Unmasking tandem site interaction in human acetylcholinesterase. Substrate activation with a cationic acetanilide substrate

Joseph L. Johnson, Bernadette Cusack, Matthew P. Davies, Abdul Fauq, Terrone L. Rosenberry

Research output: Contribution to journalArticle

60 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 acyl enzyme intermediate is produced. A conformational interaction between the A- and P-sites has recently been found to modulate ligand affinities. We now demonstrate that this interaction is of functional importance by showing that the acetylation rate constant of a substrate bound to the A-site is increased by a factor a when a second molecule of substrate binds to the P-site. This demonstration became feasible through the introduction of a new acetanilide substrate analogue of acetylcholine, 3-(acetamido)-N,N,N-trimethylanilinium (ATMA), for which a = 4. This substrate has a low acetylation rate constant and equilibrates with the catalytic site, allowing a tractable algebraic solution to the rate equation for substrate hydrolysis. ATMA affinities for the A- and P-sites deduced from the kinetic analysis were confirmed by fluorescence titration with thioflavin T as a reporter ligand. Values of a > 1 give rise to a hydrolysis profile called substrate activation, and the AChE site-specific mutant W86F, and to a lesser extent wild-type human AChE itself, showed substrate activation with acetylthiocholine as the substrate. Substrate activation was incorporated into a previous catalytic scheme for AChE in which a bound P-site ligand can also block product dissociation from the A-site, and two additional features of the AChE catalytic pathway were revealed. First, the ability of a bound P-site ligand to increase the substrate acetylation rate constant varied with the structure of the ligand: thioflavin T accelerated ATMA acetylation by a factor a2 of 1.3, while propidium failed to accelerate. Second, catalytic rate constants in the initial intermediate formed during acylation (EAP, where EA is the acyl enzyme and P is the alcohol leaving group cleaved from the ester substrate) may be constrained such that the leaving group P must dissociate before hydrolytic deacylation can occur.

Original languageEnglish (US)
Pages (from-to)5438-5452
Number of pages15
JournalBiochemistry
Volume42
Issue number18
DOIs
StatePublished - May 13 2003

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Acetylcholinesterase
Chemical activation
Acetylation
Ligands
Acylation
Substrates
Rate constants
Catalytic Domain
Hydrolysis
Acetylthiocholine
Propidium
Enzymes
Acetylcholine
acetanilide
Esters
Fluorescence
Binding Sites
Alcohols
Titration
Demonstrations

ASJC Scopus subject areas

  • Biochemistry

Cite this

Unmasking tandem site interaction in human acetylcholinesterase. Substrate activation with a cationic acetanilide substrate. / Johnson, Joseph L.; Cusack, Bernadette; Davies, Matthew P.; Fauq, Abdul; Rosenberry, Terrone L.

In: Biochemistry, Vol. 42, No. 18, 13.05.2003, p. 5438-5452.

Research output: Contribution to journalArticle

Johnson, Joseph L. ; Cusack, Bernadette ; Davies, Matthew P. ; Fauq, Abdul ; Rosenberry, Terrone L. / Unmasking tandem site interaction in human acetylcholinesterase. Substrate activation with a cationic acetanilide substrate. In: Biochemistry. 2003 ; Vol. 42, No. 18. pp. 5438-5452.
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