Pancreatic peptidases

PA S1 S01.156 Entero peptidase Potential drug target for acute pancreatitis... 

P cells of the pancreatic islets in combination with atoms of zinc, but when required to regulate blood glucose concentration, the prohormone is cleaved and functional insulin is released into the circulation along with the C-peptide. This example of post-translational processing is mediated by peptidases which are contained in the vesicles along with the proinsulin. The fusion of the secretory vesicles with the cell membrane and activation of the peptidase prior to exocytosis of the insulin are prompted by an influx of calcium ions into the P-cell in response to the appropriate stimulus. Similarly, catecholamines are synthesized and held within the cell by attachment to proteins called chromogranins. 

The carboxypeptidases are released from their inactive precursors in the pancreatic juice of animals. The most studied example is bovine carboxypeptidase A, which contains one mole of zinc per protein molecular weight of 34 500. These enzymes cleave the C-terminal amino acid residue from peptides and proteins, when the side-chain of the C-terminal residue is aromatic or branched aliphatic of l configuration. At least the first five residues in the substrate affect the activity of the enzyme. The enzyme also shows esterase activity. Esters and peptides inhibit each other competitively, indicating that the peptidase and esterase sites overlap, even if they are not the same. 

Plasma from patients with pancreatitis or other disease states may change the plasma peptidase activity—for example, certain viral diseases (30). 

Abstract The major enzymatic barrier to the absorption of macromolecules, particularly therapeutic peptides, is the pancreatic enzymes the peptidases, nucleases, lipases and esterases that are secreted in considerable quantities into the intestinal lumen and rapidly hydrolyse macromolecules and lipids. In the case of the peptidases, they work in a co-ordinated fashion, whereby the action of the pancreatic enzymes is augmented by those in the brush borders of the intestinal cells. The sloughing-off of mucosal cells into the lumen also furnishes a mixture of enzymes that are a threat to macromolecules. As the specificity and activity of the enzymes are not always predictable, during pharmaceutical development it is important to test the stability of therapeutic macromolecules, and novel macromolecular-containing or lipid-containing formulations, in the presence of mixtures of pancreatic enzymes and bile salts, or in animal intestinal washouts or ideally, aspirates of human intestinal contents. 

Figure 1.1 shows the major sites of enzyme activity in the GI tract, and we will consider each of these in turn. While most of the enzymes that hydrolyse macromolecules enter the gut in the pancreatic fluid and hence are found in the lumen of the gut, there is significant peptidase activity located on the membranes of the intestinal cells, the so-called brush border. Consideration should also be given to the enzymes that are located inside the cells of the intestinal mucosa, namely, the epithelial cells or enterocytes. This is for two reasons first, the intestinal mucosa has a turnover of 3-6 days in humans and this means that the enterocytes are constantly being sloughed-off into the lumen of the gut. Thus intracellular enzymes and brush border enzymes will be found in the lumen of the gut, though the precise quantity is difficult to assess (see later in Section 1.6). 

The GI tract of humans contain a number of enzymes involved in the hydrolysis of proteins and peptides and they are located in a number of sites. It is important to recognize that peptidase enzymes never occur alone. Throughout the GI tract, there is always a mixture of peptidases working synergistic. The main event in the intraluminal digestion of proteins consists of cleavage of polypeptides by pancreatic proteases, such as trypsin,... 

Hydrolysis of peptides and proteins in the GI tract can occur luminally, at the brash border and intracellularly. Luminal activity from the pancreatic proteases trypsin, chymotrypsin, elastase and carboxypeptidase A is mainly directed against large dietary proteins. The main enzymatic activity against small bioactive peptides is derived from the bmsh border of the enterocyte. Brash border proteases, such as aminopeptidase A and N, diaminopeptidease IV and Zn-stable Asp-Lys peptidase, preferentially cleave oligopeptides of up to 10 ammo acid residues and are particularly effective in the cleavage of tri- and tetra-peptides. 

Protein and starch digestion, on the other hand, have potent nonpancreatic compensatory mechanisms. Due to the compensatory action of salivary amylase and brush border oligosaccharidases, a substantial proportion of starch digestion can be achieved without pancreatic amylase. Similarly, protein denaturation and hydrolysis is initiated by gastric proteolytic activity (acid and pepsin) and continued by intestinal brush border peptidases, and is thus partly maintained even in the absence of pancreatic proteolytic activity. 

Biocytin is hydrolyzed by biotinidase, which acts on free or peptide-incorporated biocytin to release biotin, but has no general peptidase or esterase activity. Biotinidase is most active toward free biocytin, but it will also release biotin from biocytin-containing peptides. The activity decreases as the size of the peptide increases, so it is likely that in vivo the catabolism of biotin-containing enzymes is by proteolysis, followed by biotinidase action, rather than the release of biotin, leaving the apoenzyme as a substrate for proteolysis. Biotinidase is found in all tissues, including the pancreatic juice and intestinal mucosa. 

The specificities of the various digestive exo- and endopep-tidases suggest that they act synergistically to fulfill a major nutritional function. The concerted action of trypsin, chy-motrypsin, pepsin, and carboxypeptidases A and B facilitate and ensure formation of essential amino acids. The chemical characteristics and metalloenzyme nature of two bovine exopeptidases, lens aminopeptidase and pancreatic carboxy-peptidase A, indicate similarities in their mechanisms of action. However, the aminopeptidase exhibits an unusual type of metal ion activation not observed unth carboxy-peptidase. Chemical and physicochemical studies reveal that the latter enzyme has different structural conformations in its crystal and solution states. Moreover, various kinetic data indicate that its mode of action toward ester substrates differs from that toward peptide substrates. The active site metal atom of carboxypeptidase figures prominently in these differences. 

MEROPS identifies Kunitz-type inhibitors as families 12 and 13, yet they seem to have developed separately in evolutionary history. Families 12 and 13 are referred to as Kunitz-A and Kunitz-P for their origin from animals and plants, respectively. Aprotinin, also known as bovine pancreatic trypsin inhibitor, was one of the first protease inhibitors identified and isolated by Kraut and coworkers in 1930. The 12 family is considerably more homogenous and thought to inhibit only S1 peptidases. In contrast, the 13 family is split into two phylogenetic groups, 13A and 13B, both of which typically inhibit SI peptidases, yet members of the I3A family can also potentially inhibit the A1 family aspartyl proteases and the Cl family cysteine proteases. The first structure of an 13 inhibitor was the... 

The action of these two pancreatic exopeptidases on synthetic substrates, proteins, and peptides has been reviewed in detail by Neurath (1960). The specificity requirements which were deduced from studies with synthetic peptides have been confirmed by studies with polypeptides. The structural requirements of specific substrates for both types of carboxy-peptidase are analogous except for the nature of the amino acids which contain the free, ionized a-carboxyl group at the terminus of the substrate. Carboxypeptidase B hydrolyzes most rapidly those bonds formed by terminal lysyl and arginyl residues, whereas carboxypeptidase A hydrolyzes terminal bonds formed by a variety of aromatic, neutral, or acidic amino acids. Of the natural amino acids only carboxyl-terminal prolyl residues are resistant to the action of the enzyme. The rate of hydrolysis depends upon the nature of the side chains of the amino acids which form the susceptible bonds. Thus, differences in the rate of hydrolysis of different substrates may vary several thousandfold. The methods for application of these peptidases to hydrolysis of proteins have been discussed in detail by Canfield and Anfinsen (1963). 

Protein digestion begins in the stomach, where a proenz5nme called pepsinogen is secreted, autocatalytically converted to Pepsin A, and used for the first step of proteolysis. However, most proteolysis takes place in the duodenum as a consequence of enzyme activities secreted by the pancreas. All of the serine proteases and the zinc peptidases of pancreatic secretions are produced in the form of their respective proenzymes. These proteases are both endopeptidase and exopeptidase, and their combined action in the intestine leads to the production of amino acids, dipeptides, and tripeptides, all of which are taken up by enterocytes of the mucosal wall. How preoteolytic enzymes are regulated ... 

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