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

FEBS Letters

Sussman, Joel L.³  Silman, Israel²  Massoulié, Jean²  Le Feuvre, Yves²  Bon, Suzanne²  Ripoll, Daniel¹  Morel, Nathalie²  Faerman, Carlos⁴ 

[1]Cornell Theory Center, Cornell University, Ithaca, NY 14853, USA

[2]Laboratoire de Neurobiologie, Ecole Normale Supérieure, 75230 Paris, France

[3]Department of Structural Biology, The Weizmann Institute of Science, Rehovot 76100, Israel

[4]Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY 14853, USA

关键词: Torpedo californica;    Ω loop;    Site-directed mutagenesis;    Molecular dynamics;    Disulfide;    AChE;    acetylcholinesterase;    ACh;    acetylcholine;    ATCh;    acetylthiocholine;    MPT;    O-ethyl-S2-diisopropylaminoethyl methylphosphothionate;    DMEM;    Dulbecco's modified Eagle's medium;   

DOI  :  10.1016/0014-5793(96)00374-2

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 Ω 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 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 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.

【 授权许可】

Unknown