title: Mutation of acetylcholinesterase to enhance oxime-assisted catalytic turnover of methylphosphonates.
abstract: Selected mutagenesis of acetylcholinesterase (AChE; EC 3.1.1.7) may enable one to develop more effective scavenging agents in which AChE itself, in combination with an oxime, will complete a catalytic cycle of hydrolysis of the organophosphate by rapid conjugation followed by enhanced nucleophile-mediated hydrolysis of the phosphonyl enzyme conjugate. Through enlargement of the active site gorge of mouse AChE by mutations Y337A, F295L and F297I, we studied continuous enzymatic degradation of S(P)-cycloheptyl methylphosphonyl thiocholine (S(P)-CHMPTCh) in the presence of HI-6. Continuous hydrolysis of S(P)-CHMPTCh was measured spectrophotometrically from thiocholine released during hydrolysis with DTNB as the thiol reagent. The rates of hydrolysis expressed as moles of formed thiocholine per mole of enzyme per minute were 3.3, 0.69, 0.34 and 0.15min(-1) for F295L/Y337A, Y337A, F297I/Y337A and AChE wild-type, respectively. These rates did not depend on the initial S(P)-CHMPTCh concentration range employed. However, by increasing HI-6 concentrations, the rates approached a limiting value, indicating that oxime reactivation is the rate-limiting step in S(P)-CHMPTCh hydrolysis. Our results confirm that a mixture of a mutant enzyme and an oxime might serve as an in vivo catalytic scavenger of organophosphates.
authors.affiliation: Department of Pharmacology, University of California at San Diego, La Jolla, CA 92093-0636, USA.
authors.forename: Zrinka
authors.initials: Z
authors.lastname: Kovarik
authors.forename: Zoran
authors.initials: Z
authors.lastname: Radić
authors.forename: Harvey A
authors.initials: HA
authors.lastname: Berman
authors.forename: Palmer
authors.initials: P
authors.lastname: Taylor
mesh_terms: D000110:Acetylcholinesterase / Q000737:chemistry / Q000235:genetics; D000818:Animals; D002384:Catalysis; D002800:Cholinesterase Inhibitors / Q000737:chemistry*; D002801:Cholinesterase Reactivators / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D051379:Mice; D015394:Molecular Structure; D009154:Mutation; D009943:Organophosphorus Compounds / Q000737:chemistry*; D010091:Oximes / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013997:Time Factors
chemical_list: D002800:Cholinesterase Inhibitors; D002801:Cholinesterase Reactivators; D009943:Organophosphorus Compounds; D010091:Oximes; D011994:Recombinant Proteins; C032627:methylphosphonic acid; D000110:Acetylcholinesterase