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Site‐directed mutants designed to test back‐door hypotheses of acetylcholinesterase function
Author(s) -
Faerman Carlos,
Ripoll Daniel,
Bon Suzanne,
Le Feuvre Yves,
Morel Nathalie,
Massoulié Jean,
Sussman Joel L.,
Silman Israel
Publication year - 1996
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(96)00374-2
Subject(s) - acetylcholinesterase , torpedo , active site , shutter , homology modeling , mutant , biophysics , stereochemistry , twist , enzyme , mutagenesis , chemistry , acetylcholine , binding site , biochemistry , biology , physics , acetylcholine receptor , gene , receptor , geometry , mathematics , optics , endocrinology
The location of the active site of the rapid enzyme, acetylcholinesterase, near the bottom of a deep and narrow gorge indicates that alternative routes may exist for traffic of substrate, products or solute into and out of the gorge. Molecular dynamics suggest the existence of a shutter‐like back door near Trp 84 , a key residue in the binding site for acetylcholine, in the Torpedo californica enzyme. The homology of the Ω loop, bearing Trp 84 , with the lid which sequesters the substrate in neutral lipases displaying structural homology with acetylcholinesterase, suggests a flap‐like back door. Both possibilities were examined by site‐directed mutagenesis. The shutter‐like back door was tested by generating a salt bridge which might impede opening of the shutter. The flap‐like back door was tested by de novo insertion of a disulfide bridge which tethered the Ω loop to the body of the enzyme. Neither type of mutation produced significant changes in catalytic activity, thus failing to provide experimental support for either back door model. Molecular dynamics revealed, however, substantial mobility of the Ω loop in the immediate vicinity of Trp 84 , even when the loop was tethered, supporting the possibility that access to the active site, involving limited movement of a segment of the loop, is indeed possible.