CONFORMATIONAL INSTABILITY OF THE N-TERMINAL AND C-TERMINAL LOBES OF PORCINE PEPSIN IN NEUTRAL AND ALKALINE-SOLUTIONS

Pepsin contains, in a single chain, two conformationally homologous lo bes that are thought to have been evolutionarily derived by gene dupli cation and fusion. We have demonstrated that the individual recombinan t lobes are capable of independent folding and reconstitution into a t wo-chain pepsin or a two-chain pepsinogen (Lin, X., et al., 1992, J. B iol. Chem. 267, 17257-17263). Pepsin spontaneously inactivates in neut ral or alkaline solutions. We have shown in this study that the enzymi c activity of the alkaline-inactivated pepsin was regenerated by the a ddition of the recombinant N-terminal lobe but not by the C-terminal l obe. These results indicate that alkaline inactivation of pepsin is du e to a selective denaturation of its N-terminal lobe. A complex betwee n recombinant N-terminal lobe of pepsinogen and alkaline-denatured pep sin has been isolated. This complex is structurally similar to a two-c hain pepsinogen, but it contains an extension of a denatured pepsin N- terminal lobe. Acidification of the complex is accompanied by a cleava ge in the pro region and proteolysis of the denatured N-terminal lobe. The structural components that are responsible for the alkaline insta bility of the N-terminal lobe are likely to be carboxyl groups with ab normally high pK(a) values. The electrostatic potentials of 23 net car boxyl groups in the N-terminal domain (as compared to 19 in the C-term inal domain) of pepsin were calculated based on the energetics of inte racting charges in the tertiary structure of the domain. The groups mo st probably causing the alkaline denaturation are Asp11, Asp159, Glu4, Glu13, and Asp118. Especially, the partially buried Asp11, which inte racts with Asp159, could cause one of these two groups to have an abno rmally high pK(a) and the other an abnormally low pK(a) value. Thus, t he ionization of Asp11 at a high pH may place two negatively charged r esidues in close vicinity. This unfavorable situation may be the trigg er for the denaturation of the N-terminal lobe of pepsin.