Solvent Deuterium Oxide Isotope Effects on the Reactions of Organophosphorylated Acetylcholinesterase

Abstract

Organophosphates (OPs) are esters of substituted phosphates, phosphonates or phosphoramidates that react with acetylcholinesterase (AChE) by initially transferring the organophosphityl group to a serine residue in the enzyme active site, concomitant with loss of an alcohol or halide leaving group. With substituted phosphates, this transfer is followed by relatively slow hydrolysis of the organophosphoryl AChE, or dephosphorylation, that is often accompanied by an aging reaction that renders the enzyme irreversibly inactivated. Aging is a dealkylation that converts the phosphate triester to a diester. OPs are very effective AChE inhibitors and have been developed as insecticides and chemical warfare agents. We examined three reactions of two organophosphoryl AChEs, dimethyl- and diethylphosphorylated AChE, by comparing rate constants and solvent deuterium oxide isotope effects for hydrolysis, aging and oxime reactivation with pralidoxime (2-PAM). Our study was motivated (1) by a published x-ray crystal structure of diethylphosphorylated AChE, which showed severe distortion of the active site that was restored by the binding of pralidoxime, and (2) by published isotope effects for decarbamoylation that decreased from 2.8 for N-monomethylcarbamoyl AChE to 1.1 for N,N-diethylcarbamoyl AChE. We previously reconciled these results by proposing a shift in the rate-limiting step from proton transfer for the small carbamoyl group to a likely conformational change in the distorted active site of the large carbamoyl enzyme. This proposal was tested but was not supported in this report. The smaller dimethylphosphoryl AChE and the larger diethylphosphoryl AChE gave similar isotope effects for both oxime reactivation and hydrolysis, and the isotope effect values of about two indicated that proton transfer was rate limiting for both reactions.

Enzymes, Reacting with Organophosphorus Compounds as Detoxifiers: Diversity and Functions

“…It should be noted here that a contribution of geometries of phosphorylated adducts to the rate constants appears to be underestimated. According to the isotope effect on AChE, bulkier substituents decrease rate constants dramatically [ 81 ].…”

Phosphocreatine Improves Muscle Microcirculation During Exercise

“…The weight of rubber sleeve and iron ring for self-made weight bearing is 2.1-3.0g; A manual glass homogenizer; A model FA2004 electronic balance made by Shanghai Precision Scientific Instrument Co., Ltd. A 2P-200 oscillator; DRAON pipetting gun; Surgical instruments 721; Visible light spectrophotometer; The corresponding wavelength of the enzyme plate instrument; 1000C constant temperature water bath; Whirlpool mixer and a table centrifuge. 5…”