FASCICULIN INHIBITION OF ACETYLCHOLINESTERASE IS PREVENTED BY CHEMICAL MODIFICATION OF THE ENZYME AT A PERIPHERAL SITE

Fasciculin 2 (FAS) is a 61 amino acid peptide present in Dendroaspis a ngusticeps snake venom, with a selective and potent inhibitory activit y towards acetylcholinesterase (AChE). The specific interaction of FAS with peripheral sites present in Electrophorus electricus AChE (K-i = 0.04 nM FAS) was investigated by chemical modification with N,N-dimet hyl-2-phenylaziridinium (DPA) in the presence of active or peripheral anionic site protective agents. An enzyme was obtained that compared t o the native AChE is 10(6)-times less sensitive to FAS, is fully inhib ited by edrophonium and tacrine, and is 25-170-times less sensitive to several peripheral site ligands. Characterization of catalytic functi ons showed that K-m for acetylthiocholine was 4-fold lower in the DPA- modified enzyme, whereas K-m for phenylacetate remained the same. Valu es for k(cat) determined with both substrates were unchanged. Diminish ed catalytic efficiency reflects that hydrolysis and/or supply of cati onic substrates to the active site was affected by DPA reaction at a p eripheral 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.