ALLOSTERIC CONTROL OF ACETYLCHOLINESTERASE CATALYSIS BY FASCICULIN

The interaction of fasciculin 2 was examined with wild-type and severa l mutant forms of acetylcholinesterase (AChE) where Trp(86), which lie s at the base of the active center gorge, is replaced by Tyr, Phe, and Ala. The fasciculin family of peptides from snake venom bind to a per ipheral site near the rim of the gorge, but at a position which still allows substrates and other inhibitors to enter the gorge. The interac tion of a series of charged and uncharged carboxyl esters, alkyl phosp horyl esters, and substituted trifluoroacetophenones were analyzed wit h the wild-type and mutant AChEs in the presence and absence of fascic ulin. We show that Trp(86) is important for the alignment of carboxyl ester substrates in the AChE active center. The most marked influence of Trp(86) substitution in inhibiting catalysis is seen for carboxyl e sters that show rapid turnover. The extent of inhibition achieved with bound fasciculin is also greatest for efficiently catalyzed, charged substrates. When Ala is substituted for Trp(86), fasciculin becomes an allosteric activator instead of an inhibitor for certain substrates. Analysis of the kinetics of acylation by organophosphates and conjugat ion by trifluoroacetophenones, along with deconstruction of the kineti c constants for carboxyl esters, suggests that AChF inhibition by fasc iculin arises from reductions of both the commitment to catalysis and diffusional entry of substrate into the gorge. The former is reflected in the ratio of the rate constant for substrate acylation to that for dissociation of the initial complex. The action of fasciculin appears to be mediated allosterically from its binding site at the rim of the gorge to affect the orientation of the side chain of Trp(86) which li es at the gorge base.