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

Abstract

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 M. 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,Ntrimethylammonio)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.

Monitoring the reaction of carbachol with acetylcholinesterase by thioflavin T fluorescence and acetylthiocholine hydrolysis

“…The dependence on i and i 2 was reduced when the multiple data sets were analyzed simultaneously in the SCoP program. We previously showed that the equilibrium constants corresponding to K M and i 2 for the binding of the specific Asite ligand edrophonium in its binary complex with E and its ternary complex with E and thioflavin T could be assigned unambiguously from a similar combination of fluorescence data sets (7). However, edrophonium does not show significant binding to the P-site (equivalent to eliminating ESS P in Scheme 3 and to setting iK S −1 = 0 and eliminating i as a variable in eq.…”

“…The difference in thioflavin T fluorescence intensity between these binary and ternary complexes allows the binding of both A and P-site ligands to be quantified (7,12). To apply this approach to the reaction of carbachol with recombinant human AChE, we first compared recordings obtained with acetylthiocholine or thioflavin T. Because carbamates are poor substrates that form a slowly hydrolyzed EC intermediate (Scheme 1-Scheme 3), their reaction progress is usually monitored by changes in AChE activity with a reporter substrate like acetylthiocholine at discrete time points.…”

“…We show that the reaction of carbachol with AChE can be monitored both with acetylthiocholine as a reporter substrate and with thioflavin T (Figure 1) as a fluorescent reporter group. The fluorescence of thioflavin T is strongly enhanced when it binds to the Psite of AChE, and this fluorescence is partially quenched when a second ligand binds to the Asite to form a ternary complex (7). In addition to monitoring the course of the carbamoylation reaction, we show that these fluorescence changes allow the determination of carbachol affinities for the A-and P-sites.…”

“…When thioflavin T binds to the AChE P-site its fluorescence is enhanced nearly 1000-fold, and the enhanced fluorescence is partially quenched when a second ligand binds to the A-site in a ternary complex (7). In experiments with thioflavin T and AChE alone, the fluorescence (F) was recorded over 3 -4 min intervals in the Hi-Tech SFA 20 stopped flow apparatus described above for improved F stability.…”

“…At the base of the gorge is the acylation or A-site where residue W86 2 binds the trimethylammonium group of acetylcholine and H447, E334, and S203 participate in a triad that catalyzes the transient acylation and deacylation of S203 during each substrate turnover. The peripheral or P-site, spanned by residues W286 near the mouth of the gorge and D74 near a constriction at the boundary between the P-site and the A-site, specifically binds certain ligands like the neurotoxin fasciculin (9,10)] and the fluorescent probes propidium (6) and thioflavin T (7).…”