Molecular Pharmacology, Vol 12, 658-666, Copyright © 1976 by the American Society for Pharmacology and Experimental Therapeutics

Free Energy Relationships in the Inhibition of Acetylcholinesterase by Diethyl Phosphates

¹ Department of Chemistry, University of Colorado, Boulder, Colorado 80309

Acetylcholinesterase inhibited by diethyl phosphorofluoridate (DEP-enzyme) was reactivated by a number of phenols and hydroxyquinolines. The second-order rate constants for reactivation were divided into the known second-order rate constants for inhibition by the corresponding diethyl phosphoryl derivatives of the reactivators (conjugate inhibitors) to obtain the equilibrium constant in terms of analytical concentrations for the inhibition of the enzyme. These values were multiplied by the known value of the equilibrium constant for the hydrolysis of the DEP-enzyme, ECH (DEP-enzyme), to obtain equilibrium constants for the hydrolysis of the inhibitor, ECH (inhibitor). These values were converted to ECH (inhibitor) in terms of acidic species, ECH (inhibitor-acidic), and it was found that a linear free energy relationship was obeyed with the pK_a of the conjugate acid of the leaving group ( reactivator): log ECH (inhibitor-acidic) = 13.18 - 0.620 pK_a Using this relationship, log k_i/k_r vs. pK_a and log ECH (anal.) vs. pK_a, plots were calculated for pH 7.0. Since the DEP-enzyme falls on these curves using pK_a = 13.6 for the hydroxyl group of serine, it was concluded that there were no very substantial interactions, positive or negative, between the phosphoryl group and the protein. This is not the case with other DEP-enzymes, such as chymotrypsin, which is 4 orders of magnitude more stable. Diethyl phosphorofluoridate is much more stable than esters of oxygen leaving groups, with the same pK_a as HF. Compounds containing a sulfur leaving group are much less stable than oxygen esters of the same pK_a. A number of observations can be explained using the thermodynamic data.