BINDING OF THE NEUROTOXIN FASCICULIN-2 TO THE ACETYLCHOLINESTERASE PERIPHERAL SITE DRASTICALLY REDUCES THE ASSOCIATION AND DISSOCIATION RATE CONSTANTS FOR N-METHYLACRIDINIUM BINDING TO THE ACTIVE-SITE

The acetylcholinesterase (AChE) active site consists of a gorge 2 nm 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. Like certain other cationic ligands that bind spe cifically to the acylation site, N-methylacridinium can still interact with the acylation site in the AChE-fasciculin 2 complex. At 310 K (3 7 degrees C), the equilibrium dissociation constant K-L' for N-methyla cridinium binding to the complex was 4.0 +/- 0.7 mu M, less than an or der of magnitude larger than the K-L = 1.0 +/- 0.3 mu M for N-methylac ridinium interaction with human AChE in the absence of fasciculin 2. T o assess whether fasciculin 2 can sterically block access of a ligand to the acylation site, thermodynamic and kinetic constants for the int eraction of N-methylacridinium with AChE in the presence and absence o f fasciculin 2 were measured by fluorescence temperature jump relaxati on kinetics. During progressive titration of the enzyme with increasin g concentrations of N-methylacridinium, a prominent relaxation in the 0.1-1 ms range was observed in the absence of fasciculin 2. When exces s fasciculin 2 was added, the prominent relaxation shifted to the 0.3- 1 s range. Estimates of total AChE concentrations, K-L, or K-L' from a nalyses of relaxation amplitudes agreed well with those from equilibri um fluorescence, confirming that the relaxations corresponded to the b imolecular reactions of interest. Further analysis of the relaxation t imes in the absence of fasciculin 2 gave estimates of the N-methylacri dinium association rate constant k(12) = 8 x 10(8) M(-1) s(-1) and dis sociation rate constant k(21) = 750 s(-1) at 310 K (37 degrees C). For the AChE-fasciculin 2 complex, the corresponding constants were k(12) ' = 1.0 x 10(5) M(-1) s(-1) and k(21)' = 0.4 s(-1). Thus the rate cons tants decreased by more than 3 orders of magnitude when fasciculin 2 w as bound, consistent with a pronounced steric blockade of N-methylacri dinium ingress to and egress from the acylation site.