acetylcholine receptors (le Du et al., 1992). They inhibit AChE at subnanomolar concentrations with an apparent noncompetitive inhibition pattern, and fasciculins and propidium interfere with each other in binding to AChE (Karlsson et al., 1984; Marchot et al., 1993). Furthermore, site-specific mutants reveal that residues at the gorge rim, particularly Trp, are essential for high affinity fasciculin binding (Radic et al., 1994). These features suggest that fasciculins bind to the same peripheral site on the gorge rim defined by propidium. We confirm this point here by analyzing the kinetics of fasciculin 2 binding, and we investigate the mechanism by which this binding alters AChE-catalyzed substrate hydrolysis.


EXPERIMENTAL PROCEDURES

Materials

Human erythrocyte AChE was purified as outlined previously and active site concentrations were determined by assuming 410 units/nmol (Rosenberry and Scoggin, 1984; Roberts et al., 1987). Purified fasciculin 2 concentrations were determined by molar absorptivity ( = 4900), and stocks were mixed with bovine serum albumin (to 1 mg/ml) for storage (-20 or 4 °C) prior to dilution in experiments (Karlsson et al., 1984; Cervenansky et al., 1994).

Kinetic Model of Fasciculin 2 Binding in the Presence of an Inhibitor

The interaction of fasciculin 2 (F) with AChE (E) in the presence of an inhibitor I is given by the model in .

In , I binding to Eand EF is assumed to reach equilibrium instantaneously with dissociation constants K(I) and K, respectively. Binding of F to Eand EI is much slower and occurs with association rate constants k(F) and k and dissociation rate constants k and k, respectively. Equilibrium dissociation constants K(F) = k/k(F) and K = k/k. According to , the observed pseudo-first-order rate constant k for the approach to equilibrium is given by .

where

According to a and b, if F and I are competitive (i.e.the ternary complex EFI does not form), then k=k(F)/(1+[I]/K(I)) and k = k. If F and I are noncompetitive, then at saturating concentrations of I, k = k and k = k.

Analysis of the Rates of Approach to Equilibrium for Fasciculin 2 Binding to AChE

Association reactions were initiated by adding fasciculin 2 to AChE in pH 7 buffer (20 mM sodium phosphate, 0.02% Triton X-100, pH 7.0) and, in some cases, an inhibitor at 23 °C. To ensure that binding was measured under pseudo-first-order conditions, the concentration of fasciculin 2 was adjusted to at least eight times the concentration of AChE. At various times a 2.9-ml aliquot was removed to a cuvette, 120 µl of acetylthiocholine and 5,5`-dithiobis-(2-nitrobenzoic acid) (DTNB) were added to final concentrations of 0.5 and 0.33 mM, respectively, and a continuous assay trace was immediately recorded at 412 nm on a Cary 3A spectrophotometer. Dissociation reactions were measured by preincubating fasciculin 2 with AChE at 50 times the indicated final concentrations. The concentration of fasciculin 2 was at least eight times that of AChE except at low concentrations of fasciculin 2 where dissociation was virtually complete. After 1-4 h of incubation, 58 µl was added to 2.82 ml of buffer and inhibitor at 23 °C in a cuvette at time 0. After various times, acetylthiocholine and DTNB were added for assay as above.

Assay points v from association and dissociation reactions were fitted by the nonlinear regression analysis program Fig.P (BioSoft, version 6.0) to .

In , k is the observed pseudo-first-order rate constant for the approach to equilibrium as given in and v and v are the calculated values of v at time 0 and at equilibrium, respectively.

Equilibrium Binding of N-Methylacridinium to AChE in the Presence and Absence of Fasciculin 2

Fluorescence titrations were conducted as outlined in Rosenberry and Neumann(1977), and data were fit to a single binding site for N-methylacridinium by nonlinear regression analysis. Titrations of 0.5-2.4 µM AChE with or without 20 µM fasciculin 2 were conducted in duplicate.

Kinetic Models for Steady State Substrate Hydrolysis in the Presence of Fasciculin 2

A general model for the interaction of a substrate (S) and fasciculin 2 (F) with AChE is given in . This model includes an acylated enzyme intermediate (EA) formed from an initial enzyme-substrate complex (ES) during substrate hydrolysis, and F can bind to E, ES, and EA.

In , the equilibrium dissociation constants K = k/k, where k and k are the respective association and dissociation rate constants. It is also assumed that substrate concentrations are low enough that ESS or EAS species leading to substrate inhibition are negligible (see Rosenberry, 1975a). In the absence of fasciculin 2, the steady state hydrolysis rate v = -d [S]/dt is given by the Michaelis-Menten expression in .

In , V(max) is given by k[E], where k = k(2)k(3)/(k(2) + k(3)) and [E] is the total concentration of all enzyme species. K corresponds to Kk/k(2), where K = (k + k(2))/k(S). However, as noted under the ``Appendix,'' formulation of an appropriate equation for v when fasciculin 2 is present in is complicated by the fact that the general steady state solution for this model is too complex for useful comparison to experimental data. The conventional response to this problem has been to assume virtual equilibrium of all ligand complexes in (e.g. [ES] = [E] [S]/K(S)) by requiring that kk(2); k` ak(2); kk(2) and kak(2); kk(3) and kbk(3) (see Bernhard, 1968). When [S] and [F] are constant, the reciprocal of v is then given by (Krupka and Laidler, 1961; Barnett and Rosenberry, 1977).

In , V(max) and k are unchanged from , but K now is given by K(S)k/k(2). Quantitative information about individual equilibrium constants is obtained by first conducting reciprocal plots of 1/vversus 1/[S] at fixed inhibitor concentration [F]. predicts that these plots will be linear, and this is widely observed for a variety of inhibitors of AChE as long as [S] remains low enough to avoid substrate inhibition (see ). Slopes of these plots are calculated by linear regression analyses with appropriate weighting (here it is assumed that v has constant percent error), and a replot of these slopes against [F] corresponds to .

Nonlinear regression analysis of (with slope values weighted by the reciprocal of their variance) provides estimates of K(F) and the parameter alpha = aK(F)/K. This approach has been attractive, because and can account for most reported experimental data on AChE. However, the inequalities inherent in the virtual equilibrium assumption (noted above ) clearly are violated for a high affinity inhibitor like fasciculin 2 with the small dissociation rate constants shown under ``Results,'' and no longer is necessarily valid. A more appropriate expression for v in this situation is outlined under ``Appendix,'' and conditions are identified under which is valid.

Steady State Measurements of AChE-catalyzed Substrate Hydrolysis

Standard AChE assays (Ellman et al., 1961) were conducted as modified by Rosenberry and Scoggin(1984). One unit of AChE activity corresponds to 1 µmol of acetylthiocholine hydrolyzed per min at 23 °C as monitored by formation of the thiolate dianion of DTNB^2 (DeltaA = 14.15 mM cm). For butyrylthiocholine hydrolysis, AChE and a 10-fold molar excess of fasciculin 2 were mixed with 0.2-5.0 mM butyrylthiocholine iodide in 3.0 ml of pH 7 buffer and incubated for 10-80 min to allow equilibration of fasciculin 2 with the enzyme. DTNB was added to 0.10 mM, (^2)and the reaction was monitored at 412 nm for 0.5-5 min. Rates as low as 0.0001 DeltaA/min could be measured.

Acetylthiocholine hydrolysis rates were determined by two methods. 1) After 30-min pre-equilibration, AChE (2 nM) and fasciculin 2 (500 nM) were mixed with acetylthiocholine iodide (0.1-5 mM) and DTNB (0.10 mM) in 1.0 ml of pH 8 buffer (NaH(2)PO(4) and Na(2)HPO(4) adjusted to 100 mM phosphate and pH 8.0, 0.02% Triton X-100) and monitored as for butyrylthiocholine above. V(max)` and K` (primes indicate saturation with fasciculin 2; see ) were then obtained by weighted linear regression analysis of the reciprocal plot corresponding to . To compensate for substrate inhibition in the absence of fasciculin 2 at these acetylthiocholine concentrations, V(max) and K were obtained by nonlinear regression analysis of , where K is the substrate inhibition constant (Hodge et al., 1992).

2) For better precision in comparing rates in H(2)O and deuterium oxide (D(2)O), v was measured at 0.5 mM acetylthiocholine and V(max)/K or

V(max)`/K` was determined as the constant j from the integrated form of at low initial [S] = [S](0) (12 µM, < 0.2 K) as shown in .

To adjust j to geq0.1 m, [E] was maintained geq15 nM in the presence of 500 nM fasciculin 2 and geq30 pM in its absence. V(max) or V(max)` was then calculated from or , respectively.

Phenyl acetate hydrolysis rates were measured with pre-equilibrated AChE (3-15 nM) and fasciculin 2 (500 nM) in phenyl acetate (0.1-5 mM, leq1% methanol final) or with 60-300 pM AChE in the absence of fasciculin 2 in 1.0 ml of pH 8 buffer. Phenyl acetate reaction was monitored directly at 270 nm for 1-5 min (DeltaA = 1.40 mM cm; Rosenberry, 1975b). To improve the precision of isotope effect measurements, multiple data sets were collected, K and K` values determined from were averaged, and the data sets were subjected to a second cycle of regression analysis with these average values fixed.

Reactions in D(2)O were conducted by identical procedures except that the pH 8 buffer was adjusted to pH 8.1.(^3)AChE dilutions into D(2)O were paired with dilutions into H(2)O to maximize precision in comparing rates.


RESULTS

Fasciculin 2 Forms a 1:1 Stoichiometric Complex with the AChE Active Site in Which AChE Activity Is Inhibited

Fasciculins form high affinity complexes with AChE (Karlsson et al., 1984), and AChE activity was titrated with increasing concentrations of fasciculin 2 to demonstrate stoichiometric inhibition (Fig. 2). The titration also revealed a residual AChE activity in the presence of excess fasciculin 2, suggesting that the complex of fasciculin 2 with AChE retained some activity toward acetylthiocholine.


Figure 2: Titration of AChE with fasciculin 2. Identical AChE samples (active site concentration = [E]) were incubated with the indicated total concentrations of fasciculin 2 ([F]) in pH 7 buffer for 1 h, and small aliquots of acetylthiocholine and DTNB were added for assay as described under ``Experimental Procedures.'' Observed v were normalized to v obtained in the absence of fasciculin 2 and fitted to the equation v/v = (1 - R)(1 - [EF]/[E])+ Rwhere [EF] = [F] when [F] < [E] (-), [EF] = [E] when [F] > [E](- - -), and R is the assay activity of the EF complex relative to that of E. The lines intersect at [E] = [F], corresponding to [F] = 0.87 nM. This estimate is in reasonable agreement with an [E] of 0.64 nM calculated directly from v.



The Kinetics of Fasciculin 2 Binding to AChE Are Consistent with a Simple Equilibrium

Rates of approach to equilibrium binding of fasciculin 2 with AChE were measured by assay of aliquots with acetylthiocholine. Because of the low dissociation rate constant of the complex (Marchot et al., 1993), these rates were readily measured on a scale of m to h. Examples are shown in Fig. 3A, where 36 pM fasciculin 2 and 4 pM AChE reached an equilibrium in which the activity was about 27% of that of a corresponding control enzyme without fasciculin 2. Since Fig. 2shows that fasciculin 2 binding blocked most of the activity toward acetylthiocholine, this percentage corresponds approximately to the enzyme that remained uncomplexed. Equilibrium was approached from initial conditions in which either no AChE was bound (an association reaction) or virtually all enzyme was bound (a dissociation reaction), and the change in enzyme activity corresponded to single exponential time courses with rate constants of about 0.07 m for both initial conditions. Analysis of a series of rate constants obtained at several fasciculin 2 concentrations (Fig. 3B) indicated that the data conformed to the simple bimolecular reaction in , with an association rate constant k(F) = 2.7 10^7M s and a dissociation rate constant k = 2.9 10 s (Table 1). The equilibrium dissociation K(F) given by k/k corresponded to 11 pM. A recent report on the interaction of fasciculin 2 with mouse AChE found a similar value of K(F) but a smaller value of k(F), perhaps because of higher ionic strength conditions (Radic et al., 1994).


Figure 3: Kinetics of fasciculin 2 reaction with AChE. Panel A, the association reaction (bullet) was initiated by mixing fasciculin 2 and AChE, and the dissociation reaction () was initiated by diluting these components, to final concentrations of 36 and 4 pM, respectively. At the indicated times, aliquots were taken for assay with acetylthiocholine as outlined under ``Experimental Procedures.'' Assay points were fitted by unweighted nonlinear regression analysis to the exponential curve in to give pseudo-first-order rate constants k of 0.073 ± 0.005 m for the association reaction and 0.07 ± 0.02 m for the dissociation reaction. Assay points are shown after normalization to a control AChE activity (v = 0.0178 DeltaA/min) without fasciculin 2. Panel B, values of k obtained from 11 association reactions (bullet) and 1 dissociation reaction () as shown in panel A were plotted against the fasciculin 2 concentration according to . Points were weighted by the reciprocal of the observed variance of the k values and fit to a linear plot with slope k = k(F) and intercept k = k. Values of these constants are given in Table 1.





Fasciculin 2 Binding to AChE Is Competitively Blocked by Propidium but Not by Edrophonium

To assess the site on AChE to which fasciculin 2 binds, the effects of the peripheral site inhibitor propidium and the acylation site inhibitor edrophonium on the kinetics of fasciculin 2 binding were analyzed. According to and and , an inhibitor that binds to the same site as fasciculin 2 will be unable to form the ternary complex EFI. Because of this competitive interaction, saturating concentrations of the inhibitor will have no effect on k = k for fasciculin 2 but will decrease k to an extent inversely proportional to the inhibitor concentration (A). Precisely this type of inhibition was seen with propidium in Fig. 4, A and B. The value of k was unchanged with propidium (Table 1), and k-1 increased linearly with the propidium concentration. The value of the propidium inhibition constant K(I) = 0.31 µM obtained in Fig. 4B agreed with the K(I) = 0.6 µM determined from propidium inhibition of acetylthiocholine hydrolysis in . This agreement provides strong evidence that the binding site for which propidium and fasciculin 2 compete is the same site at which propidium inhibits substrate hydrolysis. The properties of the site-specific mutants noted under the Introduction indicate that this is the peripheral site on the rim of the active site gorge in Fig. 1.


Figure 4: Kinetics of fasciculin 2 reaction with AChE in the presence of propidium or edrophonium. Panel A, values of k in the presence of 50 µM propidium were determined as described in Fig. 3A and analyzed as in Fig. 3B. Panel B, reciprocals of k obtained from the slopes of the plots in Fig. 3B and 4A and an additional plot at 20 µM propidium (data not shown) according to were plotted against the propidium concentration and analyzed by a. The steep linear slope corresponded to K(I) = 0.31 ± 0.02 µM and indicated that k = 0. Panel C, values of k in the presence of 10 µM edrophonium were analyzed as in A. Panel D, reciprocals of k obtained from the slopes of plots in Fig. 3B, 4C, and an additional plot at 5 µM edrophonium (data not shown) were analyzed as in B. The lack of dependence on edrophonium concentration indicated that k(F) = k. In Panels A and C, the open and closed symbols represent association and dissociation reactions, respectively, as in Fig. 3B, and the dotted line corresponds to the plot without inhibitors in Fig. 3B. K(I) values obtained from steady state inhibition of acetylthiocholine hydrolysis were 0.22 ± 0.06 µM for edrophonium and 0.6 ± 0.2 µM for propidium.^5



In contrast to the observations with propidium, saturating concentrations of edrophonium had almost no effect on k for fasciculin 2 (Fig. 4, C and D and Table 1). This indicates that the rate constant for fasciculin 2 binding to its peripheral site is not significantly altered when edrophonium is bound to the acylation site at the bottom of the active site gorge. However, k for fasciculin 2 dissociation from the ternary complex EFI with edrophonium and AChE did significantly increase by 4-5-fold. The increase in k suggests a modest conformational interaction between the sites in the ternary complex.

Competition between Fasciculin 2 and Butyrylthiocholine for AChE Can Be Assessed by Both Pre-steady State and Steady State Kinetics

Because fasciculin 2 equilibrates slowly with AChE, studies of the effects of the toxin on substrate hydrolysis by the enzyme must be undertaken with care (see ``Discussion''). The slowly hydrolyzed substrate butyrylthiocholine offered a number of advantages. First, the effect of butyrylthiocholine on the kinetics of fasciculin 2 binding could be measured by techniques similar to those employed above for propidium and edrophonium. As shown in Fig. 5A and Table 1, a saturating concentration of butyrylthiocholine decreased k about 4-fold and increased k 3-4-fold relative to fasciculin 2 reactions with AChE in the absence of inhibitor. The effect on k, however, did not appear to result from competition of butyrylthiocholine for the fasciculin binding site. No differences in k were observed over butyrylthiocholine concentrations of 0.2-5 mM (data not shown), consistent with the binding of butyrylthiocholine exclusively at the acylation site. A second advantage offered by butyrylthiocholine was that steady state inhibition of its AChE-catalyzed hydrolysis by fasciculin 2 also could be measured to provide independent confirmation of several of the kinetic constants in Table 1. Slopes of reciprocal plots of 1/vversus 1/[S] at fixed inhibitor concentrations were particularly informative. According to and , if butyrylthiocholine and fasciculin 2 bind to AChE simultaneously and if the substrate can still proceed to form the acyl enzyme intermediate in this ternary complex, then these slopes will not increase linearly as the fasciculin 2 concentration increases but instead will reach a constant value. This is precisely what is observed in Fig. 5B. Fasciculin 2 is a potent inhibitor of butyrylthiocholine hydrolysis, and it induced large increases in slope. However, fasciculin 2 binding did not completely block butyrylthiocholine hydrolysis because the slope increase leveled off at high fasciculin 2 concentrations. Fitting this data to gave values of K(F) = 11 pM (Table 1) and alpha = 0.030. This K(F) agrees precisely with the K(F) obtained previously from the ratio of k to k for fasciculin 2 binding.


Figure 5: Kinetics of fasciculin 2 reaction with AChE in the presence of butyrylthiocholine. Panel A, measurements of k in the presence of 2 mM butyrylthiocholine were analyzed as in Fig. 4A. The open and closed symbols represent association and dissociation reactions, respectively, as in Fig. 3B, and the dotted line corresponds to the plot without inhibitors in Fig. 3B. Panel B, steady state inhibition of butyrylthiocholine hydrolysis by fasciculin 2. Reciprocal plots of 1/vversus 1/[S] at fixed concentrations of fasciculin 2 and one-tenth that concentration of AChE were analyzed by weighted linear regression analyses as outlined under ``Experimental Procedures.'' Slopes and intercepts of these plots were normalized to the slope and intercept of a paired control data set obtained with the same AChE concentration but in the absence of fasciculin 2. The slopes (bullet) were fit to by weighted nonlinear regression analysis (solid line) to give estimates of K(F) = 11 ± 2 pM and alpha = 0.030 ± 0.002. The intercepts () were fit to an equation of the same form as (dotted line) to establish a maximum increase of 5.6 ± 0.7 at saturating concentrations of fasciculin 2. K for butyrylthiocholine (assumed equal to K(I) in ) was 86 ± 5 µM.



Interpretation of alpha is complicated for a number of reasons. First, it includes contributions from both the fasciculin 2 affinity in the ternary complex (K) and the relative acylation rate constant in the ternary complex (a). Second, an estimate of a can be made only from k` (a), and this parameter also includes potential contributions from bk(3), the deacylation rate for the butyrylenzyme when fasiculin is bound. Third, butyrylthiocholine is a relatively poor AChE substrate, and torpedo and eel AChEs catalyze its hydrolysis with active site residues different from those utilized with better substrates (Selwood et al., 1993). However, a can be estimated if it is assumed that acylation is slower than deacylation for butyrylthiocholine (i.e.k(2) < k(3) and ak(2) < bk(3) in ). No data directly support these assumptions, but analysis of another poor AChE substrate (N-methyl-(7-dimethylcarbamoxy)quinolinium iodide) indicates that fasciculin 2 has a much greater effect on acylation than deacylation. (^4)With this assumption, the relative increase in reciprocal plot intercepts, also shown in Fig. 5B, plateaus at a value of 1/a. Inserting values of K(F) and a from Fig. 5B into the expression for alpha () leads to an estimate of 60 pM for K (Table 1). K should be identical to K which was estimated to be 147 pM from k/k for butyrylthiocholine in Table 1. The agreement within about a factor of two for these independent estimates is reasonable given the accuracy of the data and the assumptions used to estimate a.

As noted in Table 1, the ratio of k to k in the absence of inhibitors is a measure of the affinity of fasciculin 2 for the free enzyme. With saturating concentrations of noncompetitive inhibitors, this ratio reflects the affinity of fasciculin 2 in the ternary complex. From the data in Table 1, it is apparent that edrophonium and butyrylthiocholine decreased the affinity of fasciculin 2 in the ternary complex 6-14-fold relative to the fasciculin 2 affinity for free AChE. Thermodynamic considerations dictate that fasciculin 2 likewise decrease the affinities of these ligands in the ternary complex by the same amount. To investigate ligand affinity in the ternary complex by a completely different technique, we conducted fluorescence titrations of AChE with N-methylacridinium in the presence or absence of saturating amounts of fasciculin 2. N-Methylacridinium binds to the AChE acylation site and is noncompetitive with propidium (Taylor and Lappi, 1975). In the ternary complex with fasciculin 2, N-methylacridinium affinity for AChE decreased about 13-fold (Table 1), a decrease comparable to that calculated from the kinetic data for edrophonium and butyrylthiocholine.

AChE-catalyzed Hydrolysis of Acetylthiocholine and Phenyl Acetate Are Inhibited by Fasciculin 2 to About Equal Extents

Saturating amounts of fasciculin 2 did not completely block steady state butyrylthiocholine hydrolysis in Fig. 5B, indicating that enzyme acylation and deacylation still occurred when fasciculin 2 was bound to the enzyme. Similar observations were made with two very good substrates of AChE, acetylthiocholine and phenyl acetate, although technical and theoretical considerations (noted under ``Discussion'' and ``Appendix'') required that inhibition of the hydrolysis of these substrates be analyzed only at saturating concentrations of fasciculin 2. The analysis of acetylthiocholine hydrolysis in Fig. 6A showed that k`, the first-order rate constant for enzyme acylation and deacylation in the ternary complex, was about 0.5-1% of k, the corresponding rate constant in the absence of toxin. For phenyl acetate hydrolysis, k` was about 1-2% of k (Fig. 6B). The second-order rate constants k`/K` with fasciculin 2 were about 0.2-0.5% of the corresponding constants k/K without toxin for acetylthiocholine and about 1-2%, for phenyl acetate. The pattern of inhibition of both substrates thus appeared to be nearly noncompetitive, as the first- and second-order rate constants decreased to about the same extent and K` differed from K by less than a factor of 3.


Figure 6: Steady state inhibition of AChE-catalyzed hydrolysis of acetylthiocholine (panel A) and phenyl acetate (panel B) by a saturating amount of fasciculin 2. Hydrolysis rates v (DeltaA/min) were obtained at pH 8 with (bullet) or without () 500 nM fasciculin 2 as outlined under ``Experimental Procedures.'' Total AChE concentrations for the four data sets ranged from 20 pM to 3 nM, and v was normalized to 2 nM AChE for these graphs. The data set for acetylthiocholine without fasciculin 2 was fit to by nonlinear regression analysis to obtain V(max), K = 78 ± 5 µM, and K = 15 ± 2 mM. For the other three data sets, reciprocal plots of (1/vversus 1/[S]) were analyzed by weighted linear regression analysis to obtain V(max) and K or V(max)` and K` (). Data are displayed as reciprocal plots with calculated lines. Analysis of six similar data sets for phenyl acetate in the absence of fasciculin 2 gave a mean K = 1.74 ± 0.14 mM. Because of apparent substrate activation in the presence of fasciculin 2 in panel A, the three points at the highest [S] were deleted from the linear regression analysis.



Second-order Rate Constants for the Steady State Hydrolysis of Acetylthiocholine and Phenyl Acetate Show Larger D(2)O Effects When Fasciculin Is Bound

The residual AChE activities in the presence of saturating amounts of fasciculin 2 shown in Fig. 6were large enough to allow mechanistic studies. For reasons outlined under ``Discussion,'' we chose to investigate deuterium oxide (D(2)O) isotope effects on the first- and second-order rate constants. Steady state experiments similar to those in Fig. 6were conducted in parallel in H(2)O and D(2)O with 100 mM sodium phosphate buffer at pH 8.0 (to eliminate any isotope effects arising from incomplete dissociation of an essential AChE group with a pK of 6.3; Rosenberry, 1975b). In addition, rates at low acetylthiocholine concentration were compared with the integrated form of the Michealis-Menten expression in to improve precision. As shown in Table 2, the D(2)O isotope effects for k`/K` in the presence of fasciculin 2 were larger than those for k/K in its absence for both substrates. The isotope effect for k` also was larger than that for k with acetylthiocholine.




DISCUSSION

The kinetic analyses of fasciculin 2 binding presented here indicate that this toxin and propidium bind to the same peripheral site on the rim of the AChE active site gorge. The mechanism by which fasciculin 2 binding to this site inhibits substrate hydrolysis by AChE is an important focus of this paper. However, several precautions must be taken for correct analysis of steady state substrate hydrolysis in the presence of this inhibitor. 1) As in any measurement of K(I) by steady state kinetics, accurate determinations of K(F) for fasciculin 2 binding require that incubation times be long enough to achieve equilibrium and that fasciculin 2 be sufficiently in excess of AChE to permit the free fasciculin 2 concentration to be approximated by its total concentration. 2) Hydrolysis must be slow enough that substrate is not significantly depleted over the time required for fasciculin 2 binding to reach equilibrium. 3) A nonconventional solution to the steady state rate equation must be considered because of the slow approach to equilibrium fasciculin 2 binding. This point is amplified under ``Experimental Procedures'' and ``Appendix.'' We chose to investigate the dependence of inhibition on fasciculin 2 concentration only with butyrylthiocholine because of constraints imposed by points 2 and 3. For this substrate k is only about 1% of that for acetylthiocholine or phenyl acetate with human AChE (Gnagey et al., 1987), and the approach to equilibrium fasciculin 2 binding could be measured without significant substrate depletion. These measurements revealed that butyrylthiocholine at concentrations up to 5 mM bound exclusively to the AChE acylation site. The affinities of butyrylthiocholine and of edrophonium and N-methylacridinium, the other acylation site inhibitors employed in this study, in the ternary complexes with fasciculin 2 and AChE were about an order of magnitude lower than their affinities for the free enzyme. Steady state analysis of the inhibition of butyrylthiocholine hydrolysis by fasciculin 2 supported this conclusion and further revealed that hydrolysis could still proceed in the ternary complex. Saturating amounts of fasciculin 2 also failed to block completely the steady state hydrolysis of acetylthiocholine and phenyl acetate. Values of k` for the ternary complexes of these substrate were about 0.5-2% of the k values for the corresponding binary enzyme-substrate complexes (Fig. 6). We took advantage of the residual activity to obtain important mechanistic information by investigating D(2)O effects on steady state kinetic parameters.

The simplest mechanistic explanation for the relative decreases in k`/K` and k` in Fig. 6is that fasciculin 2 inhibits largely noncompetitively by slowing formation of the acyl enzyme in (i.e.a ≅ 0.01). To examine more closely the ways in which fasciculin 2 binding could interfere with substrate hydrolysis, it is useful to expand to consider additional intermediates on the catalytic pathway as shown in .

makes explicit two additional intermediates, ES(1) and ES(2). ES(1) occurs prior to the general acid base catalysis step and may involve an induced fit conformational change of the initial ES intermediate, while ES(2) occurs concomitant with general acid-base catalysis and, with carboxylic acid esters, is likely to involve formation of a tetrahedral intermediate. Solvent isotope effects provide evidence for both of these additional intermediates and indicate that their formation can be rate limiting with certain substrates (Rosenberry, 1975b; Rao et al., 1993). How could ligand binding to the peripheral site influence the reaction pathway in ? In contrast to the anionic and esteratic subsites that comprise the acylation site, the peripheral site is not an obvious feature of AChE that would be predicted by simple complementarity to acetylcholine. Its position at the rim of the active site gorge suggests that acetylcholine could bind here transiently before entering the acylation site, but it is unclear whether conformational interaction between the two sites is an important part of this process. An inhibitor bound to the peripheral site could act sterically to block access of a second ligand to the acylation site, but it could also alter the enzyme conformation in a way that would reduce reactivity at the acylation site. To illustrate how these modes of action can be distinguished with fasciculin 2, it is helpful to consider explicitly the formulations of the second-order rate constants for substrate hydrolysis in the presence () and absence () of saturating concentrations of fasciculin 2 as given by a and b.

These second-order rate constants are literally measures of the rate-limiting step at low substrate concentrations in . The rate constants k(S), k, and k(2) from are combinations of the more explicit rate constants involving the first four steps in . One useful demarcation in analyzing these combinations is the first step in which general acid base catalysis occurs. Because this step involves proton transfer, its rate constant invariably shows a D(2)O isotope effect (a decrease of 2-3-fold when D(2)O replaces H(2)O as the solvent) and often shows a pH dependence. In and a, this step is represented by k(2) because it denotes general acid base-catalyzed release of the alcohol from the ester substrate, although k(2) may in fact represent a combination of rate constants from steps III and IV in . Likewise a combines rate constants in steps 1 and II of under the general ligand association and dissociation constants k(S) and k because these steps generally do not involve proton transfer. For clarity we define a steric blockade of the active site as a reduction of both k(S) and k when fasciculin 2 is bound. In the extreme case of equal percent reductions in these rate constants, fasciculin 2 binding at the peripheral site would reduce the rates at which substrate could enter and exit the active site without changing the equilibrium affinity of the substrate in the ternary complex. However, steric blockade in principle could be the sole mechanism of interaction between the sites even if the substrate affinity did decrease, because the decrease could be due to a reduction in the AChE negative electrostatic charge in the binary and ternary complexes. This mechanism, for example, could account in principle for the decreases in ligand affinities in the ternary complex shown in Table 1. We define a conformational interaction between AChE peripheral and active sites as a change in any rate constant not compatible with a steric blockade when fasciculin 2 is bound (i.e. in a change in k(2) or k(3) or an increase in k(S) or k). These definitions permit fasciculin 2 binding at the peripheral site to have both steric and conformational effects on the AChE active site, and we argue that a clear understanding of peripheral site function requires insight into the relative contributions of these two effects.

It is difficult to conduct experiments that measure direct steric blockade due to fasciculin 2 binding. Most ligands that form ternary complexes by binding to the acylation site equilibrate in less than a second even when fasciculin 2 is bound, and no data on the rates of such equilibration are presented here. However, analysis of D(2)O effects on the second-order rate constants in a and b provides some insight into whether fasciculin 2 acts primarily through a steric or a conformational blockade. Key relationships in these constants are the ratios k(2)/k = C and ak(2)/k` = C`, termed the commitments to catalysis (see Quinn, 1987). When C is small, ES is virtually in equilibrium with Eand S, and k(2) is rate limiting for enzyme acylation. Conversely, when C is large, k/K = k(S) and the bimolecular reaction of Ewith S is rate limiting for acylation. Saturation of the peripheral site with fasciculin 2 drastically decreased k` for both substrates in Fig. 6. This decrease suggests that fasciculin 2 binding has an effect on enzyme conformation, but it does not indicate which step in the catalytic pathway is affected. According to a, a small value for C implies that k/K will include the k(2) term and show a D(2)O isotope effect, typically in the range of 2.5. This is the usual case for substrate hydrolysis by most enzymes. For AChE, however, k/K values for acetylthiocholine, phenyl acetate, and certain other substrates show only a small change in D(2)O (see Table 2), and this has led to the conclusion that k(1) or some other step prior to general acid base catalysis is rate limiting in these cases (Rosenberry, 1975b; Quinn, 1987). The D(2)O isotope effects in Table 2provided a test of whether the decreased values of k` and k`/K` in the complex of fasciculin 2 with AChE had a predominantly steric or a conformational basis. If the decreases resulted primarily from steric blockade, one would expect C` to increase and the D(2)O isotope effect on k`/K` to become smaller than that on k/K. Alternatively, if the decreased rates resulted primarily from a conformational interaction, C` should decrease and the D(2)O isotope effect on k`/K` should become larger. Table 2shows that the D(2)O isotope effects on this parameter for both substrates increased with fasciculin 2 binding, indicating that fasciculin 2 acts predominantly to alter the conformation of the active site in the ternary complex so that steps involving proton transfer during enzyme acylation are slowed.

Fasciculin 2 binding also increased the D(2)O isotope effect on k for acetylthiocholine in Table 2, suggesting greater involvement of proton transfer in the transition state for acetylthiocholine cleavage in the ternary complex. However, interpretation of this parameter is more complicated for reasons noted under ``Results,'' and we do not attempt further mechanistic conclusions. The D(2)O isotope effect on k` for phenyl acetate was less clear because of a larger error resulting from variation among AChE preparations. Three different AChE preparations were used to obtain the data in Table 2, and these preparations showed 2-3-fold differences in the maximal decreases in k observed with saturating fasciculin 2 for both substrates. These preparations showed no differences in D(2)O isotope effects in the absence of fasciculin 2 and only slight differences in its presence except for k` for phenyl acetate.

Fig. 6indicates that the decrease in k` relative to k was substantial and of the same magnitude (about 100-fold) for both the cationic substrate acetylthiocholine and the neutral substrate phenyl acetate. This observation contrasts sharply with the effects of saturating concentrations of other peripheral site ligands. Saturation of AChE with propidium resulted in much larger decreases in k for acetylthiocholine than for the neutral substrate 7-acetoxy-4-methylcoumarin (Berman and Leonard, 1990) or for phenyl acetate. (^5)Pt(terpyridine)Cl forms a covalent complex in human AChE with a histidine corresponding to residue 280. This residue is located on the rim of the active site gorge adjacent to Trp, a residue noted above to be critical to the peripheral site for propidium and fasciculin 2. With this covalent conjugate, k for acetylcholine was reduced to 9% of that with the unmodified AChE control, but k for phenyl acetate was increased to 150% of the control (Haas et al., 1992). Thus it is becoming clear that ligands which bind competitively to the same peripheral site at the rim of the AChE active site gorge nevertheless alter reactivity at the acylation site in somewhat different ways. The conformational basis for these differences remains to be explored.


APPENDIX

The general solution for the steady state velocity v from involves many more parameters than the equilibrium solution in . Since most steady state inhibition data for AChE can be fitted to , it is useful to arrange the general solution in a form that resembles this equation. To condense these solutions, the key variables are normalized as follows:

then can be written as

where

In , for example, the reciprocal plot for [F] = 0 reduces to y = 1 + x, and the plot involving competitive inhibition (I(2) = I(3) = 0) reduces to y = 1 + x (1 + I(1)).

The general solution for the initial steady state hydrolysis rate v from has been derived. (^6)A key point is to arrange the solution in a form amenable to instructive simplifications, as the general solution is too complex to be of practical use. The appropriate simplification for fasciculin 2 binding to AChE is the case when inhibitor equilibration is much slower than achievement of the enzyme-substrate steady state: k + k(2)k [F]; k` + ak(2)k; k + k(2)kk/k`; k` + ak(2)k [F] k`/k; k(3)k + k [F]; bk(3)k + k [F]; and bk(3)k + bk [F]. In this case, the ratios of [E], [ES], and [EA] approach those calculated in the virtual equilibrium case, and the general solution reduces to .

where

Analyses of a complete range of fasciculin 2 concentrations in this report is limited to the substrate butyrylthiocholine. The low value of k for this substrate (about 10^2 s; Gnagey et al., 1987) justifies the approximation that Q ≅ 1. With this assumption, collapses to the identical expression given by the virtual equilibrium model in and if C(3) = 0 (k = k(2)). Even if C(3) is allowed to range up to 0.5 (with Q = 1), computer simulations indicate that is an excellent approximation.

At saturating concentrations of fasciculin 2 (both I(1) and aQI(2) 1), reduces to the form of the Michaelis-Menten expression in , where K is replaced by K`; V(max) is replaced by V(max)` = k` [E]; and a and b hold.

This formulation is identical to the virtual equilibrium expression at high [F] in if K` is substituted for K(S)`.


FOOTNOTES

*
This work was supported by Grant NS-16577 from the National Institutes of Health and by grants from the Muscular Dystrophy Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

(^1)
The abbreviations used are: AChE, acetylcholinesterase; DTNB, 5,5`-dithiobis-(2-nitrobenzoic acid).

(^2)
DTNB appeared to have a small effect on the interaction of AChE and fasciculin 2. Addition of 0.33 mM DTNB immediately prior to fasciculin 2 gave a 20-30% decrease in the values of k measured by sequential assays even in the absence of any other ligand. Therefore, k values in the presence of butyrylthiocholine were measured by sequential aliquot assays rather than by continuous spectrophotometric recording in the presence of butyrylthiocholine and DTNB, and DTNB concentrations in steady state assays were reduced to 0.1 mM from the 0.33 mM used in standard AChE assays.

(^3)
A mixture of 1.05 mM NaH(2)PO(4) and 18.95 mM Na(2)HPO(4) gave a pH meter reading of 8.0 in H(2)O and 8.1 in D(2)O.

(^4)
J. Eastman and T. L. Rosenberry, unpublished results.

(^5)
E. W. Eckman and T. L. Rosenberry, unpublished results.

(^6)
T. L. Rosenberry, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. Daniel Quinn of the Department of Chemistry at the University of Iowa for suggesting the use of to improve the precision of D(2)O isotope effects on the hydrolysis of acetylthiocholine by AChE.


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