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

Javier Garcia-Pardo, Sebastian Tanco, Lucía Díaz, Sayani Dasgupta, Juan Fernandez-Recio, Julia Lorenzo, Francesc X. Aviles, Lloyd D. Fricker

Research output: Contribution to journalArticle

2 Citations (Scopus)

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.

Original languageEnglish (US)
Article numbere0187778
JournalPLoS One
Volume12
Issue number11
DOIs
StatePublished - Nov 1 2017

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metallocarboxypeptidases
Carboxypeptidases
carboxypeptidases
substrate specificity
Substrate Specificity
peptides
Peptides
Substrates
trans-Golgi Network
mutation
Mutation
Enzymes
enzymes
active sites
Catalytic Domain
Cell Cycle
Display devices
metallocarboxypeptidase D
cells
Membranes

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Substrate specificity of human metallocarboxypeptidase D : Comparison of the two active carboxypeptidase domains. / Garcia-Pardo, Javier; Tanco, Sebastian; Díaz, Lucía; Dasgupta, Sayani; Fernandez-Recio, Juan; Lorenzo, Julia; Aviles, Francesc X.; Fricker, Lloyd D.

In: PLoS One, Vol. 12, No. 11, e0187778, 01.11.2017.

Research output: Contribution to journalArticle

Garcia-Pardo, J, Tanco, S, Díaz, L, Dasgupta, S, Fernandez-Recio, J, Lorenzo, J, Aviles, FX & Fricker, LD 2017, 'Substrate specificity of human metallocarboxypeptidase D: Comparison of the two active carboxypeptidase domains', PLoS One, vol. 12, no. 11, e0187778. https://doi.org/10.1371/journal.pone.0187778
Garcia-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 1;12(11). e0187778. https://doi.org/10.1371/journal.pone.0187778
Garcia-Pardo, Javier ; Tanco, Sebastian ; Díaz, Lucía ; Dasgupta, Sayani ; Fernandez-Recio, Juan ; Lorenzo, Julia ; Aviles, Francesc X. ; Fricker, Lloyd D. / Substrate specificity of human metallocarboxypeptidase D : Comparison of the two active carboxypeptidase domains. In: PLoS One. 2017 ; Vol. 12, No. 11.
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