Ambenonium is a rapidly reversible noncovalent inhibitor of acetylcholinesterase, with one of the highest known affinities

A. S. Hodge, D. R. Humphrey, T. L. Rosenberry

Research output: Contribution to journalArticle

52 Citations (Scopus)

Abstract

Steady state patterns of inhibition of purified human erythrocyte acetylcholinesterase by three inhibitors were analyzed. Edrophonium acted essentially as a competitive inhibitor, whereas tacrine and ambenonium gave mixed competitive and uncompetitive inhibition with acetylthiocholine as substrate. Inhibition constants for the competitive components were 470 μM for edrophonium, 65 μM for tacrine, and 0.12 nM for ambenonium. The extremely high affinity of ambenonium permitted analysis of the rates of approach to steady state inhibition. These rates were characterized by a single exponential time course with rate constants, k(exp), that showed a linear dependence when plotted against ambenonium concentration, at fixed substrate concentration. The intercepts of these plots were independent of the substrate concentration and indicated an ambenonium dissociation rate constant of 0.013 ± 0.002 sec-1. The slope of the plot at the lowest substrate concentration approximated the ambenonium bimolecular or association rate constant and gave a value of 5.2 ± 0.6 x 107 M-1 sec-1. Three models were examined to account for the nearly linear dependence of the slopes of these plots on the substrate concentration. These models indicated that ambenonium and acetylthiocholine competed for a peripheral anionic site in the acetyl-enzyme intermediate formed during substrate hydrolysis. The apparent equilibrium dissociation constant of acetylthiocholine for this peripheral site (1.2-1.4 mM) was significantly different from that calculated from substrate inhibition data (20.1 ± 2.8 mM). We propose that acetylthiocholine can interact with the acetyl-enzyme both at the peripheral site and at the active site but that only the latter interaction inhibits substrate hydrolysis.

Original languageEnglish (US)
Pages (from-to)937-942
Number of pages6
JournalMolecular Pharmacology
Volume41
Issue number5
StatePublished - 1992
Externally publishedYes

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Acetylthiocholine
Cholinesterase Inhibitors
Edrophonium
Tacrine
Hydrolysis
Enzymes
Catalytic Domain
Erythrocytes

ASJC Scopus subject areas

  • Pharmacology

Cite this

Ambenonium is a rapidly reversible noncovalent inhibitor of acetylcholinesterase, with one of the highest known affinities. / Hodge, A. S.; Humphrey, D. R.; Rosenberry, T. L.

In: Molecular Pharmacology, Vol. 41, No. 5, 1992, p. 937-942.

Research output: Contribution to journalArticle

Hodge, A. S. ; Humphrey, D. R. ; Rosenberry, T. L. / Ambenonium is a rapidly reversible noncovalent inhibitor of acetylcholinesterase, with one of the highest known affinities. In: Molecular Pharmacology. 1992 ; Vol. 41, No. 5. pp. 937-942.
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abstract = "Steady state patterns of inhibition of purified human erythrocyte acetylcholinesterase by three inhibitors were analyzed. Edrophonium acted essentially as a competitive inhibitor, whereas tacrine and ambenonium gave mixed competitive and uncompetitive inhibition with acetylthiocholine as substrate. Inhibition constants for the competitive components were 470 μM for edrophonium, 65 μM for tacrine, and 0.12 nM for ambenonium. The extremely high affinity of ambenonium permitted analysis of the rates of approach to steady state inhibition. These rates were characterized by a single exponential time course with rate constants, k(exp), that showed a linear dependence when plotted against ambenonium concentration, at fixed substrate concentration. The intercepts of these plots were independent of the substrate concentration and indicated an ambenonium dissociation rate constant of 0.013 ± 0.002 sec-1. The slope of the plot at the lowest substrate concentration approximated the ambenonium bimolecular or association rate constant and gave a value of 5.2 ± 0.6 x 107 M-1 sec-1. Three models were examined to account for the nearly linear dependence of the slopes of these plots on the substrate concentration. These models indicated that ambenonium and acetylthiocholine competed for a peripheral anionic site in the acetyl-enzyme intermediate formed during substrate hydrolysis. The apparent equilibrium dissociation constant of acetylthiocholine for this peripheral site (1.2-1.4 mM) was significantly different from that calculated from substrate inhibition data (20.1 ± 2.8 mM). We propose that acetylthiocholine can interact with the acetyl-enzyme both at the peripheral site and at the active site but that only the latter interaction inhibits substrate hydrolysis.",
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