A partially unfolded structure of the alkaline-denatured state of pepsin and its implication for stability of the zymogen-derived protein

Pepsin, a gastric aspartic proteinase, is a zymogen-derived protein that un dergoes irreversible alkaline denaturation at pH 6-7. Detailed knowledge of the structure of the alkaline-denatured state is an important step in unde rstanding the mechanism of the formation of the active enzyme. An extensive analysis of the denatured state at pH 8.0 was performed using a variety of techniques including H-1 nuclear magnetic resonance spectroscopy and solut ion X-ray scattering. This analysis indicates that the denatured state unde r these conditions has a compact and globular conformation with a substanti al amount of secondary and tertiary structures. The data suggest that this partially structured species has a highly folded region and a flexible regi on. The NMR measurements suggest that the folded region contains His53 and is located at least partly ill the N-terminal lobe of the protein. The alka line-denatured state experiences a further reversible denaturation step at higher pH or on heating; the midpoints of the unfolding transition are DH 1 1.5 (at 25 degrees C) and 53.1 degrees C (at pH 8.0), respectively. The pre sent findings suggest that the proteolytic processing of pepsinogen has sub stantially modified the ability of the protein to fold, such that its foldi ng process cannot progress beyond the partially folded intermediate of peps in.