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

Abstract

Previously we used site-directed mutagenesis, in vitro expression, and molecular modeling to investigate the inactivation of an invertebrate acetylcholinesterase, cholinesterase 2 from amphioxus, by the sulfhydryl reagents 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM). We created the mutants C310A, C466A, C310A/C466A and C310A/F312I to assess the roles of the two cysteines and a proposal that the increased rate of inactivation previously found in an F312I mutant was due to increased access of sulfhydryl reagents to Cys310. Our results indicated that both of the cysteines could be involved in inactivation by sulfhydryl reagents, but that the cysteine near the acyl pocket was more accessible. We speculated that the inactivation of aphid AChEs by sulfhydryl reagents was due to the presence of a cysteine homologous to Cys310 and proposed that this residue could be a target for a specific insecticide. Here we reconsider this proposal.

Selective and Irreversible Inhibitors of Mosquito Acetylcholinesterases for Controlling Malaria and Other Mosquito-Borne Diseases

“…On the other hand, it has been reported that iodoacetamide-containing AChE inhibitors do not bond covalently to Ag AP-AChE [17] . Since iodoacetamide is a commonly used sulfhydryl agent, such a finding would suggest that the active-site Cys residue in Ag AP-AChE is not targetable, a conclusion advanced in a recent reconsideration of this issue [18] . Further questions regarding Cys-targeting inhibitors arise from our own observation that such inhibitors can inhibit the human AChE ( h AChE) in irreversible fashion, although this enzyme has no Cys residue near its active site [16] .…”

Novel and Viable Acetylcholinesterase Target Site for Developing Effective and Environmentally Safe Insecticides

“…This alignment [59] along with site-directed mutagenesis work with lancelet AChE [59] and the previously reported sensitivity of aphid AChEs to sulfhydryl inhibitors [60, 61] collectively led to discussions on whether Cys289 or its equivalent in other aphid AP-AChEs is located in the active site [59] and whether it could be used as a target for developing selective aphidicides [62]. …”

“…A sequence alignment of nine AChE sequences (humans, the Florida lancelet [ Branchiostoma floridae ], the electric ray [ Torpedo californica ], the African malaria mosquito, the northern house mosquito, the greenbug, the cotton aphid, the green peach aphid, and the honeybee) identified a cysteine residue, Cys289 in the greenbug AP-AChE, that is absent in human AChE but conserved in insect and lancelet AChEs [ 59 ]. This alignment [ 59 ] along with site-directed mutagenesis work with lancelet AChE [ 59 ] and the previously reported sensitivity of aphid AChEs to sulfhydryl inhibitors [ 60 , 61 ] collectively led to discussions on whether Cys289 or its equivalent in other aphid AP-AChEs is located in the active site [ 59 ] and whether it could be used as a target for developing selective aphidicides [ 62 ].…”

“…To support the cysteine-targeting hypothesis further, it is necessary to address the reported reservations about targeting the insect-specific cysteine residue [ 62 ]. If the understanding of reference [ 62 ] is correct, the concerns are about (1) the poor reaction rates of two known sulfhydryl agents, 5,5′-dithiobis(2-nitrobenzoic acid) and N -ethylmaleimide, for conjugation with Florida lancelet AP-AChE and (2) an active-site phenylalanine residue that hampers the reactions of these two reagents with the insect-specific cysteine residue. Neither 5,5′-dithiobis(2-nitrobenzoic acid) nor N -ethylmaleimide is designed for insect AP-AChEs, and their reaction rates with the lancelet enzyme and the steric hindrance experienced in the lancelet enzyme appear to be irrelevant to cysteine-targeting insecticides.…”

Thermal denaturation of wild type and mutant recombinant acetylcholinesterase from amphioxus: effects of the temperature of in vitro expression and of reversible inhibitors

“…We found that although both Cys residues could be involved in the inactivation, Cys310, near the top of the gorge was more accessible and more easily reduced by the disulfide reagents, resulting in enzyme inactivation. Many aphids, mosquitoes, and other insects have Cys residues homologous to Cys310 (Pezzementi et al, 2006; Pang, 2006a; Pang, 2006b; Rowland et al, 2008), and are sensitive to reduction by sulfhydryl reagents (Zahavi et al, 1972; Smissart, 1976), raising the possibility of this residue as a target for specific insecticides.…”