Analysis of the reaction of carbachol with acetylcholinesterase using thioflavin T as a coupled fluorescence reporter

Terrone L. Rosenberry, Leilani K. Sonoda, Sarah E. Dekat, Bernadette Cusack, Joseph L. Johnson

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acylated enzyme intermediate is produced. Carbamates are very poor substrates that, like other AChE substrates, form an initial enzyme-substrate complex with free AChE (E) and proceed to an acylated enzyme intermediate (EC), which is then hydrolyzed. However, the hydrolysis of EC is slow enough to resolve the acylation and deacylation steps on the catalytic pathway. Here, we focus on the reaction of carbachol (carbamoylcholine) with AChE. The kinetics and thermodynamics of this reaction are of special interest because carbachol is an isosteric analogue of the physiological substrate acetylcholine. We show that the reaction can be monitored with thioflavin T as a fluorescent reporter group. The fluorescence of thioflavin T is strongly enhanced when it binds to the P-site of AChE, and this fluorescence is partially quenched when a second ligand binds to the A-site to form a ternary complex. Analysis of the fluorescence reaction profiles was challenging because four thermodynamic parameters and two fluorescence coefficients were fitted from the combined data both for E and for EC. Respective equilibrium dissociation constants of 6 and 26 mM were obtained for carbachol binding to the A- and P-sites in E and of 2 and 32 mM for carbachol binding to the A- and P-sites in EC. These constants for the binding of carbachol to the P-site are about an order of magnitude larger (i.e., indicating lower affinity) than previous estimates for the binding of acetylthiocholine to the P-site.

Original languageEnglish (US)
Pages (from-to)13056-13063
Number of pages8
JournalBiochemistry
Volume47
Issue number49
DOIs
StatePublished - Dec 9 2008

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Carbachol
Acetylcholinesterase
Acylation
Fluorescence
Substrates
Thermodynamics
Enzymes
Acetylthiocholine
Ligands
Carbamates
Acetylcholine
Hydrolysis
Catalytic Domain
Binding Sites
thioflavin T
Kinetics

ASJC Scopus subject areas

  • Biochemistry

Cite this

Rosenberry, T. L., Sonoda, L. K., Dekat, S. E., Cusack, B., & Johnson, J. L. (2008). Analysis of the reaction of carbachol with acetylcholinesterase using thioflavin T as a coupled fluorescence reporter. Biochemistry, 47(49), 13056-13063. https://doi.org/10.1021/bi8015197

Analysis of the reaction of carbachol with acetylcholinesterase using thioflavin T as a coupled fluorescence reporter. / Rosenberry, Terrone L.; Sonoda, Leilani K.; Dekat, Sarah E.; Cusack, Bernadette; Johnson, Joseph L.

In: Biochemistry, Vol. 47, No. 49, 09.12.2008, p. 13056-13063.

Research output: Contribution to journalArticle

Rosenberry, TL, Sonoda, LK, Dekat, SE, Cusack, B & Johnson, JL 2008, 'Analysis of the reaction of carbachol with acetylcholinesterase using thioflavin T as a coupled fluorescence reporter', Biochemistry, vol. 47, no. 49, pp. 13056-13063. https://doi.org/10.1021/bi8015197
Rosenberry, Terrone L. ; Sonoda, Leilani K. ; Dekat, Sarah E. ; Cusack, Bernadette ; Johnson, Joseph L. / Analysis of the reaction of carbachol with acetylcholinesterase using thioflavin T as a coupled fluorescence reporter. In: Biochemistry. 2008 ; Vol. 47, No. 49. pp. 13056-13063.
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