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Crystal structure and mechanism of human carboxypeptidase O: Insights into its specific activity for acidic residues

AuthorsGarcia-Guerrero, Maria C.; García-Pardo, Javier ; Berenguer, Esther; Fernandez-Alvarez, Roberto; Barfi, Gifty B.; Lyons, Peter J.; Avilés, Francesc Xavier; Huber, Robert; Lorenzo, Julia ; Reverter, David
Protein digestion
Crystal structure
Acidic protease
Issue Date2018
PublisherNational Academy of Sciences (U.S.)
CitationProceedings of the National Academy of Sciences 115(17): E3932-E3939 (2018)
AbstractHuman metallocarboxypeptidase O (hCPO) is a recently discovered digestive enzyme localized to the apical membrane of intestinal epithelial cells. Unlike pancreatic metallocarboxypeptidases, hCPO is glycosylated and produced as an active enzyme with distinctive substrate specificity toward C-terminal (C-t) acidic residues. Here we present the crystal structure of hCPO at 1.85-Å resolution, both alone and in complex with a carboxypeptidase inhibitor (NvCI) from the marine snail Nerita versicolor. The structure provides detailed information regarding determinants of enzyme specificity, in particular Arg275, placed at the bottom of the substrate-binding pocket. This residue, located at “canonical” position 255, where it is Ile in human pancreatic carboxypeptidases A1 (hCPA1) and A2 (hCPA2) and Asp in B (hCPB), plays a dominant role in determining the preference of hCPO for acidic C-t residues. Site-directed mutagenesis to Asp and Ala changes the specificity to C-t basic and hydrophobic residues, respectively. The single-site mutants thus faithfully mimic the enzymatic properties of CPB and CPA, respectively. hCPO also shows a preference for Glu over Asp, probably as a consequence of a tighter fitting of the Glu side chain in its S1′ substrate-binding pocket. This unique preference of hCPO, together with hCPA1, hCPA2, and hCPB, completes the array of C-t cleavages enabling the digestion of the dietary proteins within the intestine. Finally, in addition to activity toward small synthetic substrates and peptides, hCPO can also trim C-t extensions of proteins, such as epidermal growth factor, suggesting a role in the maturation and degradation of growth factors and bioactive peptides.
Publisher version (URL)https://doi.org/10.1073/pnas.1803685115
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