©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Allosteric Control of Acetylcholinesterase Catalysis by Fasciculin (*)

(Received for publication, March 31, 1995; and in revised form, May 3, 1995)

Zoran Radić(§) Daniel M. Quinn (¶) Daniel C. Vellom (**) Shelley Camp Palmer Taylor

From the Department of Pharmacology, University of California San Diego, La Jolla, California 92093-0636

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The interaction of fasciculin 2 was examined with wild-type and several mutant forms of acetylcholinesterase (AChE) where Trp, which lies at the base of the active center gorge, is replaced by Tyr, Phe, and Ala. The fasciculin family of peptides from snake venom bind to a peripheral site near the rim of the gorge, but at a position which still allows substrates and other inhibitors to enter the gorge. The interaction of a series of charged and uncharged carboxyl esters, alkyl phosphoryl esters, and substituted trifluoroacetophenones were analyzed with the wild-type and mutant AChEs in the presence and absence of fasciculin. We show that Trp is important for the alignment of carboxyl ester substrates in the AChE active center. The most marked influence of Trp substitution in inhibiting catalysis is seen for carboxyl esters that show rapid turnover. The extent of inhibition achieved with bound fasciculin is also greatest for efficiently catalyzed, charged substrates. When Ala is substituted for Trp, fasciculin becomes an allosteric activator instead of an inhibitor for certain substrates. Analysis of the kinetics of acylation by organophosphates and conjugation by trifluoroacetophenones, along with deconstruction of the kinetic constants for carboxyl esters, suggests that AChE inhibition by fasciculin arises from reductions of both the commitment to catalysis and diffusional entry of substrate into the gorge. The former is reflected in the ratio of the rate constant for substrate acylation to that for dissociation of the initial complex. The action of fasciculin appears to be mediated allosterically from its binding site at the rim of the gorge to affect the orientation of the side chain of Trp which lies at the gorge base.


INTRODUCTION

The fasciculins, 61 amino acid peptides found in venom of snakes of the mamba or Dendroaspis family, are potent inhibitors of most acetylcholinesterases (EC 3.1.1.7) (AChEs)(^1)(1) . Three fasciculins have been identified to date with nearly identical amino acid sequences: fasciculin 1 (FAS1), FAS2, and FAS3. FAS3 binds to rat brain AChE with a dissociation constant one order of magnitude lower than that for FAS1 and FAS2(2) . The sequence and crystal structure of FAS1 (3) show that these toxins fall within a larger family of ``three finger'' Elapidae toxins which include erabutoxin, cardiotoxins, alpha-bungarotoxin, and alpha-cobratoxin. These latter toxins, however, do not inhibit AChE, whereas FAS at low concentrations does not block acetylcholine receptor function(4, 5) .

Torpedo and most mammalian AChEs are inhibited by FAS at picomolar concentrations, whereas the closely related butyrylcholinesterases (EC 3.1.1.8) (BuChE) require FAS concentrations approaching millimolar for inhibition(5, 6) . Three domains in AChE and butyrylcholinesterase appear responsible for their distinct substrate and inhibitor specificities(7, 8, 9, 10) . Two of them, the acyl pocket and choline-binding site of the active center, should be virtually inaccessible to FAS since they are located at the base of a narrow gorge leading to the active center of the cholinesterases. FAS, however, should access binding sites peripheral to the active center as evidenced by protection of the enzyme from FAS inhibition by micromolar concentrations of propidium and millimolar concentrations of acetylthiocholine (ATCh). This concentration range of ATCh results in substrate inhibition rather than maximal catalytic rates(2, 5) . Thus, FAS appears to be specific for a peripheral site on AChE. The high affinity of FAS is primarily a reflection of a slow dissociation rate yielding a half-time of several hours, while the rate of association between FAS and AChE is only one to two orders of magnitude slower than expected for a diffusion-controlled reaction between a peptide and a protein(2, 6) .

The mechanism of AChE inhibition by FAS might involve physical occlusion of the entrance to the active center gorge by the peptide, modification of the electrostatic field affecting substrate entry into the gorge, and/or an allosteric influence on the active center affecting the commitment to catalysis of bound substrate. The capacity of the FASbulletAChE complex to react with diisopropyl fluorophosphate (2) reveals that the active serine at the base of the gorge remains potentially accessible to reaction with substrates. In this article we analyze the possible mechanisms of inhibition by FAS by measuring kinetic constants for inhibition of wild-type and several mutant cholinesterases. In addition, we examine the capacity of FAS to affect AChE specificity for carboxyl and alkylphosphoryl ester acylation and for conjugation with substituted trifluoroacetophenones.


MATERIALS AND METHODS

Enzymes

Wild-type and mutant mouse AChEs were expressed in HEK-293 cells following transfection of the cells with the encoding cDNA as described previously(7, 8) . Cells containing the recombinant plasmids stably integrated into the DNA were used, and AChE was concentrated from the serum-free medium in which the expressing cells were grown.

Fasciculin

Purified and lyophilized FAS2 was kindly provided by Dr. Carlos Cervenansky, Instituto de Investigaciones Biologicas, Montevideo, Uruguay. Concentrations of FAS stock solutions were determined by absorbance ( = 4900 M cm)(1) .

Inhibitors

m-Tertbutyl trifluoroacetophenone (TFK) and m-trimethylammonium trifluoroacetophenone (TFK+) were synthesized as described earlier(11, 12) . Paraoxon was obtained from Sigma and echothiophate from Ayerst Laboratories Inc. (Philadelphia, PA) (Fig. ZI).


Figure ZI: Structure I.



Enzyme Activity

Hydrolysis of ATCh, phenylacetate (PA), and p-nitrophenylacetate (pNPA) was measured spectrophotometrically using the method of Ellman et al.(13) for ATCh, or measurement of phenol or p-nitrophenol release at 270 and 405 nm, respectively. Kinetic constants for hydrolysis of the above substrates by wild-type AChE and butyrylcholinesterase and mutant AChEs were determined using a nonlinear fit of the data to the following equation (modified from (14) ):

where S denotes the substrate concentration, K and K Michaelis-Menten and substrate inhibition constants, and b the productivity ratio of the ternary SES complex to the ES complex (cf. 7).

Enzyme Inhibition

Reversible inhibition constants in the picomolar range were determined upon overnight incubation of aliquots of enzymes with increasing concentrations of FAS and fitting the remaining enzyme activities to the following equation:

where v is described by(15) :

and K is a reversible inhibition constant for fasciculin, F, and is also equal to k/k(F). and were derived from :

with the constraints of F = 0 for , and KSK and F E for .

Rate constants for FAS and trifluoroacetophenone association and dissociation were determined as described earlier(6) . Rate constants for inhibition of the enzymes with paraoxon and echothiophate as well as rate constants of association of trifluoroacetophenones were determined by following the time course of the onset of inhibition using ATCh as substrate and three or more inhibitor concentrations as described earlier(16) .


RESULTS

Rate and Equilibrium Constants for Fasciculin Binding to Mutant Acetylcholinesterases

FAS inhibition of wild-type and Trp substituted AChEs, measured with ATCh and pNPA as substrates, is shown in Fig. 1. All of the enzymes were inhibited by FAS except for TrpAla, where the presence of FAS actually increased enzyme activity toward pNPA. None of the enzymes could be inhibited completely at high FAS concentrations. Residual uninhibited activities were always higher when measured with 1 mMpNPA compared to 1 or 10 mM ATCh. A nonlinear fit of experimental data to yielded K, the equilibrium dissociation constant for FAS, and beta, the fraction of residual activity reflecting partial productivity of the FES complex. These data are tabulated in Table 1. Dissociation constants for FAS inhibition, K, were indistinguishable when measured with both substrates but were increased up to 10-fold by the various mutations. Residual enzyme activities ranged from below the experimental error of detection (0.2%) in the case of wild-type AChE to 80 and 160% of the control activity in the TrpAla mutant for ATCh and pNPA, respectively. Thus, the primary effect of substitutions at Trp is on the capacity of FAS to influence catalytic parameters once bound to enzyme rather than on FAS binding per se.


Figure 1: Influence of varying FAS concentrations on the activity of wild-type and mutant mouse AChE. Enzyme activity was measured using ATCh (A) and pNPA (B) as substrates at concentrations denoted in Table 1. Theoretical curves were obtained by fitting experimental points to equation(2) . Trp at position 86 in the wild-type enzyme was replaced by Phe (F), Tyr (Y), and Ala (A). The recombinant DNA-derived enzymes were incubated overnight (14-16 h) with FAS prior to assay.





This behavior contrasts with the marked increases in K and lack of effect on beta that three substitutions at peripheral site residues, TrpArg, TyrAsn, TyrGln, have on FAS interactions with AChE. It also distinguished from the virtual lack of effect that substitutions at the acyl pocket and position 337 have on FAS inhibition(6) . With these mutants residual activities of the FAS complexes measured with ATCh were equivalent to those for wild-type enzyme. The residues at positions 286, 72, and 124 constitute part of the peripheral site and are located at the entrance of the active center gorge. They are directly accessible to FAS and might be expected to be in direct contact with FAS. Accordingly, their replacement should influence the dissociation constant of FAS. The two aromatic residues Trp and Tyr in the choline-binding site are located deep within the active center gorge and seem inaccessible for direct contact by FAS. Therefore, a minimal effect of their replacement by Ala on K of the FAS-AChE complex is not surprising. However, while TyrAla was almost totally inhibited by saturating FAS concentrations (>99%), hydrolysis of ATCh by TrpAla was only inhibited about 20% at saturating FAS concentrations. Catalytic hydrolysis of pNPA was activated in the presence of FAS.

The small increase in K arising from substitutions at Trp is due to the increase in the dissociation rate constant for FAS, k (Table 2), as previously observed for the TyrGln and AspAsn mutants(6) . Rates of FAS association are not affected by any of the mutations where the rate constants are sufficiently slow for detection by conventional measurement.



The presence of ATCh in concentrations up to 1 mM did not affect FAS association or dissociation rate constants. FAS therefore binds with the same affinity to free enzyme and enzymebulletATCh complex. Only high ATCh concentrations, sufficient to cause substrate inhibition in wild-type AChE, decrease the rates of FAS association with wild-type and mutant enzymes(6) . The decrease may be a consequence of either a modified electric field in AChE or, more likely, competition between occupation of ATCh and FAS at peripheral sites.

The Influence of Trp Substitutions on Substrate Hydrolysis

The dependences of enzyme activity on ATCh and pNPA concentrations, measured in the absence and presence of saturating FAS are shown in Fig. 2. The relatively high enzyme concentrations (2-8 nM) used in these experiments enabled detection of residual activity in the presence of FAS. Experimental data fitted to yielded the constants reported in Table 3; also included are the constants for hydrolysis of a neutral substrate PA, which has a high turnover rate.


Figure 2: Influence of fasciculin on acetylcholinesterase catalysis. A, representative plots of activity versus substrate concentration for high (ATCh) and low (pNPA) turnover substrates in the presence and absence of saturating (40-200 nM) FAS. Curves were obtained by nonlinear regression of to fit the experimental points. Fasciculin was incubated with the enzyme for at least 1 h prior to the activity measurements. B, substrate concentration dependence for catalysis of ATCh and pNPA by wild-type AChEbulletFAS complex. Data show enlarged ordinates from the top panels. The solid line was obtained by a nonlinear regression of to fit the experimental points. The dotted line shows the best fit to the data assuming Michaelis-Menten kinetics.





Substitutions of Phe, Tyr, or Ala for Trp at position 86 influence catalysis to the largest extent with the most efficient substrate ATCh. Replacements of the indole ring of Trp with other aromatic substituents reduce k/K by an order of magnitude, whereas deletion of the ring leaving the aliphatic side chain in alanine reduces k/K by two orders of magnitude. The reductions in k/K are reflected mainly in larger K values except for the TrpAla mutation where k is reduced 50-fold. However, the TrpAla enzyme shows substrate activation which will effectively increase catalysis at high substrate concentrations.

In the case of PA, a neutral but relatively high turnover substrate, k was decreased only 2-3-fold for the aromatic substituents, and 10-fold for the Ala substitution. Since the K values also decrease slightly upon substitution of Phe or Tyr for Trp, k/K is virtually unaffected. pNPA is a relatively poor substrate whose k/K for the wild-type enzyme is nearly three orders of magnitude less than that for ATCh. All three substitutions for Trp led to an increase of k/K for pNPA; this is a cumulative effect of lowering K and increasing k.

Thus, the mutations reduced k in a similar manner for the two substrates of rapid turnover, ATCh and PA, while two neutral substrates, PA and pNPA, have their K values similarly affected. k/K was affected the most for ATCh and pNPA, while for PA k was most affected.

Inhibition of Wild-type and Mutant Enzyme Substrate Hydrolysis by Fasciculin

FAS inhibition is most marked with the wild-type enzyme showing a reduction of activity by three orders of magnitude for the efficient substrates and by about 10-fold for pNPA (Table 3). Most of the reduction arises from a diminution in k. In all of the mutant enzymes, such large reductions in k associated with FAS association are not evident. Substitution of Tyr or Phe for Trp results in a smaller reduction of k/K for the two fast substrates upon FAS binding, and mainly arises from changes in K. FAS has little influence on either k or K for pNPA. In the case of the TrpAla enzyme, an apparent activation is measured for all three substrates. It is noteworthy that in the presence of FAS the mutant enzymes exhibit relatively similar catalytic parameters.

The Influence of Trp Substitutions and Fasciculin on Substrate Inhibition

Of the three carboxyl ester substrates only ATCh exhibited substrate inhibition in the range of substrate concentrations used. The upper concentration boundary for detection of catalysis is limited by the high rate of general base hydrolysis for ATCh and by the solubilities for aromatic esters. Introduction of smaller aromatic residues, Phe and Tyr, in place of Trp increased the substrate inhibition constant, K, stepwise to a value 60 times larger than the wild-type constant. By contrast for the TrpAla mutant, substrate activation was observed and no substrate inhibition was measurable (Table 3). The parameter b, reflecting the efficiency of the hydrolysis of the ternary complex (cf.), increased from 0.2 in wild-type to 0.4 for TrpPhe and to 8.2 for TrpAla, whereas for the TrpTyr mutant, ascertaining whether the mutation affects K or b is difficult owing to the lack of pronounced substrate inhibition or activation.

The binding of FAS to the wild-type and mutant enzymes completely abolished substrate inhibition by ATCh. In fact, Fig. 2B shows elements of substrate activation (b = 6.8) for hydrolysis of ATCh by the complex of wild-type AChE with bound FAS. This is similar to the activation observed for ATCh hydrolysis by the TrpAla mutant in the absence of FAS. Substrate activation should not arise as a result of FAS-ATCh competition since the FAS dissociation rate is inherently slow (cf. Table 1).

Fasciculin Inhibition of Wild-type and Mutant Enzyme Phosphorylation by Charged and Uncharged Organophosphates

Rate constants for time-dependent inhibition of the enzymes by echothiophate and paraoxon were determined in the absence and presence of saturating concentrations of FAS (Table 4). Reaction rates for enzymes in the absence of FAS followed pseudo-first-order kinetics. However, in the presence of FAS, the time courses of inhibition were frequently biphasic. The fast phase always had the greater amplitude, but the kinetics required rate constants that differ by a factor of two to eight to fit the data. This phenomenon was independent of FAS concentration in the range of 20-120 nM where enzyme concentrations were lower than 0.3 nM. Reaction rates were determined from the initial linear portion of the inhibition curves.



The largest reduction of a second-order phosphorylation constant, approaching three orders of magnitude, was produced by TrpAla substitution in reaction with echothiophate (Table 4). With the same mutation, paraoxon inhibition was affected only 3-fold. Substitution of Trp with Phe decreased the second-order inhibition constants for echothiophate and paraoxon 7- and 3-fold, respectively, while the inhibition constant for the TrpTyr mutant enzyme was not affected (Table 4). The changes in inhibition parameters were mainly a consequence of an increase in K for echothiophate and a decrease in maximal phosphorylation rate for paraoxon.

FAS association had more effect on inhibition by echothiophate with its cationic leaving group than by the neutral congener, paraoxon. The second-order inhibition rate constant for the wild-type enzymebulletFAS complex decreased 10-fold for echothiophate as a result of an increase in its K; paraoxon's overall inhibition rate constant is largely unchanged, although both K and k were decreased in a compensatory fashion in the presence of FAS. FAS binding to the TrpPhe and TrpTyr mutants only modestly affects inhibition by paraoxon, whereas echothiophate inhibition is decreased up to 10-fold as a consequence of a slower phosphorylation rate. FAS had a minimal effect on inhibition of the TrpAla mutant by paraoxon, but the second-order inhibition constant for echothiophate was increased 100-fold. This mutant enzyme shows both enhanced affinity and reactivity for the cationic organophosphate in the presence of FAS.

Fasciculin Inhibition of Wild-type and Mutant Enzyme Conjugation by Charged and Uncharged Substituted Trifluoroacetophenones

Substitution of aromatic and aliphatic residues for Trp results in destabilization of the hemiketal conjugates for both the charged and uncharged m-trimethylammonium trifluoroacetophenone and m-terbutyl trifluoroacetophenone isosteres (Table 5). These substrate analogs differ from the carboxyl and phosphoryl esters in that the reaction product is a conjugate formed without the loss of a leaving group. Recent x-ray crystallographic studies of the complex do indeed show bond distances consistent with formation of a hemiketal conjugate(17) . While mutations at position 86 are limited to 20-fold or smaller increases in the rates of dissociation of the neutral trifluoroacetophenones conjugate, the rates of dissociation of the charged conjugate are affected by one to two orders of magnitude and rates of formation up to an order of magnitude. This results in up to 1000-fold destabilization of charged conjugate for TrpAla mutant and only a 20-fold destabilization of the neutral conjugate.



Second-order inhibition rates for trifluoroacetophenones reflect both formation of a reversible complex and subsequent covalent bond formation. Since rates of conjugate formation for both isosteres and all mutants showed a linear dependence on concentration, formation of conjugates was treated as a simple bimolecular association. When correlated for the ratio of non-hydrated to hydrated species(12) , the measured second-order reaction rate constants for the non-hydrated ketones fell well in the range of rates for diffusion-limited reactions (10^9-10M min). These values are very similar to a rate of association of 4 10M min determined for the reversible inhibitor N-methylacridinium (18) and exceed k/K for ATCh. Even though NMR measurements for similar trifluoroacetophenones (19) and the crystal structure of the AChEbulletTFK complex (17) show that trifluoroacetophenones bind to the active serine covalently forming a hemiketal, the rate of the TFK reaction appears limited by diffusion in the concentration range where measurements are practical.

Bound FAS affects the reaction rates of trifluoroacetophenones for both the wild-type and mutant enzymes. Rates of conjugate formation with the wild-type and TrpPhe enzymes decreased in the fasciculin complexes, and rates of conjugate dissociation increased to a greater extent with the charged than with neutral isostere. This results in a 10-fold greater increase in K for the charged isostere. Bound FAS also affected the interaction of both trifluoroacetophenones with TrpTyr and TrpAla. While bound FAS increased Kfor TrpTyr 10-fold, the K for TrpAla mutant decreased about 2-fold. It is interesting that a 1000-fold difference in K between charged and uncharged isosteres for wild-type AChE is significantly reduced 5-100-fold when FAS is bound to the mutant or wild-type enzymes.

Bound FAS decreased rates of diffusion of the cationic TFK into the enzyme gorge by an order of magnitude. Since bound FAS may restrict diffusion-limited entry into the gorge, the reductions in rate of TFK association may reflect the gating influence of the positively charged FAS molecule toward entry of cationic substrates into the gorge. In addition, substitution of aromatic and aliphatic residues at position 86 diminishes the stability of the hemiketal conjugates formed between the substituted trifluoroacetophenones and AChE. These factors are reflected in a larger k and a smaller k(F). As the overall k(F) diminishes, diffusion is no longer rate-limiting. With the substitutions at position 86 which form the less stable complexes, FAS has a greatly diminished influence on the reaction. FAS affects rates of dissociation of the conjugate presumably by allosterically influencing the site of TFK binding. The overall effect of FAS binding is similar in magnitude to substitution of Ala for Trp.


DISCUSSION

In this study we have examined the influence of a peptidic peripheral site inhibitor, FAS 2, on the catalytic parameters of AChE. Substantial evidence has accumulated to show that FAS binds at a peripheral site to regulate substrate catalysis(1, 2, 5, 6) . We show here by examining mutations in the choline binding subsite at the base of the gorge that FAS, by acting near the rim of the gorge, controls the overall configuration of the substrate-binding site. By comparing charged and uncharged substrates, one approaches the question of whether FAS affects initial entry of substrate into the gorge or the subsequent steps of catalytic turnover of bound substrate. To this end it becomes useful to deconstruct the catalytic parameters, k and K, where possible, into primary constants. For below:

we have examined three classes of substrates designated by AB: (a) the carboxylic acid esters: ATCh, pNPA, and PA. These acetoxy esters differ about 600-fold in catalytic efficiency, k/K, with the following order ATCh>PA>pNPA; (b) the symmetric organophosphates which only differ in their leaving groups: the charged, diethoxyphosphoryl thiocholine (echothiophate), and uncharged diethoxyphosphoryl p-nitrophenol (paraoxon). These two compounds yield structurally identical covalent conjugates with the active serine. Echothiophate and paraoxon have the same leaving groups as ATCh and pNPA, respectively. In contrast to the carboxyl esters, only rates of acylation require consideration in analyzing substrate kinetics. For organophosphates K (now defined as K) is reduced to (k(1) + k(2))/k(1), and k is reduced to k(2); (c) isosteric trifluoroacetophenones which contain charged and uncharged moieties at the meta position. The conjugation reaction here only involves nucleophilic addition by Ser to form the hemiketal without loss of a leaving group. Consequently K reduces to the ratio of k and k(1). There is no substrate turnover, and k is zero.

Since the AChEbulletFAS complex remains catalytically active and susceptible to phosphorylation by diisopropyl fluorophosphate(2) , or other organophosphates (Table 4), FAS does not serve as a physical barrier to block totally substrate entry into the AChE active center gorge. Rather, binding of FAS to the enzyme appears to affect the chemical acylation step of the catalytic reaction described by k(2). This is indicated by pronounced reductions in k and k/K for the three carboxyl esters, as opposed to modest increases in their K values. Values of k and k/K for hydrolysis of ATCh and PA by human AChE in the presence of FAS decrease significantly in D(2)O buffers, while in the absence of FAS the isotope effect of D(2)O is small(20) . These findings indicate that FAS reduces the rate of ATCh and PA acylation described by k(2).

In addition, cationic ligands may find their entry to the active center diminished due to the electrostatic restrictions imposed by bound FAS. Positively charged ATCh, unlike the other two neutral substrates, has a 5-fold greater K for the AChEbulletFAS complex compared to AChE. Also, charged organophosphates and trifluoroacetophenones react with AChEbulletFAS complex at rates an order of magnitude slower than with AChE. The TrpAla enzyme is an exception where FAS accelerates an inherently slow rate of echothiophate inhibition while exerting little influence on the reaction with the neutral organophosphates.

Substitution of Trp by two other aromatic residues, Phe and Tyr, abolishes the capacity of FAS to reduce acylation rates, whereas introduction of Ala at this position results in bound FAS causing a slight increase of acylation rates for all substrates. Thus, unlike the other residues in the choline binding site (i.e. Tyr) and acyl pocket (i.e. Phe, Phe)(6) , the indole ring of Trp is linked to the mechanism of FAS inhibition. The aromatic substitutions, however, slightly enhance the increase in K induced by FAS for the two most effective substrates ATCh and PA. This suggests that an aromatic residue at position 86 influences k(1) or k in the FASbulletAChE complex. For catalytic hydrolysis of both ATCh and PA, k is likely to be increased by FAS, while we might also expect a reduction of k(1) for the charged substrate ATCh. Catalytic hydrolysis of pNPA is far slower (Table 3), and it has been suggested, based on solvent isotope effects on acylation rates, that it is rate limited by the chemical acylation step(21, 22) .

Introduction of an aliphatic residue at position 86 exerts a large reduction in the catalytic rates for both fast substrates PhAc and ATCh. With the less efficient catalysis, the influence of FAS is diminished so that the FAS complexes of these mutant enzymes have catalytic constants which approach those of wild-type enzymebulletFAS complex. The mild acceleration in catalytic rate for the TrpAla AChEbulletFAS complex is likely to be a consequence of an increase in the acylation rate constant k(2), indicating an enhanced capacity to stabilize acylation transition state upon FAS binding. This conclusion is supported by the observation that the binding of FAS increases the affinity of the TrpAla mutant for both charged and neutral trifluoroacetophenones, substrate transition state analogs. Hydrolysis of ATCh by the Trp to Ala mutant enzyme is enhanced upon binding of FAS primarily through enhancing k(2), suggesting an improved fit of the ATCh transition state in the active center gorge.

Ordentlich and colleagues (23) previously examined the TrpAla mutation on rates of catalytic hydrolysis of ATCh, pNPA, and other carboxyl esters. They also observed a dramatic reduction on ATCh hydrolysis with the TrpAla mutation and smaller effects on other substrates. They suggested that Trp is not involved in the stabilization of uncharged substrates. However, the Trp to Ala mutation accompanies a large volume change which must be accommodated by either collapse of the peptide backbone or entry of several water molecules into the gorge. Analysis of the kinetics through stepwise replacement of Trp by Tyr, Phe, and then Ala suggests that subtle changes in alignment of the associated carboxyl ester are sufficient to dramatically decrease catalysis. Moreover, occupation of the peripheral site by FAS may affect the alignment of the substrate through an allosteric influence mediated between the lip of the gorge and its base. Support for this linkage comes from previous studies of AChE structure involving circular dichroism, fluorescence spectroscopy, and site-specific mutagenesis when propidium is bound to the peripheral site(24, 25, 26) . Efficient catalysis requires an optimal alignment of substrate, and FAS exhibits its most dramatic effects on inhibition parameters for the fast substrate. When alignment is compromised through residue substitution at position 86, FAS has a far smaller effect on catalysis and can, in some cases, slightly increase k/K.

Since both Trp mutations and FAS affect substrate inhibition parameters, the binding of a second substrate molecule at the FAS site may also mediate its effect on catalysis through Trp(6, 27) . Although FAS binding and Trp substitutions occur at two different locations, they both influence the alignment of residues in the site of ATCh catalysis and prevent the most productive binding orientation of substrate. Substrate activation of the residual activity in the FAS-AChE complex (Fig. 2B), however, may be a consequence of ATCh interaction with still another site on the enzyme.

The effect of Trp substitutions on acylation by the organophosphates resembles that seen for the carboxylic acid esters. The substrate pairs of ATCh and of echothiophate and p-nitrophenyl acetate and paraoxon share identical leaving groups, and their reaction rates show similar sensitivities to Trp substitution. This underscores the importance of residue 86 in affecting the position of the leaving group. However, FAS is a less effective inhibitor of echothiophate acylation in the wild-type enzyme by at least two orders of magnitude. Compared to ATCh, the thiocholine leaving group in echothiophate is directed slightly farther away from Trp and closer to the anionic residue of Asp, due to the tetrahedral geometry around the phosphorus in echothiophate (Fig. 3). (^2)The difference in the leaving group orientations in the transition state complexes with FAS-AChE may cause Trp substitutions to affect acylation rates in markedly different manners since stabilizing forces contributed by Trp decrease with the sixth power of distance(29) . This conclusion is supported by the significantly greater acceleration of echothiophate inhibition in TrpAlabulletFAS complex as compared to the mild acceleration of ATCh hydrolysis.


Figure 3: Presumed orientation of the transition states for acylation of AChE by ATCh (black) and echothiophate (gray) obtained by molecular modeling of the acylation transition state for the reaction of the two compounds with AChE. Molecular dynamics modeling was performed as described in (28) .



The geometry of the planar trifluoroacetophenones and the positioning of the carbonyl oxygen toward the oxyanion hole should direct quaternary ammonium or tertbutyl groups to a position equivalent to that of choline in ATCh, in close apposition to Trp as confirmed by crystal structure of the charged TFKbulletAChE complex(17) . These tetrahedral adducts resemble the acylation transition state analogues of ATCh hydrolysis. That FAS affects k/K for substrates and K for trifluoroacetophenones to similar extents suggests that the major mode of FAS action is to affect stabilization of the transition state for hydrolysis of substrates.

Correlations of the free energy of association of the TFKs with AChE in the absence and presence of FAS with molar refractivities and hydrophobicities of residues at position 86 (Fig. 4) support such a mechanism. In the absence of FAS, the pK values for both TFKs increase with both molar refractivity and hydrophobicity constants for the residues at position 86 indicating that this residue is directly involved in stabilization of the TFKbulletenzyme complex. In the presence of FAS, the contributions to the energy of stabilization of the aromatic residues at position 86 remain unchanged and roughly equivalent to that of Ala. Hence FAS association at a distant location on AChE appears to negate the stabilization energy conferred by the electron-rich indole ring, perhaps by allosterically influencing its alignment.


Figure 4: Relationship between the inhibition constant of trifluoromethyl acetophenones (TFK^0 and TFK) and the molar refractivity and hydrophobicity of the side chain at residue 86 in the AChE choline subsite. Molar refractivity and hydrophobicity indexes for amino acid residues were taken from (30) and analyzed according to Nair et al.(29) . Measurements were made in the presence and in the absence of 40 nM FAS following incubation with FAS for at least 1 h.



Recently, it was suggested that products of acetylcholine hydrolysis or perhaps solvent molecules required for catalysis could exchange with bulk solvent through an opening in the AChE gorge wall behind Trp, termed the ``back door''(31, 32) . A vectoral movement of substrate and products would pose additional energetic requirements for acylation and deacylation in catalysis. Mutagenesis of residues in the region of the putative back door region has not yielded supportive evidence for this hypothesis(33) , nor is there substantive evidence for choline dissociation being a rate limitation. One might assume that diminished FAS inhibition, arising from the smaller side chain at position 86, might arise from increasing the opening at the putative back door thereby increasing the rate of exchange of ligands between bulk solvent and the active center gorge. The catalytic constants may, however, point to the opposite conclusion. FAS most effectively blocks acylation of rapidly reacting substrates and the most rapid catalysis is achieved with an indole residue at position 86. Also, rates of dissociation of positive and neutral trifluoroacetophenones increase with small side chains at position 86. The binding of FAS further increases these dissociation rates, whereas one might expect a decrease if it directly or indirectly closed the back door.

While the three-dimensional structure of AChEbulletFAS complex is still unknown, one may speculate on a possible sequence of events associated with FAS binding and enzyme inhibition. Identification of interacting residues on AChE through site-specific mutagenesis(6) , combined with an computational analysis of the interacting forces between FAS and AChE(34) , suggests that a likely area of their interaction is the most amino-terminal disulfide loop in AChE encompassing residues 69-96 (Fig. 5). This loop covers the active center gorge as a ``lid'' with Trp residing at its tip. Tyr, one of the aromatic residues that form the peripheral site, is located at the base of the loop, while the adjacent residues Tyr and Trp, which also contribute to the peripheral site, reside on the other two AChE loops. Binding of a ligand to the peripheral site could therefore serve to partly close the lid. A conformational change of a homologous loop in a lipase from Candida rugosa was shown from the crystal structure to be essential for catalysis(35) , wherein an open site is found in the active state. While there are no indications from crystallography of mobility of the homologous loop in AChE(36) , flexibility of such a loop may be required for efficient catalysis. Substitutions of two aspartates at positions 92 and 93 in Torpedo californica AChE (equivalent to Asp and Asp in mouse AChE) with neutral residues at the base of this loop yield inactive enzyme (37) and are expected to break at least two salt bridges.


Figure 5: Stereo ribbon diagram of a model of the fasciculinbulletAChE complex(34) . The first disulfide AChE loop (Cys-Cys) is represented by a bold ribbon. Side chains of Trp, active Ser, and peripheral site residues Tyr, Tyr, and Trp are displayed and labeled with their numbers. FAS represented by the gray ribbon is sitting atop the disulfide loop, while interacting with peripheral site residues.



Hence FAS inhibition may arise from an influence on two steps in the catalytic process. For cationic ligands where catalysis is efficient, FAS may serve to partially gate their entry therein diminishing k(1) in . It also exerts an allosteric influence on the alignment of all substrates in the active center gorge. This occurs in the transition state where it apparently affects the configuration of the leaving group in achieving productive acylation. Capping a potentially mobile loop which extends between the lip of the gorge, containing the peripheral site, and its base, where Trp resides, represents an attractive structural basis for an allosteric linkage. This may serve to decrease the stability of the initial complexes in the active center and/or diminish the commitment to catalysis, represented by k(2)/k(1)(22) .


FOOTNOTES

*
This work was supported by United States Public Health Service Grant GM18360 (to P. T.). 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.

§
Visiting Fellow from the Institute for Medical Research and Occupational Health, University of Zagreb, 41000 Zagreb, Croatia.

Visiting Professor: Dept. of Chemistry, University of Iowa, Iowa City, IA 52242.

**
Present address: Dept. of Chemistry, City University of New York, Brooklyn, NY 11210.

(^1)
The abbreviations used are: AChE, acetylcholinesterase; FAS, fasciculin; ATCh, acetylthiocholine; TFK, m-tertbutyl trifluoroacetophenone; TFK+, m-trimethylammonium trifluoroacetophenone; PA, phenylacetate; pNPA, p-nitrophenylacetate.

(^2)
N. Hosea, H. A. Berman, and P. Taylor, manuscript submitted.


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