SDS-induced phenoloxidase activity involves structural perturbation of the N-terminal domain in hemocyanin from Indian horseshoe crab- Tachypleus gigas: Kinetics, thermodynamics, and molecular dynamics simulation studies with catecholamines

Abstract

Hemocyanin exhibits phenoloxidase activity apart from functioning as an oxygen transporter owing to its di-copper active site that shares high structural similarities with phenoloxidases. However, the intrinsic phenoloxidase activity of the arthropod hemocyanin is quite low, as the domain containing the active site is flanked by two additional domains. Partial physical disruption of the protein conformation could enhance the phenoloxidase activity of hemocyanin. Towards this, we have activated the native Tachypleus gigas hemocyanin (TgH) with SDS micelles and have demonstrated distinct perturbations in the N-terminal domain of TgH in the presence of SDS micelles through molecular dynamics simulation. Phenoloxidase assay revealed that native TgH lacks intrinsic phenoloxidase activity with catecholamines, but exhibited manifold enhancement in the presence of 4.5 mM SDS. The kinetic study showed that TgH exhibits a higher affinity towards dopamine, with a Km value of 0.262 mM among the catecholamines. Thermodynamic studies revealed that SDS micelles facilitate the interaction between TgH and catecholamines. Further, molecular docking showed that the N-terminal domain of TgH specifically regulates the substrate access to the active site, which was validated through simulation studies where a distinct displacement in the N-terminal was observed in the presence of SDS micelles.