RESUMO
Fasciculin 2 (FAS) is a 61 amino acid peptide present in Dendroaspis angusticeps snake venom, with a selective and potent inhibitory activity towards acetylcholinesterase (AChE). The specific interaction of FAS with peripheral sites present in Electrophorus electricus AChE (Ki = 0.04 nM FAS) was investigated by chemical modification with N,N-dimethyl-2-phenylaziridinium (DPA) in the presence of active or peripheral anionic site protective agents. An enzyme was obtained that compared to the native AChE is 10(6)-times less sensitive to FAS, is fully inhibited by edrophonium and tacrine, and is 25-170-times less sensitive to several peripheral site ligands. Characterization of catalytic functions showed that Km for acetylthiocholine was 4-fold lower in the DPA-modified enzyme, whereas Km for phenylacetate remained the same. Values for Kcat determined with both substrates were unchanged. Diminished catalytic efficiency reflects that hydrolysis and/or supply of cationic substrates to the active site was affected by DPA reaction at a peripheral site. Previous data implicate Trp-279 (Torpedo AChE sequence numbering) as the residue actually involved in DPA modification. Our results strongly support FAS binding to an AChE peripheral site which partially overlaps the site of other peripheral site ligands including acetylthiocholine.