Organophosphorylation of acetylcholinesterase in the presence of peripheral site ligands - Distinct effects of propidium and fasciculin

Structural analysis of acetylcholinesterase (AChE) has revealed two sites o f ligand interaction in the active site gorge: an acylation site at the bas e of the gorge and a peripheral site at its mouth. A goal of our studies is to understand how ligand binding to the peripheral site alters the reactiv ity of substrates and organophosphates at the acylation site. Kinetic rate constants were determined for the phosphorylation of AChE by two fluorogeni c organophosphates, 7-[(diethoxyphosphoryl) oxy]-1-methylquinolinium iodide (DEPQ) and 7-[(methylethoxyphosphonyl)oxy] -4-methylcoumarin (EMPC), by mo nitoring release of the fluorescent leaving group, Rate constants obtained with human erythrocyte AChE were in good agreement with those obtained for recombinant human AChE produced from a high level Drosophila S2 cell expres sion system. First-order rate constants k(OP) were 1,600 +/- 300 min(-1) fo r DEPQ and 150 +/- 11 min(-1) for EMPC, and second-order rate constants k(O P)/K-OP were 193 +/- 13 mu M-1 min(-1) for DEPQ and 0.7-1.0 +/- 0.1 mu M-1 min(-1) for EMPC; Binding of the small ligand propidium to the AChE periphe ral site decreased K-OP/K-OP by factors of 2-20 for these organophosphates, Such modest inhibitory effects are consistent with our recently proposed s teric blockade model (Szegletes, T,, Mallender, W. D., and Rosenberry, T. L . (1998) Biochemistry 37, 4206-4216), Moreover, the binding of propidium re sulted in a clear increase in ho, for EMPC, suggesting that molecular or el ectronic strain caused by the proximity of propidium to EMPC in the ternary complex may promote phosphorylation, In contrast, the binding of the polyp eptide neurotoxin fasciculin to the peripheral site of AChE dramatically de creased phosphorylation rate constants. Values of k(OP)/K-OP were decreased by factors of 10(3) to 10(5), and k(OP) was decreased by factors of 300-4, 000, Such pronounced inhibition suggested a conformational change in the ac ylation site induced by fasciculin binding, As a note of caution to other i nvestigators, measurements of phosphorylation of the fasciculin-AChE comple x by AChE inactivation gave misleading rate constants because a small fract ion of the AChE was resistant to inhibition by fasciculin.