Investigating the structural influence of surface mutations on acetylcholinesterase inhibition by organophosphorus compounds and oxime reactivation

https://doi.org/10.1016/j.cbi.2010.03.050Get rights and content

Abstract

Organophosphates (OPs) exert their toxicity by inhibiting primarily acetylcholinesterase (AChE) and to a lesser extent butyrylcholinesterase (BChE). Binary mixtures of mammalian AChE and oximes of varying structure have been recently considered for treatment of OP poisoning as catalytic bioscavengers. In this study wild type human AChE and human AChE with residue mutations D134H, D134H_E202Q and D134H_F338A were characterized and investigated for inhibition by OPs and consequent oxime reactivation of phosphylated enzymes. The rationale for selecting these substitution positions was based on D134H being a naturally occurring single nucleotide polymorphism (SNP) in humans and that E202Q and F338A mutations slow aging of OP inhibited AChEs.

Inhibition of D134H by paraoxon and analogues of cyclosarin was 2–8 times slower than inhibition of wild type (wt), while reactivation of the paraoxon inhibited enzyme by 2PAM was 6 times faster. Both inhibition and reactivation of D134H_E202Q and D134H_F338A double mutants were up to two orders of magnitude slower than the wt indicating that introduction of the active center substitutions abolished fully the effect of the peripherally located D134H. These results indicate that selected residues outside the active center influence inhibition, reactivation and catalysis rates through longer range interactions.

Introduction

To minimize the toxicity of organophosphate (OP) inhibitors, binary mixtures of mammalian AChE and oximes of varying structure were recently considered as catalytic bioscavengers promoting the catalysis of the OP in plasma before it reacts with the target site in skeletal muscle or the nervous system [1]. Our goal is to generate site-specific mutations in AChE protein sequence in order to convert human AChE into catalytic scavenger that, when coupled with an oxime, will inactivate multiple OP molecules per one molecule of scavenger.

Human AChE wild type and D132H, D134H_E202Q, and D134H_F338A mutants were characterized and investigated for inhibition by OPs (an insecticide and nerve agent analogues; Fig. 1) and reactivation of the phosphylated enzymes with 2PAM and HI6. The rationale for selecting these substitution positions was based on D134H being a naturally occurring SNP in humans and that E202Q and F338A mutations slow aging of OP inhibited AChEs [2], [3].

Section snippets

Materials and methods

Nerve agent analogues of cyclosarin (methylphosphonic acid 3-cyano-4-methyl-2-oxo-2H-coumarin-7-yl ester cyclohexyl ester), VX (methylphosphonic acid 3-cyano-4-methyl-2-oxo-2H-coumarin-7-yl ester ethyl ester), soman (methylphosphonic acid 3-cyano-4-methyl-2-oxo-2H-coumarin-7-yl ester pinacolyl ester) were synthesized as previously described [4], as well as SP-Cycloheptyl methyl phosphonyl thiocholine (SPCHMP) and RP-Isopropyl methyl phosphonyl thiocholine (RPIPMP) [5]. O,O-Diethyl

Results and discussion

Inhibition of human D134H with paraoxon and an analogue of cyclosarin was 2–8 times slower than that of human wt (Table 1), whereas inhibition by soman and analogues of VX was not affected by D134H substitution. Introduction of additional substitutions in two double mutants further slowed inhibition up to two orders of magnitude.

2PAM reactivation of paraoxon inhibited human D134H is, on the other hand, 6 times faster than human wt (Table 2). Our previous study indicated that differential

Conflict of interest statement

None declared.

Acknowledgements

This study was supported by fellowships from IIE and The Fulbright Program, and the Scientific and Technical Research Council of Turkey to Tuba Kucukkilinc and by NIH NINDS U01NS58046 to Palmer Taylor.

Cited by (8)

  • In silico studies on the role of mutant Y337A to reactivate tabun inhibited mAChE with K048

    2015, Chemico-Biological Interactions
    Citation Excerpt :

    It has been perceived that the modification of the active center through mutagenesis can augment the catalytic hydrolysis of OP-inhibited AChE [18]. Such studies have been performed with mutant enzyme and oxime drug to achieve higher efficiency to restore free enzyme from OP poisoning [19–23]. The tabun-mutant mAChE(Y337A), however is not always very productive in terms of the reactivation process compared to their wild-type.

  • Probing the role of amino acids in oxime-mediated reactivation of nerve agent-inhibited human acetylcholinesterase

    2015, Toxicology in Vitro
    Citation Excerpt :

    Taylor et al. (1999) found that by mutating Phe to smaller aliphatic residues at positions 295 and 297, reactivation was enhanced 10- to 20-fold because this allowed greater access of the oxime. Küçükkilinç et al. (2010) showed that double mutants of Hu AChE, which contain substitution of an active center residue besides the peripheral site residue D134 had 20 times slower inhibition by paraoxon and similar magnitude of slowdown in 2-PAM reactivation of the inhibited enzyme. Cochran et al. (2011) designed a Hu AChE Y337A/F338A double mutant that showed enhanced reactivation rates over wild-type AChE and displayed no apparent aging when analogs of soman, sarin and VX were used as OP inhibitors.

  • Why has butyrylcholinesterase been retained? Structural and functional diversification in a duplicated gene

    2012, Neurochemistry International
    Citation Excerpt :

    Seven others (Y33, G115, A201, L330, R386, G390, and F446) are second shell residues. The role of other residues in influencing catalysis is not understood; however, in AChE, it has been shown that the mutation of residues outside the active centre, on the surface of the molecule, adversely influences catalytic function (Küçükkilinç et al., 2010). In view of the close similarity in structure between AChE and BChE, it is likely that the same occurs in the latter.

  • Oxime-assisted acetylcholinesterase catalytic scavengers of organophosphates that resist aging

    2011, Journal of Biological Chemistry
    Citation Excerpt :

    The naturally occurring human AChE single nucleotide polymorphism, D134H (24), when combined with the choline-binding site mutations (Y337A and F338A), did not exhibit different kinetic characteristics from the corresponding single mutants. As noted previously, mutations outside of the active center gorge could affect catalysis through long range interactions (25). To identify suitable AChE oxime pairs for catalytic bio-scavenging, reactivation rates for each mutant OP-hAChE conjugate were determined using HI-6 and 2-PAM (supplemental Fig. S1).

View all citing articles on Scopus
View full text