Marchot_1993_J.Biol.Chem_268_12458

Iodination of fasciculin 3 (FAS3) from Dendroaspis viridis venom provided us with a fully active specific probe of fasciculin binding sites on rat brain acetylcholinesterase (AChE). Binding and inhibition are concomitant, as association and inhibition rate constants k1 and ki are identical. The 125I-FAS3.AChE complex dissociates very slowly (t 1/2 = 48 h) and is characterized by a dissociation constant, Kd, of 0.4 pM. All the specific binding of 125I-FAS3 to AChE is prevented by FAS3 as from D. angusticeps venom (Kd = 0.4, 14, and 25 pM, respectively). It is also prevented by propidium iodide, BW284C51, and d-tubocurarine, which bind to peripheral anionic sites of AChE, by Ca2+ and Mg2+, known to enhance AChE activity through an allosteric phenomenon and by acetylthiocholine concentrations which lead to excess substrate inhibition of the enzyme. Diisopropyl fluorphosphate and paroxon, which inhibit AChE by phosphorylating the catalytic serine, have no effect on either the binding rate or the number of binding sites of 125I-FAS3. O-Ethyl-S2-diisopropylaminoethyl methylphosphonothionate, however, which binds irreversibly to the AChE catalytic site but reversibly to a peripheral site, induces a 130% increase in the binding rate of 125I-FAS3, without changing the total number of 125I-FAS3 binding sites. Our results demonstrate that fasciculins bind on a peripheral site of AChE, distinct from the catalytic site and, at least partly, common with the sites on which some cationic inhibitors and the substrate in excess bind. Since phosphorylation of the catalytic serine (esteratic subsite) by [1,3-3H]diisopropyl fluorophosphate can still occur on the FAS3.AChE complex, the structural modification induced by fasciculins may affect the anionic subsite of AChE catalytic site.