Mercury(II) binds to both of chymotrypsin's histidines, causing inhibition followed by irreversible denaturation/aggregation

Abstract

The toxicity of mercury is often attributed to its tight binding to cysteine thiolate anions in vital enzymes. To test our hypothesis that Hg(II) binding to histidine could be a significant factor in mercury's toxic effects, we studied the enzyme chymotrypsin, which lacks free cysteine thiols; we found that chymotrypsin is not only inhibited, but also denatured by Hg(II). We followed the aggregation of denatured enzyme by the increase in visible absorbance due to light scattering. Hg(II)-induced chymotrypsin precipitation increased dramatically above pH 6.5, and free imidazole inhibited this precipitation, implicating histidine-Hg(II) binding in the process of chymotrypsin denaturation/aggregation. Diethylpyrocarbonate (DEPC) blocked chymotrypsin's two histidines (his and his ) quickly and completely, with an IC of 35 ± 6 µM. DEPC at 350 µM reduced the hydrolytic activity of chymotrypsin by 90%, suggesting that low concentrations of DEPC react with his at the active site catalytic triad; furthermore, DEPC below 400 µM enhanced the Hg(II)-induced precipitation of chymotrypsin. We conclude that his reacts readily with DEPC, causing enzyme inhibition and enhancement of Hg(II)-induced aggregation. Above 500 µM, DEPC inhibited Hg(II)-induced precipitation, and [DEPC] >2.5 mM completely protected chymotrypsin against precipitation. This suggests that his reacts less readily with DEPC, and that chymotrypsin denaturation is caused by Hg(II) binding specifically to the his residue. Finally, we show that Hg(II)-histidine binding may trigger hemoglobin aggregation as well. Because of results with these two enzymes, we suggest that metal-histidine binding may be key to understanding all heavy metal-induced protein aggregation.

Mercury pollution in vegetables, grains and soils from areas surrounding coal-fired power plants

“…However, studies have shown that cooking reduced mercury contents in mushroom by 10% and had no effect on the mercury contents in fish 45 46 47 . These studies suggest that cooking-mediated reductions in mercury content in food may be minimal, consistent with the reports that inorganic mercury and methyl mercury irreversibly bind to cellular components such as thiol-containing proteins in cells 17 48 .…”

New Insights into Alterations in PL Proteins Affecting Their Binding to DNA after Exposure of Mytilus galloprovincialis to Mercury—A Possible Risk to Sperm Chromatin Structure?

“…Some hydrophobic residues such as Cys, Ala and Gly undergo much more conformational adaptations at the interface than others [ 35 ], and the two latter amino acids are particularly abundant in PL proteins. As is well-known, mercury is capable of binding cysteines [ 28 ] and histidines [ 36 ]. The binding of mercury ions to these amino acids may underlie the conformational changes we observed from the fluorescence analyses, but we cannot exclude that mercury could also be able to bind other amino acids in which PL proteins are richer, such as glycine and alanine [ 37 ].…”

“…Some studies have indicated that Hg binds histidines. For example, Stratton et al 2016 [ 36 ] found that Hg binds two histidines of chymotrypsin, inducing aggregation of this protein. Secondary Hg 2+ -binding sites have been suggested to also contain histidines in alpha-lactalbumin [ 53 ].…”

Novel Insights into Mercury Effects on Hemoglobin and Membrane Proteins in Human Erythrocytes

“…Additional Hg-binding sites cannot be excluded, likely enhancing the aggregation process, including histidine residues. In this respect, the Hg-induced chymotrypsin aggregate formation is reported to be mediated by Hg 2+ –histidine interaction [ 51 ].…”