Conference paper

Interaction of Ligand Binding Sites of Acetylcholinesterase

Gregory Mooser ; Department of Biochemistry, School of Dentistry, University of Southern California, Los Angeles, California 90007, USA
David S. Sigman ; Department of Biological Chemistry, Center for the Heaith Sciences, University of California, Los Angeles, California 90024, USA

Abstract

Ligand binding properties of acetylcholinesterase from Electrophorus
electricus have been investigated under equilibrium and
transient •state conditions with the reversible fluorescent probes,
N-methylacridinium (N-MAC) and bis(3-aminopyridinium)-l,10-
-decane (DAP). N-MAC binds at the active site and DAP, an analog
of decamethonium, bridges the active site and peripheral modifier
site. The probes were used to monitor the binding of ligands that
interact at one or both of these sites with resultant probe displacement.
Using a nonlinear least-squares analY'sis to reduce
fluorescent probe displacement data, d-tubocurarine was found to
bind at a site remote from the active site. In physiological ionic
strength media, d-tubocurarine binds exclusively at the peripheral
modifier site, and in low ionic strength media it binds significantly
to both the peripheral site and active site. In both ionic ,strength
media, the peripheral site was found to be the same site that interacts
with the second cationic function of bis-quaternary ammonium
compounds like decamethonium and DAP. When d-tubocurarine
occupies the peripheral site, the affinity of the enzyme for active
site ligands is decreased. There is a positive correlation between
the size of the active site ligand and the degree of d-tubocurarine
induced destabilization suggesting steric factorn may be operative.
Gallamine btnding is mutually exclusive with active site ligands.
Rapid mixing stopped flow experiments were used to determine if
this results from gallamine binding at the active site or bLnding
at the peripheral stte with resultant conformational effects mediated
to the active site. The demonstration of a transient gallamine--
enzyme--N-MAC ternary complex suggests the latter binding
pattern occurs.

Keywords

Hrčak ID:

196590

URI

https://hrcak.srce.hr/196590

Publication date:

3.12.1975.

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