Interaction of quaternary ammonium compounds with acetylcholinesterase: characterization of the active site.

The relation of the structure of 31 quaternary ammonium compounds (28 inhibitors; 3 substrate analogues) with their effects on the activity of acetylcholinesterase (EC 3.1.1.7; AChE) was studied. The compounds were structurally related to the natural substrate acetylcholine (ACh). All bear a trimethylammonium moiety as cationic head. The inhibitors include a variety of functional groups instead of an electrophilic ester group, making these substances suitable to probe the esteratic subsite. The inhibition constants and Km values were determined in kinetic experiments under steady state conditions (pH-stat method). Most of the substances acted as reversible, competitive inhibitors with KI in the range of 10(-6)-10(-3) M. The substrate analogues had Km values between (1.2-2.2) X 10(-4) M. The data allow the following main conclusions: (1) The quaternary trimethylammonium group of ACh is of high importance for substrate binding to AChE. It mediates association at the anionic site. (2) A poorer contribution to binding (two orders of magnitude lower) is attributable to the apolar methylene chain in ACh. It can be related to a hydrophobic interaction of the hydrocarbon chain at a region neighbouring the anionic site. (3) The ester group (both C = O and O) does not contribute to substrate binding. It is only responsible for reactivity.