Site-directed mutants designed to test back-door hypotheses of acetylcholinesterase function

• Published in: FEBS letters 1996-05, Vol.386 (1), p.65-71
• Main Authors: Faerman, Carlos, Ripoll, Daniel, Bon, Suzanne, Le Feuvre, Yves, Morel, Nathalie, Massoulié, Jean, Sussman, Joel L., Silman, Israel
• Format: Article
• Language: English
• Subjects: acetylcholine; acetylcholinesterase; Acetylcholinesterase - chemistry; Acetylcholinesterase - genetics; Acetylcholinesterase - physiology; acetylthiocholine; ACh; AChE; Animals; ATCh; Base Sequence; Binding Sites; Cells, Cultured - metabolism; Computer Simulation; Disulfide; Disulfides; DMEM; Dulbecco's modified Eagle's medium; Enzyme Activation; Kidney - cytology; Models, Molecular; Molecular dynamics; Molecular Sequence Data; MPT; Mutagenesis, Site-Directed; Mutation; O-ethyl-S2-diisopropylaminoethyl methylphosphothionate; Protein Conformation; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Site-directed mutagenesis; Thermodynamics; Torpedo - physiology; Torpedo californica; Ω loop
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(96)00374-2

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.