engleski

Mutation of acetylcholinesterase to enhance oxime assisted catalytic turnover of methylphosphonates

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 SP-cycloheptyl methylphosphonyl thiocholine (SP-CHMPTCh) in the presence of HI-6. Continuous hydrolysis of SP-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 0.15 min-1 for F295L/Y337A, Y337A, F297I/Y337A and AChE wild-type, respectively. These rates did not depend on the initial SP-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 SP-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.

acetylcholinesterase; reactivation; inhibition; organophosphorus compounds; oxime

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