Acetylcholinesterase: evidence that sodium ion binding at the anionic site causes inhibition of the second-order hydrolysis of acetylcholine and a decrease of its pKα as well as of deacetylation

Abstract

For bovine erythrocyte acetylcholinesterase (acetylcholine hydrolase) the Michaelis parameters of Vmax, and Km for the natural substrate acetylcholine were estimated as a function of pH and sodium chloride concentration by the pH-stat method. A single Kd for Na+ binding (K = 7 .times. 10-3 M) suffices to explain the salt dependence of Vmax/Km and the pH dependence of Vmax/Km and Vmax/Km being pH independent. This finding provides evidence for a specific effect of Na+, presumably by binding at the anionic subsite of the active center. Na+ binding causes a 50-fold decrease in kcat[catalytic rate constant]/Km as well as a decrease of 1 unit in the pKa of both kcat/Km and kcat. The intrinsic pKa in the absence of salt at 25.degree. C is .apprx. 7.5. Comparison of the degree of fit of the data to the Debeye-Huckel equation, in accordance with an alternative general salt effect and published data for sodium and potassium chlorides also favor a specific salt effect.