Chemically Denatured Structures of Porcine Pepsin using Small-Angle X-ray Scattering

Porcine pepsin is a gastric aspartic proteinase that reportedly plays a pivotal role in the digestive process of many vertebrates. We have investigated the three-dimensional (3D) structure and conformational transition of porcine pepsin in solution over a wide range of denaturant urea concentrations (0–10 M) using Raman spectroscopy and small-angle X-ray scattering. Furthermore, 3D GASBOR ab initio structural models, which provide an adequate conformational description of pepsin under varying denatured conditions, were successfully constructed. It was shown that pepsin molecules retain native conformation at 0–5 M urea, undergo partial denaturation at 6 M urea, and display a strongly unfolded conformation at 7–10 M urea. According to the resulting GASBOR solution models, we identified an intermediate pepsin conformation that was dominant during the early stage of denaturation. We believe that the structural evidence presented here provides useful insights into the relationship between enzymatic activity and conformation of porcine pepsin at different states of denaturation.

Residues of acidic chitinase cause chitinolytic activity degrading chitosan in porcine pepsin preparations

Commercially available porcine pepsin preparations have been used for the production of chitooligosaccharides with various biomedical activities. However, the origin of this activity is not well understood. Here we show that the chitosan-degrading activity is conferred by residues with chitinolytic activity of truncated forms of acidic chitinase (Chia) persisting in the pepsin preparation. Chia is an acid-stable and pepsin-resistant enzyme that degrades chitin to produce N-acetyl-D-glucosamine dimer. We found that Chia can be truncated by pepsin under stomach-like conditions while maintaining its enzymatic activity. Similarly to the full-length protein, truncated Chia as well as the pepsin preparations digested chitosan with different degrees of deacetylation (DD: 69–84%) with comparable degradation products. The efficiency was DD-dependent with a marked decrease with higher DD, indicating that the chitosan-degrading activity in the pepsin preparation is due to the chitinolytic activity rather than chitosanolytic activity. We suggest that natural or recombinant porcine Chia are suitable for producing chitooligosaccharides for biomedical purposes.

Enzyme-catalyzed domino reaction: efficient construction of spirocyclic oxindole skeleton using porcine pepsin

Pepsin from porcine gastric mucosa was used as a catalyst in the domino Knoevenagel/Michael/Michael reaction for the synthesis of spirooxindoles.