title: A partially unfolded structure of the alkaline-denatured state of pepsin and its implication for stability of the zymogen-derived protein.
abstract: Pepsin, a gastric aspartic proteinase, is a zymogen-derived protein that undergoes irreversible alkaline denaturation at pH 6-7. Detailed knowledge of the structure of the alkaline-denatured state is an important step in understanding 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 (1)H nuclear magnetic resonance spectroscopy and solution X-ray scattering. This analysis indicates that the denatured state under these conditions has a compact and globular conformation with a substantial amount of secondary and tertiary structures. The data suggest that this partially structured species has a highly folded region and a flexible region. The NMR measurements suggest that the folded region contains His53 and is located at least partly in the N-terminal lobe of the protein. The alkaline-denatured state experiences a further reversible denaturation step at higher pH or on heating; the midpoints of the unfolding transition are pH 11.5 (at 25 degrees C) and 53.1 degrees C (at pH 8.0), respectively. The present findings suggest that the proteolytic processing of pepsinogen has substantially modified the ability of the protein to fold, such that its folding process cannot progress beyond the partially folded intermediate of pepsin.
authors.affiliation: Center for Brain Experiment, National Institute for Physiological Sciences, Okazaki 444-8585, Japan. konno@nips.ac.jp
authors.forename: T
authors.initials: T
authors.lastname: Konno
authors.forename: Y O
authors.initials: YO
authors.lastname: Kamatari
authors.forename: N
authors.initials: N
authors.lastname: Tanaka
authors.forename: H
authors.initials: H
authors.lastname: Kamikubo
authors.forename: C M
authors.initials: CM
authors.lastname: Dobson
authors.forename: K
authors.initials: K
authors.lastname: Nagayama
mesh_terms: D000477:Alkylating Agents; D000818:Animals; D004792:Enzyme Precursors; D004795:Enzyme Stability; D010434:Pepsin A / Q000737:chemistry* / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding
chemical_list: D000477:Alkylating Agents; D004792:Enzyme Precursors; D010434:Pepsin A