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

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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.