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.

Butyrylcholinesterase (BChE) is inhibited by the plant growth regulator (2-chloroethyl)phosphonic acid (ethephon) as observed 25 years ago both in vitro and in vivo in rats and mice and more recently in subchronic studies at low doses with human subjects. The proposed mechanism is phosphorylation of the BChE active site at S198 by ethephon dianion. The present study tests this hypothesis directly using [(33)P]ethephon and recombinant BChE (rBChE) with single amino acid substitutions and further evaluates if BChE is the most sensitive esterase target in vitro and with mice in vivo. [(33)P]Ethephon labels purified rBChE but not enzymatically inactive diethylphosphoryl-rBChE (derivatized at S198 by preincubation with chlorpyrifos oxon) or several other esterases and proteins. Amino acid substitutions that greatly reduce rBChE sensitivity to ethephon are G117H and G117K in the oxyanion hole (which may interfere with hydrogen bonding between glycine-N-H and ethephon dianion) and A328F, A328W, and A328Y (perhaps by impeding access to the active site gorge). Other substitutions that do not affect sensitivity are D70N, D70K, D70G, and E197Q which are not directly involved in the catalytic triad. The effect of pH and buffer composition on inhibition supports the hypothesis that ethephon dianion is the actual phosphorylating agent without activation by divalent cations. Human plasma BChE in vitro and mouse plasma BChE in vitro and in vivo are more sensitive to ethephon than any other esterases detected by butyrylthiocholine or 1-naphthyl acetate hydrolysis in native-PAGE. All mouse liver esterases observed are less sensitive than plasma BChE to ethephon in vitro and in vivo. More than a dozen other esterases examined are 10-100-fold less sensitive than BChE to ethephon. Thus, BChE inhibition continues to be the most sensitive marker of ethephon exposure.