Effect of mutations within the peripheral anionic site on the stability of acetylcholinesterase.
Torpedo acetylcholinesterase is irreversibly inactivated by modifying a buried free cysteine, Cys231, with sulfhydryl reagents. The stability of the enzyme, as monitored by measuring the rate of inactivation, was reduced by mutating a leucine, Leu282, to a smaller amino acid residue. Leu282 is located within the "peripheral" anionic site, at the entrance to the active-site gorge. Thus, loss of activity was due to the increased reactivity of Cys231. This was paralleled by an increased susceptibility to thermal denaturation, which was shown to be due to a large decrease in the activation enthalpy. Similar results were obtained when either of two other residues in contact with Leu282 in Torpedo acetylcholinesterase, Trp279 and Ser291, was replaced by an amino acid with a smaller side chain. We studied the effects of various ligands specific for either the active or peripheral sites on both thermal inactivation and on inactivation by 4,4'-dithiodipyridine. The wild-type and mutated enzymes could be either protected or sensitized. In some cases, opposite effects of the same ligand were observed for chemical modification and thermal denaturation. The mutated residues are within a conserved loop, W279-S291, at the top of the active-site gorge, that contributes to the peripheral anionic site. Theoretical analysis showed that Torpedo acetylcholinesterase consists of two structural domains, each comprising one contiguous polypeptide segment. The W279-S291 loop, located in the first domain, makes multiple contacts with the second domain across the active-site gorge. We postulate that the mutations to residues with smaller side chains destabilize the conserved loop, thus disrupting cross-gorge interactions and, ultimately, the entire structure.
Morel
N
N
Laboratoire de Neurobiologie Cellulaire et Moléculaire, Centre National de la Recherche Scientifique, Unité de Recherche Mixte 8544, Ecole Normale Supérieure, Paris, France.
Bon
S
S
Greenblatt
H M
HM
Van Belle
D
D
Wodak
S J
SJ
Sussman
J L
JL
Massoulié
J
J
Silman
I
I
D000110:Acetylcholinesterase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D004795:Enzyme Stability; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D014101:Torpedo
D000110:Acetylcholinesterase