Substrate binding to the peripheral site of acetylcholinesterase initiatesenzymatic catalysis. Substrate inhibition arises as a secondary effect

Two sites of ligand interaction in acetylcholinesterase (AChE) were first d emonstrated in ligand binding studies and later confirmed by crystallograph y, site-specific mutagenesis, and molecular modeling: an acylation site at the base of the active site gorge and a peripheral site at its mouth. We re cently introduced a steric blockade model which demonstrated how small peri pheral site ligands such as propidium may inhibit substrate hydrolysis [Sze gletes, T., Mallender, W. D., and Rosenberry, T. L. (1998) Biochemistry 37, 4206-4216]. In this model, the only effect of a bound peripheral site liga nd is to decrease the association and dissociation rate constants for an ac ylation site ligand without altering the equilibrium constant for ligand bi nding to the acylation site. Here, we first provide evidence that not only rate constants for substrates but also dissociation rate constants for thei r hydrolysis products are decreased by bound peripheral site ligand. Previo us reaction schemes for substrate hydrolysis by AChE were extended to inclu de product dissociation steps, and acetylthiocholine hydrolysis rates in th e presence of propidium under nonequilibrium conditions Even simulated with assigned rate constants in the program SCoP. We next showed that cationic substrates such as acetylthiocholine and 7-acetoxy-N-methylquinolinium (M7A ) bind to the peripheral site as well as to the acylation site. The neuroto xin fasciculin was used to report specifically on interactions at the perip heral site. Analysis of inhibition of fasciculin association rates by these substrates revealed K-s values of about 1 mM for the peripheral site bindi ng of acetylthiocholine and 0.2 mM for the binding of M7A. The AChE reactio n scheme was further extended to include substrate binding to the periphera l site as the initial step in the catalytic pathway. Simulations of the ste ric blockade model with this scheme were in reasonable agreement with obser ved substrate inhibition for acetylthiocholine and M7A and with mutual comp etitive inhibition in mixtures of acetylthiocholine and M7A. Substrate inhi bition was explained by blockade of product dissociation when substrate is bound to the peripheral site. However, our analyses indicate that the prima ry physiologic role of the AChE peripheral site is to accelerate the hydrol ysis of acetylcholine at low substrate concentrations.