dc.contributor |
Barcelona Supercomputing Center |
dc.contributor.author |
García-Pardo, Javier |
dc.contributor.author |
Tanco, Sebastian |
dc.contributor.author |
Diaz, Lucia |
dc.contributor.author |
Dasgupta, Sayani |
dc.contributor.author |
Fernandez-Recio, Juan |
dc.contributor.author |
Lorenzo, Julia |
dc.contributor.author |
Aviles, Francesc X. |
dc.contributor.author |
Fricker, Lloyd D. |
dc.date |
2017-11-13 |
dc.identifier.citation |
García-Pardo, J. [et al.]. Substrate specificity of human metallocarboxypeptidase D: Comparison of the two active carboxypeptidase domains. "PLoS ONE", 13 Novembre 2017, vol. 12, núm. 11. |
dc.identifier.citation |
1932-6203 |
dc.identifier.citation |
10.1371/journal.pone.0187778 |
dc.identifier.citation |
29131831 |
dc.identifier.uri |
http://hdl.handle.net/2117/112434 |
dc.language.iso |
eng |
dc.publisher |
Public Library of Science |
dc.relation |
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0187778 |
dc.relation |
info:eu-repo/grantAgreement/ES/PE2013-2016/BIO2013-44973-R |
dc.relation |
info:eu-repo/grantAgreement/ES/PE2013-2016/BIO2016-78057-R |
dc.relation |
info:eu-repo/grantAgreement/ES/PE2013-2016/BIO2016-79960-R |
dc.rights |
Attribution-NonCommercial-NoDerivs 4.0 Spain |
dc.rights |
info:eu-repo/semantics/openAccess |
dc.rights |
http://creativecommons.org/licenses/by-nc-nd/4.0/es/ |
dc.subject |
Àrees temàtiques de la UPC::Enginyeria biomèdica |
dc.subject |
Protein engineering |
dc.subject |
Metallocarboxypeptidase D (CPD) |
dc.subject |
Carboxypeptidase domains |
dc.subject |
Enginyeria de proteïnes |
dc.title |
Substrate specificity of human metallocarboxypeptidase D: Comparison of the two active carboxypeptidase domains |
dc.type |
info:eu-repo/semantics/publishedVersion |
dc.type |
info:eu-repo/semantics/article |
dc.description.abstract |
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. |
dc.description.abstract |
This work was funded by the Spanish Ministry of Innovation and Competitiveness grants BIO2013-44973-R and BIO2016-78057-R (to FXA), by Plan Estatal grant number BIO2016-79960-R from the Spanish Ministry of Economy and Competitiveness (to JFR), and by grant R01-DA004494 from the United States’ National Institute of Health (to LDF). |
dc.description.abstract |
Peer Reviewed |