Kinetic evidence for the allosteric substrate inhibition of human erythrocyte acetylcholinesterase

Abstract

Kinetic experiments described in this study were carried out with an electrophoretically and immunologically homogeneous acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) preparation isolated from human erythrocyte membranes. At low concentration of substrates, the acetylcholinesterasecatalyzed reaction follows Michaelis-Menten kinetics. In comparative studies using acetylthiocholine and acetylcholine as substrates, the corresponding K m values were 150 ± 30 μM and 200 ± 20 μM, respectively. At low concentrations of both substrates, Hill plots indicated the existence of either a single or multiple independent active site(s). The inhibition mechanism of acetylcholinesterase by high concentration of substrates were studied by utilizing a new kinetic parameter, Δ, which allows discrimination between the competitive and uncompetitive types of substrate inhibitions (Wang, C.-S. (1977) Eur. J. Biochem. 78, 568–574). This kinetic approach provided evidence that the inhibition of acetylcholinesterase by excess substrate was effected by its interaction with multiple allosteric sites on the enzyme.