Characterization of the enzymatic properties of the first and second domains of metallocarboxypeptidase D

Carboxypeptidase D (CPD) contains three domains with homology to other meta llocarboxypeptidases. To further characterize the various domains, we const ructed a series of point mutants with a critical active site Glu of duck CP D 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 t hird domain is inactive toward dansyl-Phe-Ala-Arg. CPD removed only the C-t erminal Lys or Arg from peptides, with the first domain more efficient towa rd Arg and the second domain more efficient toward Lys, Peptides containing Pro in the penultimate position were poorly cleaved by either domain. Clea vage of a peptide with Ala in the penultimate position was most efficient, with the relative order Ala greater than or equal to Met > Ser, Phe > Tyr > Trp > Thr greater than or equal to Gln, Asp, Leu, Gly >> Pro for CPD with both domains active. There were only minor differences between the first an d 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 do main was optimally active at pH 5.0-6.5. Thus, the first and second carboxy peptidase domains have complementary enzyme activities. Furthermore, the fi nding that CPD with both domains active shows a broad activity to a wide ra nge of substrates is consistent with a role for this enzyme in the processi ng of many proteins that transit the secretory pathway.