Advances in metallo‐procarboxypeptidases

Abstract

Our knowledge on the structure and functionality of pancreatic carboxypeptidases is rapidly expanding to include that of their zymogen forms. The recent application of fast and mild isolation procedures, together with modern molecular genetic and biochemical-biophysical characterization approaches, has provided a clearer view of the basic structures and functional states in which these zymogens occur, and their evolutionary relationships. The same holds for related metallocarboxypeptidases, either in the pro or active forms, that have been isolated and characterized in non-digestive fluids and tissues, where they probably play an important role in protein and peptide processing. The determination of the three-dimensional structure of the A and B pancreatic zymogens has revealed the molecular determinants of their inactivity and proteolytic activation. The folding of their 95-residue activation segment in a globular N-terminal domain (74-81 residues) and in a connecting region (20-14 residues), and the specific contacts of these pieces with the substrate binding sites of the enzyme, are important factors in zymogen inhibition. On the other hand, the different length of the a-helical connecting region and the stability of its contacts with the enzyme account for the different activation properties of A and B zymogens.The hydrolysis of the peptide bond at the C-terminus of peptides and proteins in living organisms is performed by enzymes belonging either to the metallo-carboxypeptidase family or to the serine-carboxypeptidase family. The latter contain a reactive Ser residue at the active center and other catalytic residues which resemble and have a similar role to those of the Ser-endopeptidases superfamily [I -31. The residues and conformation of the active center of metallocarboxypeptidases are different, having one atom of Zn2 + directly involved in the catalytic mechanism. The present review refers to this latter class of carboxypeptidases and to their precursors.Pancreatic carboxypeptidases, the digestive enzymes involved in the hydrolysis of alimentary proteins from their Cterminal, are the best known enzymes among metallocarboxypeptidases and their occurrence and fundamental properties are very well documented [4 - (EC 3.4.17.10); CPM, membrane-bound carboxypeptidase M (EC 3.4.17.12); CPN, plasma carboxypeptidase N or kininase I (EC 3.4.17.3). specificity between the A form (CPA, with preference for aliphatic C-terminal residues) and the B form (CPB, with preference for basic C-terminal residues), as well as the tertiary structure and mechanism of action of carboxypeptidase A are well known. A number of non-digestive pancreatic-like carboxypeptidases have also been reported in the recent literature, expanding the field of interest in metallo-carboxypeptidases. In contrast to the active enzymes, and for many years, information on the inactive precursor forms of synthesis and storage, procarboxypeptidases A and B (pro-CPA and pro-CPB), has been limited.The lability of these precursors and their occurrence...

The activation pathway of procarboxypeptidase B from porcine pancreas: Participation of the active enzyme in the proteolytic processing

“…This has allowed a complete description of the process and shown that the first proteolytic action of trypsin can also take place on a second target bond. Structural considerations that take into account the three-dimensional structures of the A and B forms of the proenzymes lead us to propose that the differences in conformation at the region that connects the globular activation domain to the enzyme are the main responsible elements for the differences observed in the activation processes of both proenzymes.Keywords: activation domain; activation segment; inhibition mechanism; limited proteolysis; procarboxypeptidase; zymogen Porcine pancreatic secretion contains considerable amounts of the precursor forms of the two major pancreatic carboxypeptidases: AI and B. Procarboxypeptidase A1 (pro-CPA1) is found in the monomeric state and as a member of a binary complex with proproteinase E, whereas procarboxypeptidase B (pro-CPB) is found only in the monomeric state (Aviles et al, 1993). The oligomeric association is the most common occurrence of pro-CPAs (Yamasaki et al, 1963;Uren & Neurath, 1972;Kobayashi et al, 1978;Oppezzo et al, 1994), and it has been observed that the quaternary structure affects their activation behavior (Puigserver & Desnuelle, 1977;Kobayashi et al, 1981; Puigserver et al, 1986;Chapus et al, 1987).…”

“…The activation process of pancreatic pro-CPs stands out among those of other pancreatic zymogens because of the length of the N-terminal pro-pieces (also called activation segments): 94 residues for the AI form and 95 residues for the B form; other important digestive zymogens (e.g., trypsinogens or proelastases) have much shorter activation pro-pieces. This differential characteristic suggests that the activation peptides of pro-CPs might provide the conformational elements that modulate the activation process of these proenzymes.Sequential data are available for a number of pro-CPs from different species (for a review, see Aviles et al, 1993). On average, the percentage of sequence identity between pro-CPA1 (403 residues) and pro-CPB (402 residues) is about 45%.…”

“…Sequential data are available for a number of pro-CPs from different species (for a review, see Aviles et al, 1993). On average, the percentage of sequence identity between pro-CPA1 (403 residues) and pro-CPB (402 residues) is about 45%.…”

Crystallization and preliminary X‐ray analysis of the ternary complex of procarboxypeptidase A from bovine pancreas

“…The occurrence of these proenzymes usually is a control mechanism for the biological activity, achieved by limited proteolysis of these proforms upon arrival at their operating milieu. This is the case for pancreatic carboxypeptidases A and B, digestive exopeptidases that belong to the family of metallo exoproteases [1]. They are involved in the hydrolysis of alimentary proteins from their C-terminal ends, and are produced in the pancreas of vertebrates as inactive precursor forms, the procarboxypeptidases A and B (PCPA and PCPB, respectively).…”

“…They are involved in the hydrolysis of alimentary proteins from their C-terminal ends, and are produced in the pancreas of vertebrates as inactive precursor forms, the procarboxypeptidases A and B (PCPA and PCPB, respectively). One of the remarkable properties of pancreatic PCPA is its occurrence as a monomer, as a binary non-covalent complex with proproteinase E (BPE) or chymotrypsinogen C (CTGC), or as a ternary complex (TC) with both proenzymes [1].…”