Thioflavin T Is a Fluorescent Probe of the Acetylcholinesterase Peripheral Site That Reveals Conformational Interactions between the Peripheral and Acylation Sites

Three-dimensional structures of acetylcholinesterase (AChE) reveal a narrow and deep active site gorge with two sites of ligand binding, an acylation site at the base of the gorge, and a peripheral site near the gorge entrance. Recent studies have shown that the peripheral site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, but the question of whether the peripheral site makes other contributions to the catalytic process remains open. A possible role for ligand binding to the peripheral site that has long been considered is the initiation of a conformational change that is transmitted allosterically to the acylation site to alter catalysis. However, evidence for conformational interactions between these sites has been difficult to obtain. Here we report that thioflavin T, a fluorophore widely used to detect amyloid structure in proteins, binds selectively to the AChE peripheral site with an equilibrium dissociation constant of 1.0 μm. The fluorescence of the bound thioflavin T is increased more than 1000-fold over that of unbound thioflavin T, the greatest enhancement of fluorescence for the binding of a fluorophore to AChE yet observed. Furthermore, when the acylation site ligands edrophonium or m-(N,N,N-trimethylammonio)trifluoroacetophenone form ternary complexes with AChE and thioflavin T, the fluorescence is quenched by factors of 2.7–4.2. The observation of this partial quenching of thioflavin T fluorescence is a major advance in the study of AChE for two reasons. First, it allows thioflavin T to be used as a reporter for ligand reactions at the acylation site. Second, it indicates that ligand binding to the acylation site initiates a change in the local AChE conformation at the peripheral site that quenches the fluorescence of bound thioflavin T. The data provide strong evidence in support of a conformational interaction between the two AChE sites. Three-dimensional structures of acetylcholinesterase (AChE) reveal a narrow and deep active site gorge with two sites of ligand binding, an acylation site at the base of the gorge, and a peripheral site near the gorge entrance. Recent studies have shown that the peripheral site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, but the question of whether the peripheral site makes other contributions to the catalytic process remains open. A possible role for ligand binding to the peripheral site that has long been considered is the initiation of a conformational change that is transmitted allosterically to the acylation site to alter catalysis. However, evidence for conformational interactions between these sites has been difficult to obtain. Here we report that thioflavin T, a fluorophore widely used to detect amyloid structure in proteins, binds selectively to the AChE peripheral site with an equilibrium dissociation constant of 1.0 μm. The fluorescence of the bound thioflavin T is increased more than 1000-fold over that of unbound thioflavin T, the greatest enhancement of fluorescence for the binding of a fluorophore to AChE yet observed. Furthermore, when the acylation site ligands edrophonium or m-(N,N,N-trimethylammonio)trifluoroacetophenone form ternary complexes with AChE and thioflavin T, the fluorescence is quenched by factors of 2.7–4.2. The observation of this partial quenching of thioflavin T fluorescence is a major advance in the study of AChE for two reasons. First, it allows thioflavin T to be used as a reporter for ligand reactions at the acylation site. Second, it indicates that ligand binding to the acylation site initiates a change in the local AChE conformation at the peripheral site that quenches the fluorescence of bound thioflavin T. The data provide strong evidence in support of a conformational interaction between the two AChE sites. acetylcholinesterase m-(N,N,N-trimethylammonio)trifluoroacetophenone Acetylcholinesterase (AChE)1 hydrolyzes the neurotransmitter acetylcholine at extremely high catalytic rates (1Rosenberry T.L. Meister A. Advances in Enzymology. John Wiley 14: 1989-1997Crossref PubMed Scopus (335) Google Scholar) and x-ray crystallography (3Sussman J.L. Harel M. Frolow F. Oefner C. Goldman A. Toker L. Silman I. Science. 1991; 253: 872-879Crossref PubMed Scopus (2426) Google Scholar) have revealed a narrow active site gorge some 20 Å deep with two separate ligand binding sites. During catalytic hydrolysis, the substrate acyl group is transferred briefly to residue Ser-200 2Throughout this paper we number residues according to the Torpedo AChE sequence. For example, Trp-84 and Ser-200 in this sequence correspond to Trp-86 and Ser-203, respectively, in mammalian AChE. in the acylation site at the bottom of the gorge. This site contains residues involved in a catalytic triad (His-440, Glu-327, Ser-200) and Trp-84, which binds to the trimethylammonium group of acetylcholine. The peripheral site near the mouth of the gorge includes, among others, residues Asp-72 and Trp-279. Recent investigations have shown that the peripheral site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site (4Szegletes T. Mallender W.D. Thomas P.J. Rosenberry T.L. Biochemistry. 1999; 38: 122-133Crossref PubMed Scopus (152) Google Scholar, 5Tara S. Elcock A.H. Kirchhoff P.D. Briggs J.M. Radic Z. Taylor P. McCammon J.A. Biopolymers. 1998; 46: 465-474Crossref PubMed Google Scholar, 6Mallender W.D. Szegletes T. Rosenberry T.L. Biochemistry. 2000; 39: 7753-7763Crossref PubMed Scopus (125) Google Scholar). The question of whether the peripheral site plays additional roles in the catalytic process has long been of interest. Changeux (7Changeux J.-P. Mol. Pharmacol. 1966; 2: 369-392PubMed Google Scholar) was among the first to appreciate that AChE contained two distinct ligand binding sites and that there may be allosteric interactions between ligands bound at these sites involving conformational changes in the protein molecule. Ligands bound to the peripheral site can inhibit or accelerate reactions at the acylation site, and these effects have often been attributed to conformational interactions between the sites (8Hucho F. Jarv J. Weise C. Trends Pharmacol. Sci. 1991; 12: 422-426Abstract Full Text PDF PubMed Scopus (36) Google Scholar, 9Barak D. Ordentlich A. Bromberg A. Kronman C. Marcus D. Lazar A. Ariel N. Velan B. Shafferman A. Biochemistry. 1995; 34: 15444-15452Crossref PubMed Scopus (82) Google Scholar). However, direct evidence for such conformational interactions has been difficult to obtain. For example, three-dimensional structures of AChE complexes with two ligands specific for the acylation site, edrophonium and the cationic trifluoromethylketone TMTFA, showed no changes in the structure of the peripheral site (10Harel M. Schalk I. Ehret-Sabatier L. Bouet F. Goeldner M. Hirth C. Axelsen P.H. Silman I. Sussman J.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9031-9035Crossref PubMed Scopus (845) Google Scholar, 11Harel M. Quinn D.M. Nair H.K. Silman I. Sussman J.L. J. Am. Chem. Soc. 1996; 118: 2340-2346Crossref Scopus (342) Google Scholar). In this report we show that thioflavin T (Fig. 1) binds specifically to the AChE peripheral site and is one of the most useful fluorescent probes of AChE yet discovered. This fluorophore frequently is used to detect amyloid structure in proteins (12LeVine III, H. Wetzel R. Methods in Enzymology. Academic Press, Orlando, FL1999: 274-284Google Scholar), but the three-dimensional structure of the AChE peripheral site shows no indication of the extensive β sheet structure characteristic of amyloid. The intense fluorescence of thioflavin T bound to AChE is partially quenched by the binding of acylation site ligands in ternary complexes, and this quenching appears to result from a conformational interaction between the two sites. Recombinant human AChE (13Mallender W.D. Szegletes T. Rosenberry T.L. J. Biol. Chem. 1999; 274: 8491-8499Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) was purified as outlined previously, and active site AChE concentrations were determined by assuming 450 units/nmol (6Mallender W.D. Szegletes T. Rosenberry T.L. Biochemistry. 2000; 39: 7753-7763Crossref PubMed Scopus (125) Google Scholar). 3One unit of AChE activity corresponds to 1 umol of acetylthiocholine hydrolyzed/min under standard pH-stat assay conditions, and these conditions correspond to maximal AChE activity at pH 8 (31Rosenberry T.L. Scoggin D.M. J. Biol. Chem. 1984; 259: 5643-5652Abstract Full Text PDF PubMed Google Scholar). Our conventional spectrophotometric assay at 412 nm is conducted in pH 7 buffer with 0.5 mm acetylthiocholine, conditions that result in 4.8 ΔA412 nm/min with 1 nm AChE (or about 76% of the maximal activity). Thioflavin T chloride (Sigma) concentrations were assigned as 70% of the dry weight. Thioflavin T chloride recrystallized from water gave an extinction coefficient ε412 nm of 36,000m−1 cm−1. Concentrations of propidium iodide (Calbiochem) were determined with an extinction coefficient ε493 nm of 5900m−1 cm−1(2Taylor P. Lappi S. Biochemistry. 1975; 14: 1989-1997Crossref PubMed Scopus (335) Google Scholar), and concentrations of edrophonium chloride (ethyl(3-hydroxyphenyl)dimethylammonium chloride; Sigma) were determined with ε271 nm of 3400m−1 cm−1from the dry weight (14Sharp T.R. Rosenberry T.L. J. Biochem. Biophys. Methods. 1982; 6: 159-172Crossref PubMed Scopus (13) Google Scholar). TMTFA (kindly provided by Dr. Daniel Quinn, University of Iowa) concentrations were calibrated by titration with AChE. Substrate hydrolysis rates ν were measured in buffer (20 mm sodium phosphate and 0.02% Triton X-100 at pH 7.0) at 25 °C of of thioflavin T, acetylthiocholine, and a of in a of 1.0 assay Biochem. Pharmacol. PubMed Scopus Google Scholar) was used to of the of at 412 nm nm B. Biochem. PubMed Scopus Google for on a and substrate concentrations were for substrate from hydrolysis this was that acetylthiocholine concentrations were at to substrate in the of (4Szegletes T. Mallender W.D. Thomas P.J. Rosenberry T.L. Biochemistry. 1999; 38: 122-133Crossref PubMed Scopus (152) Google Scholar). In this a for by peripheral site ligands is in In this can to of the and For example, a ternary with substrate at the acylation site and at the peripheral site by the The acylation is by a a in this ternary of at concentrations were a result with (4Szegletes T. Mallender W.D. Thomas P.J. Rosenberry T.L. Biochemistry. 1999; 38: 122-133Crossref PubMed Scopus (152) Google Scholar, T. Mallender W.D. Rosenberry T.L. Biochemistry. 1998; PubMed Google Scholar), and of these were by that that ν has a constant of these were to 1 by with by the of their T. Mallender W.D. Rosenberry T.L. Biochemistry. 1998; PubMed Google Scholar). 1 is the equilibrium dissociation constant for with and the is the of the constant with to that in the of T. Mallender W.D. Rosenberry T.L. Biochemistry. 1998; PubMed Google Scholar). thioflavin T can form a ternary with a and the of in the of to the of when is is by P. B. Mallender W.D. R. J. D. Rosenberry T.L. Mol. Pharmacol. 2000; Google Scholar). In this is the equilibrium dissociation constant for with is the equilibrium dissociation constant for with and is the equilibrium dissociation constant for with the The concentrations and to the for substrate was from the ν at acetylthiocholine, a the of for acetylthiocholine T. Mallender W.D. Rosenberry T.L. Biochemistry. 1998; PubMed Google Scholar), to the that substrate hydrolysis was The fluorescence of thioflavin T or propidium is when these ligands to the AChE peripheral site. was on a in 20 mm sodium phosphate buffer 7.0) and 0.02% Triton X-100 at 1 Thioflavin T fluorescence was measured with at 450 nm and from to nm with and of fluorescence was with at nm and from to nm with of nm (2Taylor P. Lappi S. Biochemistry. 1975; 14: 1989-1997Crossref PubMed Scopus (335) Google Scholar, P. B. Mallender W.D. R. J. D. Rosenberry T.L. Mol. Pharmacol. 2000; Google Scholar). under the fluorescence were and fluorescence contributions from in the buffer and were In some of were by to P. B. Mallender W.D. R. J. D. Rosenberry T.L. Mol. Pharmacol. 2000; Google Scholar). In is the fluorescence coefficient for is the fluorescence coefficient for bound is the is the ligand is the equilibrium dissociation and were by to with as the and or with the as the and to and in some In these of were by assuming constant In other was to interactions in ternary complexes involving thioflavin T and a ligand in fluorescence In this the of thioflavin T at the peripheral site by of AChE is by the dissociation constant in the of an acylation site ligand and when the acylation site. was the binding of to was to equilibrium with dissociation of to was when was TMTFA and with and dissociation and The Inc., was to the to T. Mallender W.D. Rosenberry T.L. Biochemistry. 1998; PubMed Google Scholar). This with and of at concentrations The and as as were the as the the fluorescence to the edrophonium concentrations to the equilibrium the fluorescence coefficient for the ternary TMTFA, was and the the of fluorescence at nm to the to two additional and A conventional of thioflavin T of AChE is shown in concentrations of thioflavin T the of the in and a of these thioflavin T shows a (Fig. with a of in In the equilibrium of is than 1 when However, we have shown that a a more and in this is than 1 when T. Mallender W.D. Rosenberry T.L. Biochemistry. 1998; PubMed Google Scholar). The substrate when the bound peripheral site ligand the of substrate to the acylation site, an we have The of of for thioflavin T is to for the peripheral site propidium and T. Mallender W.D. Rosenberry T.L. Biochemistry. 1998; PubMed Google Scholar), that with bound thioflavin is than In to a by thioflavin T, the data in a constant for thioflavin T of μm. that thioflavin T binds to the peripheral site, we measured the of two ligands to inhibit thioflavin T binding in a substrate hydrolysis assay P. B. Mallender W.D. R. J. D. Rosenberry T.L. Mol. Pharmacol. 2000; Google Scholar). is specific for the AChE peripheral site (2Taylor P. Lappi S. Biochemistry. 1975; 14: 1989-1997Crossref PubMed Scopus (335) Google Scholar), edrophonium is specific for the AChE acylation site (10Harel M. Schalk I. Ehret-Sabatier L. Bouet F. Goeldner M. Hirth C. Axelsen P.H. Silman I. Sussman J.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9031-9035Crossref PubMed Scopus (845) Google Scholar), and substrate hydrolysis in the of thioflavin T, as shown by the in of these ligands was with thioflavin T such that no ternary the in ν correspond to the in This is the with propidium in A. The of thioflavin T in the ternary by a of to in the a change that was from at the ligand concentrations In the of thioflavin T was no in the ternary with edrophonium and in the and the with was on the for edrophonium in B. data that thioflavin T binds to the AChE peripheral site in a that is with The fluorescence of thioflavin T was when it bound to AChE. at nm and an at nm to of thioflavin T bound to amyloid at at III, H. Wetzel R. Methods in Enzymology. Academic Press, Orlando, FL1999: 274-284Google Scholar). appreciate the fluorescence enhancement of thioflavin T when bound to the peripheral site of we it to the fluorescence enhancement with propidium bound to AChE. to this propidium has been the fluorophore with the fluorescence enhancement with and it is widely used as a reporter of ligand at the AChE peripheral site (2Taylor P. Lappi S. Biochemistry. 1975; 14: 1989-1997Crossref PubMed Scopus (335) Google Scholar, 9Barak D. Ordentlich A. Bromberg A. Kronman C. Marcus D. Lazar A. Ariel N. Velan B. Shafferman A. Biochemistry. 1995; 34: 15444-15452Crossref PubMed Scopus (82) Google Scholar, P. B. Mallender W.D. R. J. D. Rosenberry T.L. Mol. Pharmacol. 2000; Google Scholar). However, when AChE was with concentrations of the two the fluorescence for thioflavin T bound to AChE was than that for propidium was about 7 with a of for propidium AChE P. J. Lappi S. J. Mol. Pharmacol. thioflavin T be a more and fluorescent reporter of ligand interactions with AChE than propidium has The titration in gave a for thioflavin T binding of in with the in this titration and with a of AChE a in the T fluorescent were conducted with a of thioflavin T and concentrations of AChE and with P. B. Mallender W.D. R. J. D. Rosenberry T.L. Mol. Pharmacol. 2000; Google Scholar) a of was with thioflavin T in this titration the for thioflavin T increased by at a of to a ternary of thioflavin T and edrophonium can form with AChE a in of ligand for AChE site. whether the binding of an acylation site ligand the fluorescence of bound thioflavin T. first the of edrophonium concentrations and a in the fluorescence as edrophonium the acylation site of the data to gave for thioflavin T for edrophonium that were in with from and no change in the of these ligands for AChE in the ternary in from However, the binding of edrophonium the fluorescence of thioflavin T in the ternary by a of The observation of this partial quenching of thioflavin T fluorescence is a major advance in the study of AChE for two reasons. First, it allows thioflavin T to be used as a reporter for ligand reactions at the acylation site. Second, it indicates that ligand binding to the acylation site the or the of thioflavin T bound to the peripheral site, a that we to the first we TMTFA, which a with Ser-200 in the acylation site that is an of the by acetylcholine M. Quinn D.M. Nair H.K. Silman I. Sussman J.L. J. Am. Chem. Soc. 1996; 118: 2340-2346Crossref Scopus (342) Google Scholar, U. R. M. Biophys. PubMed Scopus Google Scholar, H.K. Quinn D.M. J. Am. Chem. Soc. 1993; Scopus Google Scholar). The high of TMTFA in this allows the of binding to be over a of TMTFA for of TMTFA and thioflavin T concentrations were and reactions shown in The were to with the this of rates the TMTFA concentrations were high to for was at as determined T. Mallender W.D. Rosenberry T.L. Biochemistry. 1998; PubMed Google Scholar), and were A of was First, a of 1.0 for thioflavin T and an enhancement in fluorescence of bound thioflavin T of were with data in and Second, the of thioflavin T and TMTFA in the ternary with AChE were at as high as the in their complexes in with the for edrophonium in of TMTFA binding by bound thioflavin T was the of the was than the in constant for TMTFA when propidium was bound to the peripheral site T. Mallender W.D. Rosenberry T.L. Biochemistry. 1998; PubMed Google Scholar). the binding of TMTFA the fluorescence of thioflavin T in the ternary by a of The of this quenching appears to be than that with edrophonium in that the quenching of bound thioflavin T in ternary complexes with AChE may on the structure of the acylation site A quenching than that with edrophonium or TMTFA was when Ser-200 was with a group that the acylation site ligand be cationic to the fluorescence of bound thioflavin T. The that the fluorescence of the T is partially quenched when a ligand binds to the acylation site is of and In this fluorescence change can be to report on reactions at the acylation site. this with TMTFA in but the reactions of to be measured over as as the partial quenching on the binding of acylation site ligands is the evidence to of conformational interaction between the peripheral and acylation sites of AChE. such conformational interaction has been widely to for a of indicates that evidence for such interactions is A is that ligand binding to the peripheral site the conformation of the acylation site to the efficiency of acylation or by This has been to for of substrate hydrolysis by peripheral site ligands D. Ordentlich A. Bromberg A. Kronman C. Marcus D. Lazar A. Ariel N. Velan B. Shafferman A. Biochemistry. 1995; 34: 15444-15452Crossref PubMed Scopus (82) Google Scholar) when the substrate can to the peripheral site, for substrate at high substrate concentrations Z. Taylor P. Mol. Pharmacol. 1991; 39: Google Scholar). by Taylor and Radic P. Radic Z. Pharmacol. 34: PubMed Scopus Google Scholar), ligand with the peripheral site may of a cationic substrate to the acylation site by substrate or by between ligand and an allosteric interaction can be contributions from these other factors be the we have that bound peripheral site ligands the of substrates as as of other ligands the acylation site. propidium the and dissociation for the acylation site ligands A and TMTFA by factors of have this T. Mallender W.D. Rosenberry T.L. Biochemistry. 1998; PubMed Google Scholar) and shown that in the for substrates and the dissociation for the of substrate hydrolysis to for the of substrate hydrolysis by peripheral site ligands (4Szegletes T. Mallender W.D. Thomas P.J. Rosenberry T.L. Biochemistry. 1999; 38: 122-133Crossref PubMed Scopus (152) Google Scholar, T. Mallender W.D. Rosenberry T.L. Biochemistry. 1998; PubMed Google Scholar) as as for substrate (4Szegletes T. Mallender W.D. Thomas P.J. Rosenberry T.L. Biochemistry. 1999; 38: 122-133Crossref PubMed Scopus (152) Google Scholar). in is a and it can have no on substrate or hydrolysis by substrates that with AChE However, to in for AChE were with a cationic and a an additional of interaction (6Mallender W.D. Szegletes T. Rosenberry T.L. Biochemistry. 2000; 39: 7753-7763Crossref PubMed Scopus (125) Google Scholar, W.D. Szegletes T. Rosenberry T.L. J. Biol. Chem. 1999; 274: 8491-8499Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). that an with a group with propidium ligands were in their sites in AChE (13Mallender W.D. Szegletes T. Rosenberry T.L. J. Biol. Chem. 1999; 274: 8491-8499Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), and we have attributed these in to that ligands in the ternary (6Mallender W.D. Szegletes T. Rosenberry T.L. Biochemistry. 2000; 39: 7753-7763Crossref PubMed Scopus (125) Google Scholar, W.D. Szegletes T. Rosenberry T.L. J. Biol. Chem. 1999; 274: 8491-8499Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). that such is a in ternary complexes involving and A or yet ligand in the ternary complexes to than in the complexes T. Mallender W.D. Rosenberry T.L. Biochemistry. 1998; PubMed Google Scholar). have that the from interactions between these cationic ligands T. Mallender W.D. Rosenberry T.L. Biochemistry. 1998; PubMed Google Scholar). In data that the binding of a peripheral site ligand to AChE can result in interaction that substrate hydrolysis or ligand at the acylation site. peripheral site ligands propidium and we no evidence of an additional allosteric conformational that contributes to of conformational interaction between the peripheral and acylation sites of AChE have from two other First, the of a group to Ser-200 in the acylation site by the binding of to the peripheral site Taylor P. Biochemistry. PubMed Scopus Google Scholar). The attributed these to an in of the to of in the of the by the binding of the peripheral site Second, bound peripheral site ligands can accelerate the of with Ser-200 in the acylation site. the acylation of AChE by S. Mol. Pharmacol. 6: Google Scholar), and peripheral site propidium and increased the first for with AChE (6Mallender W.D. Szegletes T. Rosenberry T.L. Biochemistry. 2000; 39: 7753-7763Crossref PubMed Scopus (125) Google Scholar, W.D. Szegletes T. Rosenberry T.L. J. Biol. Chem. 1999; 274: 8491-8499Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, Z. Taylor P. Chem. Biol. 1999; PubMed Scopus Google Scholar). This constant the of the group to Ser-200 in the or ternary complexes to respectively, in these effects can be attributed to a conformational change in the acylation site by ligand binding to the peripheral site, other be among is as of the with first when propidium is bound was shown by to with AChE in a way that partially with the propidium binding site (13Mallender W.D. Szegletes T. Rosenberry T.L. J. Biol. Chem. 1999; 274: 8491-8499Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). have that of the to this in the ternary in the to the group and the constant (6Mallender W.D. Szegletes T. Rosenberry T.L. Biochemistry. 2000; 39: 7753-7763Crossref PubMed Scopus (125) Google Scholar). In that the group to Ser-200 is to partially with the peripheral site. the of and propidium were for this AChE and for a interaction between the ligands is for there to be changes to the ligand remains a when conformational interactions between the peripheral and acylation sites However, of peripheral site ligands and with showed the Z. Taylor P. Chem. Biol. 1999; PubMed Scopus Google Scholar). is to the in of these it appears to a conformational change that on ligand binding to the peripheral site. Our data the first to that ligand binding to the acylation site can alter the conformation of the peripheral site. The partial quenching of the fluorescence of bound thioflavin T when a ligand binds to the acylation site result from fluorescence there is no between thioflavin T and of the acylation site ligands edrophonium or However, the fluorescence of thioflavin T has been shown to on P. A. Biochemistry. 1998; PubMed Scopus Google Scholar), with a for a of fluorescent Biochemistry. Scopus Google J. Chem. 1982; Scopus Google Scholar). show increased fluorescence when high to a to the AChE peripheral site the of thioflavin T and for is the binding of an acylation site ligand partially this fluorescence in the ternary but the quenching to an in the of bound thioflavin T by a change in the local AChE conformation that is in the acylation site. The of thioflavin T when bound to the AChE peripheral site remains to be but it is that to the acylation site ligand for the structures of bound edrophonium and bound TMTFA show that ligands near the base of the acylation site, from the that the peripheral site (10Harel M. Schalk I. Ehret-Sabatier L. Bouet F. Goeldner M. Hirth C. Axelsen P.H. Silman I. Sussman J.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9031-9035Crossref PubMed Scopus (845) Google M. Quinn D.M. Nair H.K. Silman I. Sussman J.L. J. Am. Chem. Soc. 1996; 118: 2340-2346Crossref Scopus (342) Google Scholar). Furthermore, these ligands show no in in their ternary complexes with no one to support this conformational interaction with additional but the conformational changes involved to be structures of AChE and the show a of Å between and no changes in the structure of the peripheral site M. Quinn D.M. Nair H.K. Silman I. Sussman J.L. J. Am. Chem. Soc. 1996; 118: 2340-2346Crossref Scopus (342) Google Scholar). of AChE with a at Ser-200 gave no change in when propidium was bound to the peripheral site, a change was with or the of which to a the peripheral site M. S. J. P. Radic Z. Z. Biophys. 1999; PubMed Scopus Google Scholar). we that the quenching of the fluorescence of thioflavin T by acylation site ligands is the to that is to reveal a conformational interaction between the two sites. This interaction may provide a for bound peripheral site ligands to accelerate acylation site to for of and of human AChE and to Dr. and for with TMTFA