García Pardo, JavierTanco, Sebastián MartínDíaz, LucíaDasgupta, SayaniFernández Recio, JuanLorenzo, JúliaAvilés, Francesc XavierFricker, Lloyd D.2018-07-132018-07-132017Garcia-Pardo J, Tanco S, Díaz L, Dasgupta S, Fernandez-Recio J, Lorenzo J et al. Substrate specificity of human metallocarboxypeptidase D: Comparison of the two active carboxypeptidase domains. PLoS One. 2017 Nov 13;12(11):e0187778. DOI: 10.1371/journal.pone.01877781932-6203http://hdl.handle.net/10230/35150Metallocarboxypeptidase 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.application/pdfeng© 2017 Garcia-Pardo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.info:eu-repo/semantics/articlehttp://dx.doi.org/10.1371/journal.pone.0187778Peptide librariesThyroglobulinSequence alignmentEnzymesRecombinant proteinsSerum albumininfo:eu-repo/semantics/openAccess