AbstractMost eukaryotic aspartic protease zymogens are synthesized as a single polypeptide chain that contains two distinct homologous lobes and a pro peptide, which is removed upon activation. In pepsinogen, the pro peptide precedes the N‐terminal lobe (designated pep) and the C‐terminal lobe (designated sin). Based on the three‐dimensional structure of pepsinogen, we have designed a pepsinogen polypeptide with the internal rearrangement of domains from pro‐pep‐sin (native pepsinogen) to sin‐pro‐pep. The domain‐rearranged zymogen also contains a 10‐residue linker designed to connect sin and pro domains. Recombinant sin‐pro‐pep was synthesized in Escherichia coli, refolded from 8 M urea, and purified. Upon acidification, sin‐pro‐pep autoactivates to a two‐chain enzyme. However, the emergence of activity is much slower than the conversion of the single‐chain zymogen to a two‐chain intermediate. In the activation of native pepsinogen and sin‐pro‐pep, the pro region is cleaved at two sites between residues 16P and 17P and 44P and 1 successively, and complete activation of sin‐pro‐pep requires an additional cleavage at a third site between residues IP and 2P. In pepsinogen activation, the cleavage of the first site is rate limiting because the second site is cleaved more rapidly to generate activity. In the activation of sin‐pro‐pep, however, the second site is cleaved slower than the first, and cleavage of the third site is the rate limiting step. The reason for these differences is the result of the presence of activation intermediates bearing pro peptide 1P‐16P, which is still covalently attached to the sin domain after the first and second cleavages. This peptide is known to have affinity to the enzyme moiety. Its presence apparently prevents the full expression of proteolytic activity, which catalyzes the cleavage of sites 2 and 3. A mechanism of intramolecular cleavage of site 1 is proposed that involves the local conformational change only near site 1 in the N‐terminal region of the pro peptide.