Characterization of the Enzymatic Properties of the First and Second Domains of Metallocarboxypeptidase D

Abstract

Carboxypeptidase D (CPD) contains three domains with homology to other metallocarboxypeptidases. To further characterize the various domains, we constructed a series of point mutants with a critical active site Glu of duck CPD converted to Gln. The proteins were expressed in the baculovirus system, purified to homogeneity, and characterized. Point mutations within both the first and second domains eliminated enzyme activity, indicating that the third domain is inactive toward dansyl-Phe-Ala-Arg. CPD removed only the C-terminal Lys or Arg from peptides, with the first domain more efficient toward Arg and the second domain more efficient toward Lys. Peptides containing Pro in the penultimate position were poorly cleaved by either domain. Cleavage of a peptide with Ala in the penultimate position was most efficient, with the relative order Ala > Met > Ser, Phe > Tyr > Trp > Thr > Gln, Asp, Leu, Gly Ͼ Ͼ Ͼ Ͼ Pro for CPD with both domains active. There were only minor differences between the first and the second domains regarding the influence of the penultimate amino acid. The first domain was optimally active at pH 6.3-7.5, whereas the second domain was optimally active at pH 5.0 -6.5. Thus, the first and second carboxypeptidase domains have complementary enzyme activities. Furthermore, the finding that CPD with both domains active shows a broad activity to a wide range of substrates is consistent with a role for this enzyme in the processing of many proteins that transit the secretory pathway.

Characterization of the Molecular Basis of the Drosophila Mutations in Carboxypeptidase D

“…6A). Because the constructs did not contain the third CP-like domain or transmembrane domain, they were expected to be secreted into the media, as previously found for duck CPD constructs containing just the first two CP domains (28). Upon Western blot analysis, a protein of 110 kDa was detected in the media from cells expressing constructs containing wild type CP domain 2 attached to either the 1A or the 1B domain (Fig.…”

“…Misexpressed Sog reduces the space between longitudinal veins 3 and 4, which is similar to the phenotype of the Drosophila CPD mutants (58). Because CPD is able to remove C-terminal Lys and Arg residues from a large number of peptides (5,28) and is present in the same compartments as furin (43,45,59), it is likely that CPD also processes those proteins initially cleaved by furin and related endopeptidases. Although it is not known if the removal of C-terminal basic residues is required for the production of the active form of any of these peptides, the fact that svr PG33 mutants die in the larval stage and the svr 1 and svr poi mutants have altered wing shape suggests that at least some of the peptides processed by CPD require this step for the generation of the biologically active form.…”

“…In both duck and Drosophila, the first domain is more active at neutral pH, whereas the second domain is optimally active at pH 5-6 (16, 17, 28). Protein containing both domains together showed a broader pH optimum (17,28).…”

“…The functional significance of the three CP-like domains in CPD is not clear. One proposal is that the distinct pH optima of the first two domains enable CPD to be active throughout the secretory pathway, which ranges from neutral to acidic pH values (17,28). In both duck and Drosophila, the first domain is more active at neutral pH, whereas the second domain is optimally active at pH 5-6 (16, 17, 28).…”

Identification of Novel Genes That Modify Phenotypes Induced by Alzheimer's β-Amyloid Overexpression in Drosophila

“…taining multiple carboxypeptidase domains and it has been proposed that this special structure increases the range of pH at which it can be active, ensuring activity throughout the secretory pathway (Novikova et al 1999). The Drosophila silver protein contains three carboxypeptidase domains, a transmembrane domain, and a cytosolic tail (Sidyelyeva and Fricker 2002).…”