US EPA

662086 

Journal Article 

Abstract 

The Use Of The Inhibitory Rate Constant Ki To Characterize Inhibition Of Acetylcholinesterase By Organophosphates 

Rosenfeld, C; Sultatos, LG 

2006 

1 

Toxicologist
ISSN: 0731-9193 

TOX/6001752 

90 

1-S 

248 

English 

For over four decades the inhibitory constant ki has been used to quantify to inhibitory capacity of organophosphates (and other anticholinesterases) towards acetylcholinesterase. More recently, results from this laboratory have indicated that ki for an organophosphate can vary over a wide range of inhibitor concentrations, thereby prompting the current investigation into the applicability this rate constant. Incubation of human recombinant acetylcholinesterase (3-5 pM) with paraoxon ((O,O-diethyl O-(p-nitrophenyl) phosphate)(0.017-100 nM) revealed a decreasing ki with increasing inhibitor, until a plateau was reached at about 10 nM paraoxon. Co-incubation of paraoxon with p-nitrophenyl acetate and enzyme yielded an inhibitor Kd = 339 nM, and a k2 = 51.8 h-1. Optimization of a computer model based on an Ordered Uni Bi mechanism for phosphorylation of acetylcholinesterase determined k1 to be 0.5 nM-1h-1, and k-1 to be 170 h-1. These values were used in a comparison of the Ordered Uni Bi model versus a ki model. Interestingly, the ki model was accurate only at equilibrium (or near equilibrium), and when the inhibitor concentration was well below its Kd (pseudo first order conditions). These results indicate that the often-utilized large inhibitor concentrations for ki determinations could yield erroneous values. Furthermore, the changing ki as a function inhibitor is not an artifact resulting from inappropriate inhibitor concentrations. 

Research Support, N.I.H., Extramural; Humans; Phosphoric Acid Esters/*TOXICITY/CHEMISTRY; Cholinesterase Inhibitors/*TOXICITY/CHEMISTRY; Recombinant Proteins/CHEMISTRY; Acetylcholinesterase/CHEMISTRY; Models, Biological; NO CAS RN 

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