Substrate specificity of human metallocarboxypeptidase D: Comparison of the two active carboxypeptidase domains

• Published in: PloS one Vol. 12; no. 11; p. e0187778
• Main Authors: Garcia-Pardo, Javier, Tanco, Sebastian, Díaz, Lucía, Dasgupta, Sayani, Fernandez-Recio, Juan, Lorenzo, Julia, Aviles, Francesc X, Fricker, Lloyd D
• Format: Journal Article Publication
• Language: English
• Published: United States Public Library of Science 13.11.2017; Public Library of Science (PLoS)
• Subjects: Amino Acid Sequence; Amino acids; Biochemistry; Biology; Biology and Life Sciences; Bortezomib - chemistry; Carboxypeptidase D; Carboxypeptidase domains; Catalytic Domain; Cell surface; Compounds; Drosophila; Enginyeria biomèdica; Enginyeria de proteïnes; Enzymes; Genetic aspects; Geographical variations; Golgi apparatus; HEK293 Cells; Humans; Hydrogen ions; Hydrogen-Ion Concentration; Insects; Kinetics; Life sciences; Mammals; Metallocarboxypeptidase D (CPD); Molecular docking; Molecular Docking Simulation; Mutation; Peptides; Peptides - chemistry; pH effects; Physical Sciences; Physiological aspects; Point Mutation; Polypeptides; Properties; Proteases; Protein engineering; Proteins; Proteins - chemistry; Proteins - genetics; Proteins - metabolism; Research and Analysis Methods; Substrate Specificity; Substrates; Àrees temàtiques de la UPC; Belgium; United States; Spain; New York; United States > US
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0187778

Metallocarboxypeptidase D (CPD) is a membrane-bound component of the trans-Golgi network that cycles to the cell surface through exocytic and endocytic pathways. Unlike other members of the metallocarboxypeptidase family, CPD is a multicatalytic enzyme with three carboxypeptidase-like domains, although only the first two domains are predicted to be enzymatically active. To investigate the enzymatic properties of each domain in human CPD, a critical active site Glu in domain I and/or II was mutated to Gln and the protein expressed, purified, and assayed with a wide variety of peptide substrates. CPD with all three domains intact displays >50% activity from pH 5.0 to 7.5 with a maximum at pH 6.5, as does CPD with mutation of domain I. In contrast, the domain II mutant displayed >50% activity from pH 6.5-7.5. CPD with mutations in both domains I and II was completely inactive towards all substrates and at all pH values. A quantitative peptidomics approach was used to compare the activities of CPD domains I and II towards a large number of peptides. CPD cleaved C-terminal Lys or Arg from a subset of the peptides. Most of the identified substrates of domain I contained C-terminal Arg, whereas comparable numbers of Lys- and Arg-containing peptides were substrates of domain II. We also report that some peptides with C-terminal basic residues were not cleaved by either domain I or II, showing the importance of the P1 position for CPD activity. Finally, the preference of domain I for C-terminal Arg was validated through molecular docking experiments. Together with the differences in pH optima, the different substrate specificities of CPD domains I and II allow the enzyme to perform distinct functions in the various locations within the cell.