Mechanisms of cholinesterase inhibition by inorganic mercury

Abstract

The poorly known mechanism of inhibition of cholinesterases by inorganic mercury (HgCl2) has been studied with a view to using these enzymes as biomarkers or as biological components of biosensors to survey polluted areas. The inhibition of a variety of cholinesterases by HgCl2 was investigated by kinetic studies, X‐ray crystallography, and dynamic light scattering. Our results show that when a free sensitive sulfhydryl group is present in the enzyme, as in Torpedo californica acetylcholinesterase, inhibition is irreversible and follows pseudo‐first‐order kinetics that are completed within 1 h in the micromolar range. When the free sulfhydryl group is not sensitive to mercury (Drosophila melanogaster acetylcholinesterase and human butyrylcholinesterase) or is otherwise absent (Electrophorus electricus acetylcholinesterase), then inhibition occurs in the millimolar range. Inhibition follows a slow binding model, with successive binding of two mercury ions to the enzyme surface. Binding of mercury ions has several consequences: reversible inhibition, enzyme denaturation, and protein aggregation, protecting the enzyme from denaturation. Mercury‐induced inactivation of cholinesterases is thus a rather complex process. Our results indicate that among the various cholinesterases that we have studied, only Torpedo californica acetylcholinesterase is suitable for mercury detection using biosensors, and that a careful study of cholinesterase inhibition in a species is a prerequisite before using it as a biomarker to survey mercury in the environment.

Enzymatic and biochemical characterization of Bungarus sindanus snake venom acetylcholinesterase

“…This may be due to the absence of a free sulfhydryl group. According to Frasco et al (28), when a free sulfhydryl group is absent in the enzyme (Drosophila melonogaster acetylcholinesterase and human serum butyrycholinesterase) inhibition by mercury will occur in the millimolar range, while in the presence of a free sulfhydryl group (Electrophorus electricus), the inhibition will only require a micromolar concentration (28). It is possible that the inhibitory effects of metals on venom acetylcholinesterase are due to the formation of inactive enzyme aggregate (29).…”

Effect of ions on the activity of brain acetylcholinesterase from tropical fish

“…These two regions are mutually responsive to ligand-dependent conformational changes and it was proposed by other authors that occupancy of peripheral site induces movements in the loop which in turn modify the orientation of the key tryptophan residue present in the choline binding site of the active center [23,[38][39][40]. It suggested that conformational alterations from the binding of Hg 2+ to the loop and the other three peripheral mercury-binding sites reported by Frasco et al [10] could allosterically be transmitted to the choline binding locus of the active center interfering in substrate binding and, inversely, the substrate binding to peripheral (substrate inhibition site) and active sites, in increasing concentrations, could hinder Hg 2+ binding to AChE loop since the position of residues in this region undergo a modification becoming more exposed to the solution after peripheral site occupancy [39,40]. The ion would be displaced from the loop and not from the active center therefore mimicking the behavior of a competitive inhibitor.…”

“…Several studies reported inhibition of AChE activity by ions [9][10][11]. AChE activation by Ca 2+ , Mg 2+ , Al 3+ has also been reported [12,13].…”

“…However, Frasco et al [10] reported that no Hg 2+ ion was attached to the anionic subsite (choline binding site or ammonium binding site) of the AChE active center in the three-dimensional structure of the enzyme and therefore could not be a competitive inhibitor. Nevertheless, the effects of Hg 2+ binding could present competitive-like consequences.…”

“…: E. electricus AChE) or only one in a position (not conserved) buried or accessible through the solution but not always capable to react with thiol agents [10,34,35]. Investigations in the binding sites of Hg 2+ to human BChE, observed no mercury bound to sulfhydryl groups in crystal structure and the only free accessible cysteine was persulfured (Cys-S-SH) and not easily susceptible to reduction [10]. Moreover, arsenic was not listed in the second group of Tomlinson but was also regarded as a free -SH ligand.…”