Naslov
Dissociation constants for oxime binding to free and phosphylated cholinesterases

Autori
Kovarik, Zrinka

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

Izvornik
International Medical Chemical Defense Conference 2005, Program/Abstracts / Szinicz, Ladislaus - München : Bundeswehr Institute of Pharmacology and Toxicology, 2005

Skup
International Medical Chemical Defense Conference 2005

Mjesto i datum
München, Njemačka, 26.04.2005. - 28.04.2005

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
acetylcholinesterase; butyrylcholinesterase; organophosphorus compounds; oxime; inhibition; reactivation

Sažetak
Oximes are reversible inhibitors of acetylcholinesterase (AChE ; EC 3.1.1.7) and butyrylcholinesterase (BChE ; EC 3.1.1.8), but as strong nucleophiles, they are also effective reactivators of phosphylated AChE and BChE. Since a high affinity for binding to the free enzyme does not mean a high oxime efficacy in reactivation, relevant dissociation constants were determined and compared. The mouse recombinant enzymes were inhibited by the pyridinium oximes 2-PAM (2-(hydroxyiminomethyl)-1-methylpyridinium chloride) and HI-6 ((1-(2&#8217; -hydroxyiminomethyl-1'-pyridinium)-3-(4''-carbamoyl-1''-pyridinium)-2-oxapropane dichloride)) to determine enzyme-oxime dissociation constants. Dimethoxy-phosphorylated enzymes (resulting from the inhibition by DDVP ; O, O-dimethyl-O-(2, 2-dichlorovinyl phosphate) were reactivated by 2-PAM and HI-6, and dimethoxy-phosphoryl-enzyme-oxime dissociation constants (KOX), maximum reactivation constants (kmax) and the second order rate constants (kr) were determined. In attempting to determine the amino acid residues within the active site gorge involved in the interaction with the oximes, selective mutants of mouse AChE (Y337A, F295L, F297I) were subjected to inhibition and reactivation, and their affinities were compared with those of AChE w.t. and BChE. The change of binding free energy (&#61508; &#61508; G0mut-wt) was calculated from dissociation constants as a quantitative measure of involvement of the residues in oxime binding to the free or phosphorylated enzyme. Enzyme-oxime dissociation constants (Ki) for the catalytic site of AChE w.t. were 150 and 47&micro ; ; ; ; ; M, of BChE 320 and 23 &micro ; ; ; ; ; M for 2-PAM and HI-6, respectively. Introduced mutations lowered oxime binding affinities for both oximes, and Ki were 590 and 87 &micro ; ; ; ; ; M for Y337A, 650 and 110 &micro ; ; ; ; ; M for F295L/Y337A, and 1700 and 180 &micro ; ; ; ; ; M for F297I/Y337A, for 2-PAM and HI-6, respectively. A high extent of reactivation with HI-6 and 2-PAM was achieved in reactivation of all conjugates. Both oximes reactivated the BChE conjugate at a similar rate (kr(HI-6)=420 min-1M-1 ; kr(2-PAM)=360 min-1M-1), but about 4-fold faster by HI-6 and 1.5-fold faster by 2-PAM than the AChE w.t. conjugate. Up to 10-fold slower reactivation of mutants by both oximes than of AChE w.t. was primarily the result of increased KOX and similar kmax. KOX for mutants was 2-20 fold higher for HI-6 and 1.4&#8211; 3 fold higher for 2-PAM than the respective Ki. Despite introduced mutations in AChE, which correspond to residues found in BChE active site, affinities for the oximes binding to the free or phosphylated enzyme did not approximate BChE affinities. From the calculated change of free energy of binding 2-PAM or HI-6 to the free enzyme, it seems that mutations Y337A and F297I affected binding with a cumulative effect, since the calculated change of energy was nearly doubled for F297I/Y337A relative to the single mutation Y337A, or to F295L/Y337A. Oxime binding to phosphylated enzymes is most stabilized with mutation F295L (&#61508; &#61508; G0mut-wt<0), but probably due to the very slow kmax the mutation did not achieve enhancement in the overall reactivation rate as compared to the wild type enzyme. (Supported by grant No: 22014, Ministry of Science, Education and Sports, Croatia)

Izvorni jezik
Engleski

Znanstvena područja
Kemija

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