Rearranging the domains of pepsinogen.
Most 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 1P 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Lin
X
X
Protein Studies Program, Oklahoma Medical Research Foundation, Oklahoma City 73104, USA.
Koelsch
G
G
Loy
J A
JA
Tang
J
J
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D001483:Base Sequence; D004789:Enzyme Activation; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010435:Pepsinogens / Q000737:chemistry* / Q000378:metabolism; D015202:Protein Engineering; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
D010435:Pepsinogens; D016282:Aspartic Acid Endopeptidases