Binding of the neurotoxin fasciculin 2 to the acetylcholinesterase peripheral site drastically reduces the association and dissociation rate constants for N-methylacridinium binding to the active site

Terrone L. Rosenberry, Carl Roland Rabl, Eberhard Neumann

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

The acetylcholinesterase (ACHE) active site consists of a gorge 2 nm deep that is lined with aromatic residues. A serine residue near the base of the gorge defines an acylation site where an acyl enzyme intermediate is formed during the hydrolysis of ester substrates. Residues near the entrance to the gorge comprise a peripheral site where inhibitors like propidium and fasciculin 2, a snake neurotoxin, bind and interfere with catalysis. Like certain other cationic ligands that bind specifically to the acylation site, N-methylacridinium can still interact with the acylation site in the AChE- fasciculin 2 complex. At 310 K (37 °C), the equilibrium dissociation constant K(L)' for N-methylacridinium binding to the complex was 4.0 ± 0.7 μM, less than an order of magnitude larger than the K(L) = 1.0 ± 0.3 μM for N-methylacridinium interaction with human AChE in the absence of fasciculin 2. To assess whether fasciculin 2 can sterically block access of a ligand to the acylation site, thermodynamic and kinetic constants for the interaction of N-methylacridinium with AChE in the presence and absence of fasciculin 2 were measured by fluorescence temperature jump relaxation kinetics. During progressive titration of the enzyme with increasing concentrations of N-methylacridinium, a prominent relaxation in the 0.1-1 ms range was observed in the absence of fasciculin 2. When excess fasciculin 2 was added, the prominent relaxation shifted to the 0.3-1 s range. Estimates of total AChE concentrations, K(L), or K(L)' from analyses of relaxation amplitudes agreed well with those from equilibrium fluorescence, confirming that the relaxations corresponded to the bimolecular reactions of interest. Further analysis of the relaxation times in the absence of fasciculin 2 gave estimates of the N-methylacridinium association rate constant k(12) = 8 x 108 M-1 s-1 and dissociation rate constant k(21) = 750 s-1 at 310 K (37 °C). For the AChE-fasciculin 2 complex, the corresponding constants were k(12)' = 1.0 x 10s M-1 s-1 and k(21)' = 0.4 s-1. Thus the rate constants decreased by more than 3 orders of magnitude when fasciculin 2 was bound, consistent with a pronounced steric blockade of N-methylacridinium ingress to and egress from the acylation site.

Original languageEnglish (US)
Pages (from-to)685-690
Number of pages6
JournalBiochemistry
Volume35
Issue number3
DOIs
StatePublished - Jan 23 1996
Externally publishedYes

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Neurotoxins
Acetylcholinesterase
Rate constants
Catalytic Domain
Association reactions
Acylation
Fluorescence
fasciculin
N-methylacridine
Ligands
Kinetics
Snakes
Propidium
Enzymes
Catalysis
Titration
Thermodynamics
Relaxation time
Serine
Hydrolysis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Binding of the neurotoxin fasciculin 2 to the acetylcholinesterase peripheral site drastically reduces the association and dissociation rate constants for N-methylacridinium binding to the active site. / Rosenberry, Terrone L.; Rabl, Carl Roland; Neumann, Eberhard.

In: Biochemistry, Vol. 35, No. 3, 23.01.1996, p. 685-690.

Research output: Contribution to journalArticle

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title = "Binding of the neurotoxin fasciculin 2 to the acetylcholinesterase peripheral site drastically reduces the association and dissociation rate constants for N-methylacridinium binding to the active site",
abstract = "The acetylcholinesterase (ACHE) active site consists of a gorge 2 nm deep that is lined with aromatic residues. A serine residue near the base of the gorge defines an acylation site where an acyl enzyme intermediate is formed during the hydrolysis of ester substrates. Residues near the entrance to the gorge comprise a peripheral site where inhibitors like propidium and fasciculin 2, a snake neurotoxin, bind and interfere with catalysis. Like certain other cationic ligands that bind specifically to the acylation site, N-methylacridinium can still interact with the acylation site in the AChE- fasciculin 2 complex. At 310 K (37 °C), the equilibrium dissociation constant K(L)' for N-methylacridinium binding to the complex was 4.0 ± 0.7 μM, less than an order of magnitude larger than the K(L) = 1.0 ± 0.3 μM for N-methylacridinium interaction with human AChE in the absence of fasciculin 2. To assess whether fasciculin 2 can sterically block access of a ligand to the acylation site, thermodynamic and kinetic constants for the interaction of N-methylacridinium with AChE in the presence and absence of fasciculin 2 were measured by fluorescence temperature jump relaxation kinetics. During progressive titration of the enzyme with increasing concentrations of N-methylacridinium, a prominent relaxation in the 0.1-1 ms range was observed in the absence of fasciculin 2. When excess fasciculin 2 was added, the prominent relaxation shifted to the 0.3-1 s range. Estimates of total AChE concentrations, K(L), or K(L)' from analyses of relaxation amplitudes agreed well with those from equilibrium fluorescence, confirming that the relaxations corresponded to the bimolecular reactions of interest. Further analysis of the relaxation times in the absence of fasciculin 2 gave estimates of the N-methylacridinium association rate constant k(12) = 8 x 108 M-1 s-1 and dissociation rate constant k(21) = 750 s-1 at 310 K (37 °C). For the AChE-fasciculin 2 complex, the corresponding constants were k(12)' = 1.0 x 10s M-1 s-1 and k(21)' = 0.4 s-1. Thus the rate constants decreased by more than 3 orders of magnitude when fasciculin 2 was bound, consistent with a pronounced steric blockade of N-methylacridinium ingress to and egress from the acylation site.",
author = "Rosenberry, {Terrone L.} and Rabl, {Carl Roland} and Eberhard Neumann",
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T1 - Binding of the neurotoxin fasciculin 2 to the acetylcholinesterase peripheral site drastically reduces the association and dissociation rate constants for N-methylacridinium binding to the active site

AU - Rosenberry, Terrone L.

AU - Rabl, Carl Roland

AU - Neumann, Eberhard

PY - 1996/1/23

Y1 - 1996/1/23

N2 - The acetylcholinesterase (ACHE) active site consists of a gorge 2 nm deep that is lined with aromatic residues. A serine residue near the base of the gorge defines an acylation site where an acyl enzyme intermediate is formed during the hydrolysis of ester substrates. Residues near the entrance to the gorge comprise a peripheral site where inhibitors like propidium and fasciculin 2, a snake neurotoxin, bind and interfere with catalysis. Like certain other cationic ligands that bind specifically to the acylation site, N-methylacridinium can still interact with the acylation site in the AChE- fasciculin 2 complex. At 310 K (37 °C), the equilibrium dissociation constant K(L)' for N-methylacridinium binding to the complex was 4.0 ± 0.7 μM, less than an order of magnitude larger than the K(L) = 1.0 ± 0.3 μM for N-methylacridinium interaction with human AChE in the absence of fasciculin 2. To assess whether fasciculin 2 can sterically block access of a ligand to the acylation site, thermodynamic and kinetic constants for the interaction of N-methylacridinium with AChE in the presence and absence of fasciculin 2 were measured by fluorescence temperature jump relaxation kinetics. During progressive titration of the enzyme with increasing concentrations of N-methylacridinium, a prominent relaxation in the 0.1-1 ms range was observed in the absence of fasciculin 2. When excess fasciculin 2 was added, the prominent relaxation shifted to the 0.3-1 s range. Estimates of total AChE concentrations, K(L), or K(L)' from analyses of relaxation amplitudes agreed well with those from equilibrium fluorescence, confirming that the relaxations corresponded to the bimolecular reactions of interest. Further analysis of the relaxation times in the absence of fasciculin 2 gave estimates of the N-methylacridinium association rate constant k(12) = 8 x 108 M-1 s-1 and dissociation rate constant k(21) = 750 s-1 at 310 K (37 °C). For the AChE-fasciculin 2 complex, the corresponding constants were k(12)' = 1.0 x 10s M-1 s-1 and k(21)' = 0.4 s-1. Thus the rate constants decreased by more than 3 orders of magnitude when fasciculin 2 was bound, consistent with a pronounced steric blockade of N-methylacridinium ingress to and egress from the acylation site.

AB - The acetylcholinesterase (ACHE) active site consists of a gorge 2 nm deep that is lined with aromatic residues. A serine residue near the base of the gorge defines an acylation site where an acyl enzyme intermediate is formed during the hydrolysis of ester substrates. Residues near the entrance to the gorge comprise a peripheral site where inhibitors like propidium and fasciculin 2, a snake neurotoxin, bind and interfere with catalysis. Like certain other cationic ligands that bind specifically to the acylation site, N-methylacridinium can still interact with the acylation site in the AChE- fasciculin 2 complex. At 310 K (37 °C), the equilibrium dissociation constant K(L)' for N-methylacridinium binding to the complex was 4.0 ± 0.7 μM, less than an order of magnitude larger than the K(L) = 1.0 ± 0.3 μM for N-methylacridinium interaction with human AChE in the absence of fasciculin 2. To assess whether fasciculin 2 can sterically block access of a ligand to the acylation site, thermodynamic and kinetic constants for the interaction of N-methylacridinium with AChE in the presence and absence of fasciculin 2 were measured by fluorescence temperature jump relaxation kinetics. During progressive titration of the enzyme with increasing concentrations of N-methylacridinium, a prominent relaxation in the 0.1-1 ms range was observed in the absence of fasciculin 2. When excess fasciculin 2 was added, the prominent relaxation shifted to the 0.3-1 s range. Estimates of total AChE concentrations, K(L), or K(L)' from analyses of relaxation amplitudes agreed well with those from equilibrium fluorescence, confirming that the relaxations corresponded to the bimolecular reactions of interest. Further analysis of the relaxation times in the absence of fasciculin 2 gave estimates of the N-methylacridinium association rate constant k(12) = 8 x 108 M-1 s-1 and dissociation rate constant k(21) = 750 s-1 at 310 K (37 °C). For the AChE-fasciculin 2 complex, the corresponding constants were k(12)' = 1.0 x 10s M-1 s-1 and k(21)' = 0.4 s-1. Thus the rate constants decreased by more than 3 orders of magnitude when fasciculin 2 was bound, consistent with a pronounced steric blockade of N-methylacridinium ingress to and egress from the acylation site.

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