Engineering resistance to ‘aging’ of phosphylated human acetylcholinesterase Role of hydrogen bond network in the active center

Abstract

Recombinant human acetylcholinesterase (HuAChE) and selected mutants (E202Q, Y337A, F,450A) were studied with respect to catalytic activity towards charged and noncharged substrates, phosphylation with organophosphorus (OP) inhibitors and subsequent aging of the OP-conjugates. Amino acid E450, unlike residues E202 and Y337, is not within interaction distance from the active center. Yet, the bimolecular rates of catalysis and phosphylation are 30~100 fold lower for both FA50A and E202Q compared to Y337A or the wild type and in both mutants the resulting OP-conjugates show striking resistance to aging. It is proposed that a hydrogen bond network, that maintains the functional architecture of the active center, involving water molecules and residues E202 and E450, is responsible for the observed behaviour.

Repositioning the Catalytic Triad Aspartic Acid of Haloalkane Dehalogenase: Effects on Stability, Kinetics, and Structure

“…This may lead to conformational perturbations as was observed in phospholipase A 2 (Li & Tsai, 1993;Kumar et al, 1994). The contribution of buried hydrogen-bonding networks to protein stability was also demonstrated in acetylcholinesterase (Ordentlich et al, 1993), barnase (Chen et al, 1993), and lactate dehydrogenase (Nobbs et al, 1994). In addition, the loss of the negative charge in the D260N mutant may have affected the delicate charge balance in the active site and further destabilized the structure of the catalytic site.…”

Electrostatic Attraction by Surface Charge does not Contribute to the Catalytic Efficiency of Acetylcholinesterase

“…Recombinant HuAChEs expressed in 293 cells are secreted into the growth medium as soluble T-type homo-oligomers (composed mainly of dimers; Velan et al, 1991bVelan et al, , 1993. The various secreted AChE polypeptides were quantified by AChE protein determination relying on specific ELISAs (Shafferman et al, 1992a) and by activesite titrations (Ordentlich et al, 1993b) using the organophosphonate MEPQ (Levy and Ashani, 1986). Enzyme quantitations by these two methods were in good agreement (± 10%).…”

Usage potential of acetylcholinesterase as a bioscavenger in organophasphate poisoning

“…[16,17]. Sonrasında fare asetilkolinesterazında yaratılan Glu202Gln mutasyonunun soman ile yaşlan-maya büyük ölçüde dayanıklı olduğu bildirilmiştir [18].…”

“…Hidrofobik etkileşimleri bulunan diğer rezidular ise kolin bağlama bölgesinde bulunan Tyr337 ve Phe338 olup, OPların imidazol halkasını stabilize ederek yaşlanmaya katkıda bulunmaktadır. Bu reziduların alanin ile değiş-tirilmesi sonucu yaşlanmada belirgin yavaşlamalar elde edilmiştir [15,17,19]. Tyr337Ala mutasyonuna ek olarak açil cebinde bulunan, aromatik halkaya sahip Phe295 rezidusunun alifatik yan zincire sahip Leu ile yer değiştirilmesi sonucu fare AChE'ının HI6 aracılı reaktivasyonu yaklaşık 120 kat hızlanmıştır [19].…”