Most eukaryotic aspartic protease zymogens are synthesized as a single
polypeptide chain that contains two distinct homologous lobes and a p
ro peptide, which is removed upon activation. In pepsinogen, the pro p
eptide precedes the N-terminal lobe (designated pep) and the C-termina
l lobe (designated sin). Based on the three-dimensional structure of p
epsinogen, 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 link
er designed to connect sin and pro domains. Recombinant sin-pro-pep wa
s synthesized in Escherichia coil, refolded from 8 M urea, acid purifi
ed. Upon acidification, sin-pro-pep autoactivates to a two-chain enzym
e. However, the emergence of activity is much slower than the conversi
on of the single-chain zymogen to a two-chain intermediate. In the act
ivation of native pepsinogen and sin-pro-pep, the pro region is cleave
d at two sites between residues 16P and 17P and 44P and 1 successively
, and complete activation of sin-pro-pep requires an additional cleava
ge at a third site between residues 1P and 2P. In pepsinogen activatio
n, the cleavage of the first site is rate limiting because the second
site is cleaved more rapidly to generate activity. In the activation o
f sin-pro-pep, however, the second site is cleaved slower than the fir
st, and cleavage of the third site is the rate limiting step. The reas
on for these differences is the result of the presence of activation i
ntermediates bearing pro peptide 1P-16P, which is still covalently att
ached to the sin domain after the first and second cleavages. This pep
tide is known to have affinity to the enzyme moiety. Its presence appa
rently prevents the full expression of proteolytic activity, which cat
alyzes the cleavage of sites 2 and 3. A mechanism of intramolecular cl
eavage of site 1 is proposed that involves the local conformational ch
ange only near site 1 in the N-terminal region of the pro peptide.