Substrate Binding to the Peripheral Site Occurs on the Catalytic Pathway of Acetylcholinesterase and Leads to Substrate Inhibition
Two sites of ligand interaction in acetylcholinesterase (AChE) were first demonstrated in ligand binding studies (7) and later confirmed by crystallography, site-specific mutagenesis and molecular modeling: an acylation site at the base of the gorge, and a peripheral site at its mouth. Here we address the question of how ligand binding to the peripheral site alters AChE catalytic activity. In the traditional AChE catalytic pathway (Scheme 1 below), the initial enzyme-substrate complex ES proceeds to an acylated enzyme intermediate EA which is then hydrolyzed to product P and E (2). When a peripheral site inhibitor (I) is included, typical analysis of inhibition patterns assumes S and I binding are at equilibrium (3; 4). We avoid these equilibrium assumptions here for the first time in the AChE literature by solving the appropriate differential rate equations with the simulation program SCoP. In particular, we examine the simple hypothesis that the only effect of peripheral site inhibitors like propidium is to impose a steric blockade that decreases association and dissociation rate constants for substrates and other acylation site ligands without altering their ratio, the equilibrium association constant. We test this hypothesis by examining the effect of peripheral site ligands on the rate constants for the binding of acylation site inhibitors and on the inhibition of substrate hydrolysis. We turn next to the questions of whether acetylthiocholine itself can bind to the peripheral site and whether this binding is of significance on the catalytic pathway. The acetylthiocholine affinity for the peripheral site is determined by measuring acetylthiocholine inhibition of the association rate k on for the slowly equilibrating peripheral site ligand fasciculin 2. Our re-suits indicate that failure to achieve equilibrium has a profound impact on the classical interpretation of AChE inhibition and indeed alters mechanistic conclusions. A physiological role of substrate binding to the peripheral site in the catalytic pathway is also suggested.
KeywordsSubstrate Binding Substrate Inhibition AChE Inhibition High Substrate Concentration Peripheral Site
Unable to display preview. Download preview PDF.