Inactivation of an invertebrate acetylcholinesterase by sulfhydryl reagents: the roles of two cysteines in the catalytic gorge of the enzyme

Abstract

We have used site-directed mutagenesis and molecular modeling to investigate the inactivation of an invertebrate acetylcholinesterase (AChE), ChE2 from amphioxus, by the sulfhydryl reagents 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM), creating various mutants, including C310A and C466A, and the double mutants C310A/C466A and C310A/F312I, to assess the relative roles of the two cysteines and a proposal that the increased rate of inactivation in the F312I mutant is due to increased access to Cys310. Our results suggest that both cysteines may be involved in inactivation by sulfhydryl reagents, but that the cysteine in the vicinity of the acyl pocket is more accessible. We speculate that the inactivation of aphid AChEs by sulfhydryl reagents is due to the presence of a cysteine homologous to Cys310. We also investigated the effects of various reversible cholinergic ligands, which bind to different subsites of the active site of the enzyme, on the rate of inactivation by DTNB of wild type ChE2 and ChE2 F312I. For the most part the inhibitors protect the enzymes from inactivation by DTNB. However, a notable exception is the peripheral site ligand propidium, which accelerates inactivation in the wild type ChE2, but retards inactivation in the F312I mutant. We propose that these opposing effects are the result of an altered allosteric signal transduction mechanism in the F312I mutant compared to the wild type ChE2.

Inactivation of an invertebrate acetylcholinesterase by sulfhydryl reagents: A reconsideration of the implications for insecticide design

“…The k i values of DTNB and NEM for inactivation of wild type ChE2 are 19.77 and 11.08 M −1 min −1 , respectively (Pezzementi et al, 2006). In contrast, the k i values for the carbamate and organophosphate insecticides propoxur, aldicarb, paraoxon, and dichlorvos range from 1.03-4.89 × 10 5 , approximately 5,000 to 50,000 times greater (Pezzementi et al, 2003).…”

“…We propose that the inactivation rates of wild type amphioxus ChE2 by sulfhydryl reagents are so slow because it is difficult for the reagents to gain access to the acyl pocket, where the susceptible cysteine (Cys310) is located. In our original alignment, we did not show both of the phenylalanines of the acyl pockets of the susceptible invertebrate AChEs, but only one, corresponding to Phe290 in Torpedo AChE (Pezzementi et al, 2006). Such a presentation could have been interpreted to mean that invertebrate acyl pockets are defined by a single phenylalanine, making the cysteine accessible to reduction by sulfhydryl reagents.…”

“…Given the sensitivity of aphid AChE to inactivation by sulfhydryl reagents, Smissaert (1976) raised the possibility of whether one could design "specific aphidical thiol reagents." Indeed, we proposed that a high affinity bifunctional cholinergic reagent that reacts transiently with the active site serine, and thus becomes inactivated and non-toxic as a traditional covalent AChE inhibitor, but reacts irreversibly with the acyl pocket cysteine could be a candidate for a selective aphidicide or insecticide (Pezzementi et al, 2006). Pang has more recently made similar proposals, but with a reversible inhibitor, and extending the approach to other invertebrates (2006,2006).…”

“…They concluded, "acetylcholinesterase from aphids contains an essential SH group located at the active site serine or at an area greatly influencing this site." We proposed that the sensitivity of aphid AChE to inactivation by sulfhydryl reagents was due to the presence of a cysteine homologous to Cys310 in the acyl pocket of ChE2 (Pezzementi et al, 2006). At that time, we also proposed that the aphid AChEs containing the conserved cysteine, Myzus persicae, Aphis gossypii, and Schizapus gramium, (Gao et al, 2002;Li and Han, 2002;Nabeshima et al, 2003), as well as the AChEs from the mosquitoes Anopheles gambia and Culex pipiens (Weill et al, 2002), and the honeybee Apis mellifera (Shapira et al, 2001), which also possess this cysteine, should be sensitive to sulfhydryl reagents.…”

“…We then conducted a more detailed study of the inactivation of ChE2 by the sulfhydryl reagents DTNB and N-ethylmaleimide (NEM), using site-directed mutagenesis, in vitro expression, and molecular modeling to investigate the inactivation of wild type ChE2, the site-directed mutants C310A, C466A, and F312I, and the double mutants C310A/C466A and C310A/F312I, to assess the relative roles of the two cysteines (Pezzementi et al, 2006). Our results suggested that both cysteines were involved in inactivation by sulfhydryl reagents, but that the cysteine in the acyl pocket was more accessible.…”