Molecular dynamics simulations to investigate the thermal unfolding behaviors of N-carbamyl-D-amino acid amidohydrolase

The thermal unfolding behaviors of N-carbamyl-D-amino acid amidohydrolase (DCase) were investigated by 1 ns molecular dynamics simulations in explicit water with temperature jump technique. Rather than a symmetrical event, the unfolding of the two subunits of the dimeric DCase follows different pathways. Subunit A loses its secondary structure integrity from the outermost layer 1 to the innermost layer 4, whereas subunit B unfolds first from its interior layer 3, where the active site pocket is located. Hence, the active site from subunit B is less stable than that from subunit A. Furthermore, the interfacial hydrophobic region is expanded, implying that the dimeric DCase tends to dissociate into the enzymatically inactive monomeric form. It further indicates that the interior layers lose their secondary structure integrity, resulting in destruction of the active site geometry. Thus, it is recommended to construct several disulfide bonds in the interfacial hydrophobic region to “lock” these two subunits in order to maintain its enzymatically active dimeric form.