A Single Amino Acid Substitution, Gly117His, Confers Phosphotriesterase (Organophosphorus Acid Anhydride Hydrolase) Activity on Human Butyrylcholinesterase

Abstract

The G117H mutant of human butyrylcholinesterase (EC 3.1.1.8) was expressed in Chinese hamster ovary cells. Substitution of Gly 117 with His to make the G117H mutant endowed butyrylcholinesterase with the ability to catalyze the hydrolysis of organophosphate esters. G117H was still able to hydrolyze butyrylthiocholine, benzoylcholine, and o-nitrophenyl butyrate, but in addition it had acquired the ability to hydrolyze the antiglaucoma drug echothiophate and the pesticide paraoxon. Wild-type butyrylcholinesterase was irreversibly inhibited by echothiophate and paraoxon, but G117H regained 100% activity within 2-3 min following reaction with these compounds. On a polyacrylamide gel, the same bands that stained for activity with butyrylthiocholine also stained for activity with echothiophate. G117H is the only enzyme known that hydrolyzes echothiophate. Diethoxyphosphorylated G117H aged with a half-time of 5.5 h, a rate 600 times slower than the rate of hydrolysis. Echothiophate and paraoxon were hydrolyzed with the same kcat of 0.75 min-1. This calculates to a rate acceleration of 100,000-fold for hydrolysis of echothiophate and paraoxon by the G117H mutant compared to the nonenzymatic rate.

Crystal Structures of Human Carboxylesterase 1 in Covalent Complexes with the Chemical Warfare Agents Soman and Tabun,

“…Inspired by the identification of cholinesterase mutants with favorable features in relation to organophosphate binding and hydrolysis (15,17,51,52), the structures of hCE1 in complexes with nerve agents presented here may be useful in developing this enzyme into a protective nerve agent hydrolase. Use of enzymes as a prophylaxis for OP exposure carries significant benefits beyond current chemical treatments.…”

Free Energy Perturbation (FEP) Simulation on the Transition States of Cocaine Hydrolysis Catalyzed by Human Butyrylcholinesterase and Its Mutants

“…Finally, about 20 different naturally occurring mutants of human BChE have been identified, and there is no evidence that these mutants are antigenic. 20 However, the catalytic activity of the native form of this plasma enzyme against naturally occurring (-)-cocaine is low. For example, the plasma half-life time of (-)-cocaine is ∼45-90 min, long enough for cocaine crossing the blood-brain barrier (BBB) to reach central nervous system (CNS) to make its reinforcing effects.…”

Generation of Recombinant Human Ache Op-Scavengers With Extended Circulatory Longevity

“…We have also demonstrated the ability to generate novel enzymes that are more efficient in OP scavenging through the combined effects of improved activity toward the OP-agents together with higher resistance to the aging process Shafferman et al, 1996). Such studies, by us and others (see review in Taylor and Radic, 1994; Hosea et al, 1995;Millard et al, 1995;Hosea et al, 1996;Lockridge et al, 1997), reveal the structural and mechanistic determinants of AChE activity that are essential for the design of true OP hydrolytic activity into the AChE mold. Information regarding the potential diversity of aging mechanisms is essential for further elucidation of the role of AChE active center in these processes, as part of the development of effective treatment for human intoxication by organophosphorus agents, like certain insecticides and nerve agents.…”