Cu2+-dependent hydrolysis of O-hexyl 2,5-dichlorophenyl phosphoramidate by reptile sera.

This study shows the EDTA-resistant, Ca2+ and Cu2+-dependent hydrolysis of O-hexyl 2,5-dichlorophenyl phosphoramidate (HDCP) compound in reptiles sera determined by spectrophotometry UV/Vis and chiral chromatography. Samples of ten reptile species were incubated with aliquot of 100 or 400 µM HDCP in presence of 100 or 300 µM Cu2+, or 2.5 mM Ca2+ or 5 mM EDTA at 37 °C for 30-60 min. The results shown an activator effect of Cu2+ on HDCP hydrolysis in freshwater turtles sera (Trachemys scripta, Chelydra serpentina and Macrochelys temminckii) because the levels of 2,5-dichlorophenol (DCP; product hydrolysis) were similar (∼37 µM DCP) to chicken serum (positive control group). The marine turtles (Chelonia mydas and Eretmochelys imbricata) and crocodiles (Crocodylusacutus and Crocodylus moreletii) showed ∼50% less HDCPase activity (13-17 µM DCP) compared to the HDCPase activity of the freshwater turtle species. Terrestrial reptile species (snakes and lizards) showed around 25% of activity (7-13 µM DCP) with both copper concentrations. These Cu2+-dependent hydrolysis were stereospecific to R(+)-HDCP (p˂0.05) in the three freshwater turtle species that showed similar hydrolysis to the chicken serum. However, the Ca2+ did not show a significant activating effect on the HDCPase activity (1-8 µM DCP) in any reptile serum. Their hydrolysis levels were very similar to those of EDTA-resistant activity. The present study demonstrates a Cu2+-dependent A-esterase (HDCPase) activity in turtles and points serum albumin as the cuproprotein responsible for this activity, reinforcing its N-terminal sequence (DAEH) as a catalytic center.

Computational study to find the goat serum albumin (GSA) binding site as A-esterase.

A-esterases are a classical term applied to enzymatic activity of the proteins by a mechanism not involving intermediate covalent phosphorylation, but requiring a divalent cation cofactor. Recently, a copper-dependent A-esterase activity has been identified in goat serum albumin (GSA) on the organophosphorus insecticide trichloronate. This hydrolysis was identified ex vivo with spectrophotometry and chromatography techniques. Albumin mechanism of action and catalytic site as Cu2+-dependent A-esterase are still unknown. Therefore, to know the copper bind to albumin is relevant. N-terminal sequence has been reported as the high affinity site for this cation, due to the histidine in position 3. The aim of this work in silico is to explore how occurs this metallic binding and active the esterase catalytic function. The GSA crystallized structure (PDB: 5ORI) was chosen for molecular docking and dynamics. A site-directed docking, for N-terminal site and a blind docking was done with trichloronate as ligand. Root-mean-square deviation and frequency plot was calculated to find the most frequent predicted structure and visualize the amino acids involved in binding site. The affinity energy in the blind docking (-5.80 kcal/mol) is almost twice lower than site-directed docking (-3.81 kcal/mol) and N-terminal amino acids do not appear in the most repeated structure binding site, suggesting that the protein has a site with higher affinity to the trichloronate ligand. His145 could be involved in the binding site as has been reported in previous studies.

Copper(II)-dependent hydrolysis of trichloronate by turkey serum albumin.

Trichloronate is a racemic organophosphonothioate insecticide that induced delayed neuropathic in hens and human. The avian are species with great susceptibility to organophosphorus poisoning due to their low levels of A-esterases. However, a significant copper-dependent A-esterase activity has been identified in chicken and turkey serum. This study aims at characterizing the trichloronate enantiomers hydrolysis by serum and albumin from chicken (CSA) and turkey (TSA) in the presence of copper by chiral chromatography. A significant Cu2+-dependent hydrolysis for both trichloronate enantiomers (38%) was observed in turkey serum and TSA (p < 0.05). The hydrolysis is stereoselective for (-)-trichloronate (p < 0.05). TSA incubation (200 µg) showed residual values of 56 µM and 20 µM of (+)-trichloronate and (-)-trichloronate, respectively; while the chicken serum and CSA presented a slight hydrolysis (1-7%) of both enantiomers. This copper-dependent hydrolysis and stereoselectivity of trichloronate by TSA was proportional to the incubation time. The increase of TSA in the assay (200-3000 µg) in the presence of 100 µM copper did not significantly increase the levels of hydrolysis and stereoselectivity, an opposite effect was observed for turkey serum (100-200 mL), which totally inhibited this copper-dependent activity of both isomers. The present study evidences an A-esterase activity of TSA on a thio form OP compound, which is stereoselective and activated by copper.