Naslov
Choline binding site mutations improve HI-6 assisted reactivation of the VX-acetylcholinesterase conjugate

Autori
Maček Hrvat, Nikolina ; Radić, Zoran ; Taylor, Palmer ; Kovarik, Zrinka

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

Izvornik
Book of Abstracts of the Congress of the Croatian Society of Biochemistry and Molecular Biology "The Interplay of Biomolecules", HDBMB 2014 / Katalinić, M. ; Kovarik, Z. - Zagreb : The Croatian Society of Biochemistry and Molecular Biology, 2014

ISBN
978-953-95551-5-1

Skup
The Congress of the Croatian Society of Biochemistry and Molecular Biology "The Interplay of Biomolecules", HDBMB 2014

Mjesto i datum
Zadar, Hrvatska, 24.09.2014. - 27.09.2014

Vrsta sudjelovanja
Poster

Vrsta recenzije
Domaća recenzija

Ključne riječi
choline binding site; Y337A/F338A; pseudo-catalytic bioscavenger

Sažetak
Nerve agent organophosphates (OPs) represent a threatening means of potential terrorism due to their inhibition of acetylcholinesterase (AChE, EC 3.1.1.7), which can lead to death. AChE inhibition can, among other solutions, be counteracted by administering purified human AChE mutants to OP exposed individuals. These mutant enzymes were designed with the aim to accelerate reactivation and press the fast reactivation of the OP and AChE catalytic serine conjugates. Mutants, when combined with oxime reactivators, could act as pseudo-catalytic bioscavengers, degrading OPs with a turnover before phosphylating native endogenous AChE. The AChE active site is a gorge composed of catalytic triade, oxyanion whole, choline binding site, acyl pocket, and peripheral site. We focused on the choline-binding site which has a role in binding the choline moiety of the substrate. HI-6 is a very effective reactivator of VX-inhibited AChE so we investigated the effect of the introduced mutations by in vitro kinetic experiments using HI-6. Even though Y337A was 4 times more quickly inhibited by VX and the Y337A-VX conjugate exhibited a slightly higher binding affinity for HI-6 than wt or Y337A/F338A, it was evident that the Y337A/F338A mutation increased the rate of nucleophilic displacement of the phosphonyl-moiety from the active site serine (k+2) for 5.5 and 13 fold compared to wt AChE and Y337A, respectively. These results singled out the double mutant as a potential pseudo-catalytic bioscavenger in cases of VX poisoning. Therefore, to test the bioscavenger potential of Y337A/F338A in more realistic conditions, we performed ex vivo experiments. Hydrolysis of VX was followed in human whole blood (hWB) or hWB supplemented with Y337A or Y337A/F338A ; inhibited by tenfold higher VX concentration and subsequently treated with 1 mM HI-6. We observed that 95 % of total cholinesterase (ChE) activity was restored within 15 min when supplementing with Y337A/F338A and in terms of observed first order reactivation rate (kobs), three times faster than in the case of supplementing hWB with Y337A or using only hWB when just 50 % of total ChE activity was recovered. Consequently, we conclude that the joint influence of the F338 and Y337 mutations of the choline binding site has a key role in effective reactivation and that Y337A/F338A mutant could act as a pseudo-catalytic bioscavenger candidate in VX exposure. Supported by the CounterACT Program, NIH OD and NINDS (Grants U01 NS058046 and R21NS072086).

Izvorni jezik
Engleski

Znanstvena područja
Kemija

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