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J. Biol. Chem., Vol. 276, Issue 26, 23282-23287, June 29, 2001
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From the § Department of Pharmacology and Program in
Neurosciences, Mayo Foundation for Medical Education and Research, Mayo
Clinic Jacksonville, Jacksonville, Florida 32224 and the
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.
Thioflavin T Is a Fluorescent Probe of the Acetylcholinesterase
Peripheral Site That Reveals Conformational Interactions between the
Peripheral and Acylation Sites*
,, and
Centro de Regulación Celular y Patología,
Departamento de Biología Celular y Molecular, Facultad de
Ciencias Biológicas, Pontificia Universidad Católica de
Chile, Alameda 340, 114-D Santiago, Chile
*
This work was supported by Grant NS-16577 from the National
Institutes of Health, Grant DAMD 17-98-2-8019 from the United States
Army Medical Research Acquisition Activity, grants from the Muscular
Dystrophy Association of America (to T. L. R.), Grant FONDAP N
13980001 and a Presidential Chair in Science from the Chilean
Government (to N. C. I.), and Grant FONDECYT N 4000030 (to
G. V. D.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
904-953-7375; Fax: 904-953-7370; E-mail: rosenberry@mayo.edu.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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