Phosphobutyrylcholinesterase:  Phosphorylation of the Esteratic Site of Butyrylcholinesterase by Ethephon [(2-Chloroethyl)phosphonic Acid] Dianion

Abstract

Ethephon [(2-chloroethyl)phosphonic acid] has two seemingly unrelated types of biological activity. It is a major agrochemical absorbed by crops, slowly releasing ethylene as a plant growth regulator. Ethephon also inhibits the activity of plasma butyrylcholinesterase (BuChE) in humans, dogs, rats, and mice. This is totally unexpected for an ionized phosphonic acid (mostly the dianion at physiological pH), in contrast to the classical inhibitors (nonionized triester phosphates) which phosphorylate serine at the active site. This study tests the hypothesis that ethephon (as the dianion) also acts as a phosphorylating agent in inhibiting BuChE activity. The sensitivity of plasma BuChE to ethephon (90 min preincubation at 25 degrees C) is greatest for humans, dogs, and mice (IC(50) = 6-23 microM), intermediate for chickens, rabbits, rats, and guinea pigs (IC(50) = 26-53 microM), and lowest for pigs and horses (IC(50) = 92-172 microM). The IC(50) decreases linearly with time on a log-log scale to values of 0.15-0. 3 microM for human, dog, and horse BuChE at 24 h. The inhibition rate is generally related to ethephon concentration, consistent with a bimolecular reaction, e.g., phosphorylation. The extent of inhibition of the esteratic activity of BuChE by ethephon is directly proportional to the extent of inhibition of [(3)H]diisopropyl phosphorofluoridate ([(3)H]DFP) postlabeling which is not reversible on removing the ethephon, either directly or after further incubation for 24 h at 25 degrees C. These observations strongly suggest that ethephon, as DFP, phosphorylates human plasma BuChE at Ser-198 of the esteratic site, or more generally, the formation of a phosphobutyrylcholinesterase. With human plasma BuChE, (2-bromoethyl)- and (2-iodoethyl)phosphonic acids have lower affinities for the site than ethephon but higher phosphorylation rate constants, consistent with their relative hydrolysis rates at pH 7.4 (phosphorylation of water). (2-Chlorohexyl)phosphonic acid is a poor inhibitor, perhaps being too reactive with water. Thus, potency differences for ethephon and its analogues with BuChE of various species depend on both the affinities and phosphorylation rates, i.e., the binding and reactivity of the (2-haloalkyl)phosphonic acid dianion in the esteratic site.

New insights on molecular interactions of organophosphorus pesticides with esterases

“…BuChE was the first secondary target in OP poisoning to be recognized and continues to be of interest of to monitor OP exposure by determining plasma activity inhibition and is generally more sensitive to OP esters than AChE. Ethephon inhibits plasma BuChE with no apparent ill effects on mice, rats, dogs and humans (Haux et al, 2000;Haux et al, 2002). Moreover, animals with inhibited BChE have enhanced sensitivity to succinylcholine, mivacurium and aspirin (Sparks et al, 1999).…”

Organophosphate Toxicology: Safety Aspects of Nonacetylcholinesterase Secondary Targets

“…BChE is also the most sensitive enzyme to ethephon, which undergoes inhibition in vitro and in vivo forming phosphoryl-BChE (Scheme 1) (48,49). Ethephon inhibits plasma BChE in subchronic studies with mice, rats, dogs, and humans with no apparent ill effects (48,49). Animals with inhibited BChE are of enhanced sensitivity to succinylcholine, mivacurium, and aspirin (71).…”

The subchronic toxic effects of plant growth promoters in mice

“…E has been shown to inhibit cholinesterase and particularly plasma butyrylcholinesterase, in rats, mice, dogs and humans in vivo. Most of the findings of the studies in literature were on this mechanism of chemical (3,4,19). Moreover, related studies with ethephon were very limited.…”