REARRANGING THE DOMAINS OF PEPSINOGEN

Citation
Xl. Lin et al., REARRANGING THE DOMAINS OF PEPSINOGEN, Protein science, 4(2), 1995, pp. 159-166
Citations number
18
Categorie Soggetti
Biology
Journal title
ISSN journal
09618368
Volume
4
Issue
2
Year of publication
1995
Pages
159 - 166
Database
ISI
SICI code
0961-8368(1995)4:2<159:RTDOP>2.0.ZU;2-O
Abstract
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.