Rapid unfolding of pig pancreas α-amylase: Kinetics, activity and structure evolution

alpha-Amylase plays an important role in food processing and in-vivo digestion. Many biological functions of alpha-amylase are affected by unfolding. The pre-steady-state rapid unfolding kinetics of alpha-amylase remains unknown. In this study, the rapid unfolding kinetics of porcine pancreatic alpha-amylase (PPA) with guanidine hydrochloride (GdmHCl) were investigated by stopped-flow spectroscopy. Structural characterization of PPA by fluorescence spectroscopy, and molecular dynamics simulation showed that the unfolding process of PPA might start from the internal active center, where the beta-sheet structure was destroyed, followed by the exposure of hydrophobic amino acid residues. Further research revealed that GdmHCl denaturized PPA not by complexing with PPA. The surrounding H-bond network of water was changed by GdmHCl. This research improves our understanding of the unfolding kinetics of the PPA on the microsecond scale. It also provides the evidence experimentally of the surrounding water contribution to protein denaturization.

Automated summary

This study investigated the rapid unfolding kinetics of porcine pancreatic alpha-amylase (PPA) with guanidine hydrochloride (GdmHCl) using stopped-flow spectroscopy. The results showed that the unfolding process of PPA may start from the internal active center, leading to the exposure of hydrophobic amino acid residues. Further research revealed that GdmHCl denaturized PPA not by complexing with PPA, but by changing the surrounding H-bond network of water.