FASCICULIN-2 BINDS TO THE PERIPHERAL SITE ON ACETYLCHOLINESTERASE ANDINHIBITS SUBSTRATE HYDROLYSIS BY SLOWING A STEP INVOLVING PROTON-TRANSFER DURING ENZYME ACYLATION

The acetylcholinesterase active site consists of a gorge 20 Angstrom d eep that is lined with aromatic residues, A serine residue near the ba se of the gorge defines an acylation site where an acyl enzyme interme diate is formed during the hydrolysis of ester substrates, Residues ne ar the entrance to the gorge comprise a peripheral site where inhibito rs like propidium and fasciculin 2, a snake neurotoxin, bind and inter fere with catalysis, We report here the association and dissociation r ate constants for fasciculin 2 interaction with the human enzyme in th e presence of ligands that bind to either the peripheral site or the a cylation site, These kinetic data confirmed that propidium is strictly competitive with fasciculin 2 for binding to the peripheral site, In contrast, edrophonium, N-methylacridinium, and butyrylthiocholine boun d to the acylation site and formed ternary complexes with the fascicul in a-bound enzyme in which their affinities were reduced by about an o rder of magnitude from their affinities in the free enzyme. Steady sta te analysis of the inhibition of substrate hydrolysis by fasciculin 2 revealed that the ternary complexes had residual activity, For acetylt hiocholine and phenyl acetate, saturating amounts of the toxin reduced the first-order rate constant k(cat) to 0.5-2% and the second-order r ate constant k(cat)/K-app to 0.2-2% of their values with the uninhibit ed enzyme, To address whether fasciculin 2 inhibition primarily involv ed steric blockade of the active site or conformational interaction wi th the acylation site, deuterium oxide isotope effects on these kineti c parameters were measured, The isotope effect on k(cat)/K-app increas ed for both substrates when fasciculin 2 was bound to the enzyme, indi cating that fasciculin 2 acts predominantly by altering the conformati on of the active site in the ternary complex so that steps involving p roton transfer during enzyme acylation are slowed,