title: Conformational instability of the N- and C-terminal lobes of porcine pepsin in neutral and alkaline solutions.
abstract: Pepsin contains, in a single chain, two conformationally homologous lobes that are thought to have been evolutionarily derived by gene duplication and fusion. We have demonstrated that the individual recombinant lobes are capable of independent folding and reconstitution into a two-chain pepsin or a two-chain pepsinogen (Lin, X., et al., 1992, J. Biol. Chem. 267, 17257-17263). Pepsin spontaneously inactivates in neutral or alkaline solutions. We have shown in this study that the enzymic activity of the alkaline-inactivated pepsin was regenerated by the addition of the recombinant N-terminal lobe but not by the C-terminal lobe. These results indicate that alkaline inactivation of pepsin is due to a selective denaturation of its N-terminal lobe. A complex between recombinant N-terminal lobe of pepsinogen and alkaline-denatured pepsin has been isolated. This complex is structurally similar to a two-chain pepsinogen, but it contains an extension of a denatured pepsin N-terminal lobe. Acidification of the complex is accompanied by a cleavage in the pro region and proteolysis of the denatured N-terminal lobe. The structural components that are responsible for the alkaline instability of the N-terminal lobe are likely to be carboxyl groups with abnormally high pKa values. The electrostatic potentials of 23 net carboxyl groups in the N-terminal domain (as compared to 19 in the C-terminal domain) of pepsin were calculated based on the energetics of interacting charges in the tertiary structure of the domain. The groups most probably causing the alkaline denaturation are Asp11, Asp159, Glu4, Glu13, and Asp118.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Protein Studies Program, Oklahoma Medical Research Foundation, Oklahoma City.
authors.forename: X
authors.initials: X
authors.lastname: Lin
authors.forename: J A
authors.initials: JA
authors.lastname: Loy
authors.forename: F
authors.initials: F
authors.lastname: Sussman
authors.forename: J
authors.initials: J
authors.lastname: Tang
mesh_terms: D000818:Animals; D004563:Electrochemistry; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D010434:Pepsin A / Q000737:chemistry* / Q000378:metabolism; D010435:Pepsinogens / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry; D012996:Solutions; D013552:Swine
chemical_list: D010435:Pepsinogens; D011994:Recombinant Proteins; D012996:Solutions; D010434:Pepsin A