Newly Observed Spontaneous Activation of Ethephon as a Butyrylcholinesterase Inhibitor

Abstract

The plant growth regulator ethephon (2-chloroethylphosphonic acid) inhibits human butyrylcholinesterase (BChE) by making a covalent adduct on the active site serine 198. Our goal was to extend earlier studies on ethephon inhibition. Addition of freshly prepared ethephon to BChE in buffered medium, at pH 7.0 and 22 °C, resulted in no inhibition initially. However, inhibition developed progressively over 60 min of incubation. Preincubation of ethephon in pH 7-9 buffers increased its initial inhibitory potency. These observations indicated that ethephon itself was not the inhibitor. About 3% of the initial ethephon could be trapped as a BChE adduct. Mass spectral analysis of the active site peptide from inhibited BChE showed that the inhibitor added a mass of 108 Da to the active site serine on peptide FGES198AGAAS. This result rules out a previous hypothesis that ethephon adds HPO3 to BChE (added mass of 80 Da). To accommodate these observations, we propose that in aqueous media at neutral to slightly alkaline pH about 3% of the ethephon is converted (t1/2 = 9.9 h at pH 7.0) into a cyclic oxaphosphetane which is the actual BChE inhibitor forming the 2-hydroxyethylphosphonate adduct on BChE at Ser198 while about 97% of the ethephon breaks down to ethylene (t1/2 = 11-48 h at pH 7.0) which is responsible for plant growth regulation.

Characterization of the Transient Oxaphosphetane BChE Inhibitor Formed from Spontaneously Activated Ethephon

“…1,2 Even more surprising, ethephon is not direct-acting but undergoes spontaneous activation in aqueous alkaline solutions to the activated BChE inhibitor. 1 The ethephon-inhibited BChE is covalently derivatized with a transfer of the phosphorus from ethephon.…”

“…1,2 Even more surprising, ethephon is not direct-acting but undergoes spontaneous activation in aqueous alkaline solutions to the activated BChE inhibitor. 1 The ethephon-inhibited BChE is covalently derivatized with a transfer of the phosphorus from ethephon. 2 The active site Ser198 contains a mass adduct of 108 Da thought to be the 2-hydroxyethylphosphonate ( C ) derivative which forms from the ring-opening of 2-oxo-2-hydroxy-1,2-oxaphosphetane (the proposed activated inhibitor, B ).…”

“…2 The active site Ser198 contains a mass adduct of 108 Da thought to be the 2-hydroxyethylphosphonate ( C ) derivative which forms from the ring-opening of 2-oxo-2-hydroxy-1,2-oxaphosphetane (the proposed activated inhibitor, B ). 1 Degradation of B in aqueous media in the absence of BChE by electrophilic attack on the phosphorus by water or phosphate would produce the hydrolysis product C or the phospholysis product 2-hydroxyethylphosphonyl phosphate ( E ), respectively. The reported 3–5 mol % trapping of B by BChE 1 suggests that the formation of B likely competes with the formation of phosphate ( D ) and ethylene from A .…”

“…1 Degradation of B in aqueous media in the absence of BChE by electrophilic attack on the phosphorus by water or phosphate would produce the hydrolysis product C or the phospholysis product 2-hydroxyethylphosphonyl phosphate ( E ), respectively. The reported 3–5 mol % trapping of B by BChE 1 suggests that the formation of B likely competes with the formation of phosphate ( D ) and ethylene from A . All reactions are expected to be irreversible.…”

“…Aliquots were diluted to 0.11 mM and assayed as reported 1 for BChE inhibitory potency immediately after preparation and after 20 h. Preincubation of ethephon at high concentration in phosphate or carbonate buffer gave nearly the same activation to a BChE inhibitor (Supporting Information). …”