Naslov
Acetylcholinesterase mutant - oxime-assisted catalytic scavengers of organophosphonates

Autori
Kovarik, Zrinka ; Radić, Zoran ; Simeon, Vera ; Reiner, Elsa ; Taylor, Palmer

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
VIII International Meeting on Cholinesterases: Program and Abstracts / Talesa, Vincenzo N. ; Antognelli, Cinzia - Perugia : Universita degli Studi di Perugia, 2004, 22-22

Skup
VIII International Meeting on Cholinesterases

Mjesto i datum
Perugia, Italija, 26.09.2004. - 30.09.2004

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
acetylcholinesterase; organophosphate; oxime; detoxification

Sažetak
Mutagenesis of acetylcholinesterase (AChE ; EC 3.1.1.7) should 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 inhibition followed by rapid nucleophile-mediated hydrolysis of the phosphonyl enzyme conjugate. We enlarged the active site gorge of mouse AChE with mutations Y337A, F295L and F297I, and studied the reactivation of phosphonylated mutants by the oxime HI-6, as well as the continuous enzymatic hydrolysis of the SP-cycloheptyl methylphosphonyl thiocholine (SP-CHMPTCh) in the presence of HI-6. The mutants reacted rapidly with SP-CHMPTCh, and rates of HI-6 reactivation were enhanced up to 130-fold. Continuous hydrolysis of SP-CHMPTCh was measured spectrophotometrically by determining the thiocholine released during hydrolysis with DTNB as the thiol reagent. The medium contained 0.5  M enzyme, 20  M SP-CHMPTCh, 1 mM HI-6 and 0.33 mM DTNB in 0.1 M phosphate buffer, pH 7.4, at 22  C. Under these experimental conditions, the increase in thiocholine concentration was linear with time until hydrolysis was completed. The rates of hydrolysis expressed as moles of released thiocholine per mole of enzyme per minute reaction time were 3.3, 0.69, 0.34 and 0.15 for F295L/Y337A, Y337A, F297I/Y337A and AChE wild-type, respectively. These rates did not depend on the initial SP-CHMPTCh concentration. However, by increasing the HI-6 concentration the rates approached a limiting value, indicating that oxime reactivation is probably the rate-limiting step in SP-CHMPTCh hydrolysis. These results confirm that a mixture of a mutant enzyme and an oxime might serve as a catalytic scavenger in protection and treatment of organophosphate exposure. Variations in oxime structure and further refinements of AChE mutations should improve the catalytic potential of the scavenger. (Supported by DAMD 17-02-2-25).

Izvorni jezik
Engleski

Znanstvena područja
Kemija

POVEZANOST RADA