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(Received for publication, April 16, 1997, and in revised form, June 19, 1997)
From the Departments of To explore the role that surface and active
center charges play in electrostatic attraction of ligands to the
active center gorge of acetylcholinesterase (AChE), and the influence
of charge on the reactive orientation of the ligand, we have studied
the kinetics of association of cationic and neutral ligands with the active center and peripheral site of AChE. Electrostatic influences were reduced by sequential mutations of six surface anionic residues outside of the active center gorge (Glu-84, Glu-91, Asp-280, Asp-283, Glu-292, and Asp-372) and three residues within the active center gorge
(Asp-74 at the rim and Glu-202 and Glu-450 at the base). The peripheral
site ligand, fasciculin 2 (FAS2), a peptide of 6.5 kDa with a net
charge of +4, shows a marked enhancement of rate of association with
reduction in ionic strength, and this ionic strength dependence can be
markedly reduced by progressive neutralization of surface and active
center gorge anionic residues. By contrast, neutralization of surface
residues only has a modest influence on the rate of cationic
m-trimethylammoniotrifluoroacetophenone (TFK+)
association with the active serine, whereas neutralization of residues
in the active center gorge has a marked influence on the rate but with
little change in the ionic strength dependence. Brownian dynamics
calculations for approach of a small cationic ligand to the entrance of
the gorge show the influence of individual charges to be in
quantitative accord with that found for the surface residues. Anionic
residues in the gorge may help to orient the ligand for reaction or to
trap the ligand. Bound FAS2 on AChE not only reduces the rate of
TFK+ reaction with the active center but inverts the ionic
strength dependence for the cationic TFK+ association with
AChE. Hence it appears that TFK+ must traverse an
electrostatic barrier at the gorge entry imparted by the bound FAS2
with its net charge of +4.
Volume 272, Number 37,
Issue of September 12, 1997
pp. 23265-23277
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
DISTINCTIONS BETWEEN ACTIVE CENTER LIGANDS AND
FASCICULIN
,
§
and
Pharmacology and of
§ Chemistry and Biochemistry, University of California
San Diego, La Jolla, California 92093-0636 and the
¶ Department of Chemistry, University of Iowa,
Iowa City, Iowa 52242
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