Title : Organophosphorus acid anhydride hydrolase activity in human butyrylcholinesterase: synergy results in a somanase - Millard_1998_Biochemistry_37_237
Author(s) : Millard CB , Lockridge O , Broomfield CA
Ref : Biochemistry , 37 :237 , 1998
Abstract :

Organophosphorus acid anhydride (OP) "nerve agents" are rapid, stoichiometric, and essentially irreversible inhibitors of serine hydrolases. By placing a His near the oxyanion hole of human butyrylcholinesterase (BChE), we made an esterase (G117H) that catalyzed the hydrolysis of several OP, including sarin and VX [Millard et al. (1995) Biochemistry 34, 15925-15930]. G117H was limited, however, because it was irreversibly inhibited by pinacolyl methylphosphonofluoridate (soman); soman is among the most toxic synthetic poisons known. This limitation of G117H has been overcome by a new BChE (G117H/E197Q) that combines two engineered features: spontaneous dephosphonylation and slow aging (dealkylation). G117H/E197Q was compared with the single mutants BChE G117H and E197Q. Each retained cholinesterase activity with butyrylthiocholine as substrate, although kcat/Km decreased 11-, 11- or 110-fold for purified G117H, E197Q, or G117H/E197Q, respectively, as compared with wild-type BChE. Only G117H/E197Q catalyzed soman hydrolysis; all four soman stereoisomers as well as sarin and VX were substrates. Phosphonylation and dephosphonylation reactions were stereospecific. Double mutant thermodynamic cycles suggested that the effects of the His and Gln substitutions on phosphonylation were additive for PSCR or PRCR soman, but were cooperative for the PSCS stereoisomer. Dephosphonylation limited overall OP hydrolysis with apparent rate constants of 0.006, 0.077, and 0.128 min-1 for the PR/SCR, PSCS, and PRCS soman stereoisomers, respectively, at pH 7.5, 25 degrees C. We conclude that synergistic protein design converted an archetypal "irreversible inhibitor" into a slow substrate for the target enzyme.

PubMedSearch : Millard_1998_Biochemistry_37_237
PubMedID: 9425044
Gene_locus related to this paper: human-BCHE

Related information

Mutation E197Q_human-BCHE    G117H\/E197Q_human-BCHE    G117H_human-BCHE    G117K_human-BCHE
Inhibitor Sarin    Soman    VX
Substrate Paraoxon
Gene_locus human-BCHE

Citations formats

Millard CB, Lockridge O, Broomfield CA (1998)
Organophosphorus acid anhydride hydrolase activity in human butyrylcholinesterase: synergy results in a somanase
Biochemistry 37 :237

Millard CB, Lockridge O, Broomfield CA (1998)
Biochemistry 37 :237