SITE-DIRECTED MUTANTS DESIGNED TO TEST BACK-DOOR HYPOTHESES OF ACETYLCHOLINESTERASE FUNCTION

The location of the active site of the rapid enzyme, acetylcholinester ase, near the bottom of a deep and narrow gorge indicates that alterna tive routes may exist for traffic of substrate, products or solute int o 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 Omega loop, bearing Trp(84), with the lid which sequesters the su bstrate in neutral lipases displaying structural homology with acetylc holinesterase, 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 enzy me, Neither type of mutation produced significant changes in catalytic activity, thus failing to provide experimental support for either bac k door model, Molecular dynamics revealed, however, substantial mobili ty of the Omega loop in the immediate vicinity of Trp(84), even when t he loop was tethered, supporting the possibility that access to the ac tive site, involving limited movement of a segment of the loop, is ind eed possible.