Carboxypeptidase is an enzyme synthesized in the pancreas and secreted into the small intestine. This enzyme hydrolyzes the first peptide or amide bond at the carboxyl or C-terminal end of proteins and peptides. It has a stronger preference for those amino acids that have aromatic or branched hydrocarbon chains.
Carboxypeptidases (CPs) from N/E subfamily follow the Prohormone convertases (PC) action and further process the peptides, hormones and growth factors precursors. As CPs are active toward the C-terminal basic amino acids, their substrates are precursors which transit the secretory pathways and require proteolytic processing at specific sites with multiple basic residues. PCs initially process precursors at C-terminus of the basic amino acids, and carboxypeptidases then would remove Lys and Arg from the intermediates.
Many biologically active peptides are amidated by peptidylglycine α-amidating monooxygenase (PAM) after proteolytic processing. PAM in mammals consists of two enzymes, translated from one mRNA, peptidylglycine- α-hydroxylating monooxygenase (PHM) and peptidyl-α-hydroxyglycine α-amidating lyase (PAL), sequential actions of those are required for obtaining amidated peptides and hormones.
Metallocarboxypeptidases in mammals
Metallocarboxypeptidases perform a large variety of biological functions, varying from food digestion to neuropeptide biosynthesis, and are important players in proteolytic conversion of many secreted proteins and peptides into active mature forms from initially synthesized inactive precursors. CPs require Zn2+ in the active site for catalysis. They consist of three subfamilies: A/B subfamily, N/E subfamily and Nnal/CCP subfamily. Such division is based on the amino acid sequences similarities. Members of the A/B subfamily are involved in the protein digestion, whereas proteins of the N/E subfamily selectively remove specific residues from the peptide processing intermediates, affecting biological properties of the substrate. The function of a recently discovered Nnal and related proteins that comprise another subfamily is remain unknown, however, it was proposed that CCPs may participate in proteasomal product degradation and possibly are involved in the selective processing of cytosolic proteins.
CPs of the N/E subfamily remove C-terminal residues from their substrate proteins and peptides after the members of subtilisin family, such as prohormone convertases (PCI and PC2) and furin initially cleave at the C-terminal site of the substrate basic residues. CPs substrates include neurotransmitters, growth factors, hormones, some receptors, including an insulin, and a viral proteins.
N/E subfamily members
N/E subfamily members play more biosynthetic role in producing mature forms of peptides and peptide hormones in secretory pathways. There are eight members of this subfamily in vertebrates, which includes five active CPs (CPN, CPE, CPZ, CPD, CPM) and three CPs which do not show any enzymatic activity towards known substrates (CPX1, CPX2, AEBP1). All members of N/E subfamily have 80 amino acids transthyretin-like subdomain, which participates in protein folding, and N-terminal signal peptide, but in contrast to A/B subfamily, N/E subfamily members do not have "pro" region.
Inactive CPX1, CPX2 and AEBP1 share slightly higher sequence homology between each other, than with other N/E subfamily members. They have an N-terminal discoidin-like domain, which is not present in the other metallocarboxypeptidases. Their CP domains lack one or more critical residues for CP activity or substrate binding, which questioning their function as carboxypeptidases. The mRNA distribution was found in the placenta for CPX1 and in the heart and the other tissues for CPX2 and AEBP1.
CPN and CPM are constitutively active enzymes that function extracellularly and prefer neutral pH optima. CPN is made in the liver and present in plasma as a four-unit protein complex, containing two molecules of CPN and two molecules of glycoprotein. CPM is bound to the membrane by a glycosyl-phosphatidylinositol anchor and is present in placenta and lung, as well as on white blood cells surface; CPM soluble forms are present in various fluids. CPM and CPN functions may include inactivation or specificity alteration of kinins and anaphylatoxins. Also these CPs can regulate plasminogen binding to cells as CPU or process peptide hormones which are not fully processed in secretory pathways by CPE.
CPZ is an active CP and it is present on the cell surface despite the absence of a transmembrane domain. CPZ is the only metallocarboxypeptidase with a cystein-rich domain which is conserved between rat and human and shows 20-35% sequence similarity with Drosophila and mammalian Frizzled proteins, which are the receptors for Wingless/Wnt family members. CPZ is highly abundant in embryonic tissues and its expression patterns overlap with Wnt members' pattern, and is much less present in adult tissues, which is consistent with its role in development together with Wnt signaling. CPZ is active at neutral pH towards standard CP substrates but unlikely to have overlapping functions with other N/E family members. Possible CPZ functions include antagonizing Wnt signaling by binding Wnts in the extracellular environment; may activate/inactivate Wnts proteins by removing C-terminal Arg and Lys; it may enhance extracellular matrix resident endoprotease activity. CPZ was shown to modulate Wnt signaling thus regulating the development of skeletal system elements in chicken and activates Wnt-4 protein, containing C-terminal Arg and binding to CPZ cystein-rich domain.
Sidyelyeva, G. (2010). Carboxypeptidase D: A multitasking enzyme involved in Drosophila viability, development, behavior, and memory. Yeshiva University.