Site-directed mutants designed to test back-door hypotheses of acetylcholinesterase function.
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 Trp84, a key- residue in the binding site for acetylcholine, in the Torpedo californica enzyme. The homology of the omega loop, bearing Trp84, 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 omega 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 omega loop in the immediate vicinity of Trp84, 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.
Faerman
C
C
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY 14853, USA.
Ripoll
D
D
Bon
S
S
Le Feuvre
Y
Y
Morel
N
N
Massoulié
J
J
Sussman
J L
JL
Silman
I
I
D000110:Acetylcholinesterase / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured / Q000378:metabolism; D003198:Computer Simulation; D004220:Disulfides; D004789:Enzyme Activation; D007668:Kidney / Q000166:cytology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013816:Thermodynamics; D014101:Torpedo / Q000502:physiology
D004220:Disulfides; D011994:Recombinant Proteins; D000110:Acetylcholinesterase