Carboxypeptidase, pancreatic

There are two main classes of proteolytic digestive enzymes (proteases), with different specificities for the amino acids forming the peptide bond to be hydrolyzed. Endopeptidases hydrolyze peptide bonds between specific amino acids throughout the molecule. They are the first enzymes to act, yielding a larger number of smaller fragments, eg, pepsin in the gastric juice and trypsin, chymotrypsin, and elastase secreted into the small intestine by the pancreas. Exopeptidases catalyze the hydrolysis of peptide bonds, one at a time, fi"om the ends of polypeptides. Carboxypeptidases, secreted in the pancreatic juice, release amino acids from rhe free carboxyl terminal, and aminopeptidases, secreted by the intestinal mucosal cells, release amino acids from the amino terminal. Dipeptides, which are not substrates for exopeptidases, are hydrolyzed in the brush border of intestinal mucosal cells by dipeptidases. 

Some of the pancreatic enzymes in the lumen include pancreatic amylase, pancreatic lipase, elastase, trypsin, a-chymotrypsin, and carboxypeptidase A. For example, the aspirin derivatives aspirin phenylalanine ethyl ester, aspirin phenyllactic ethyl ester, and aspirin phenylalanine amide have been studied as substrates for carboxypeptidase A [67,68], with the phenylalanine ethyl ester derivative proving to be the best substrate. This study indicated that the carboxypeptidase A may serve as a reconversion site for many drug derivatives. 

Zinc is a microelement essential for proper functioning of the human body. The level of daily demand for zinc was established as 13 to 16 mg (Ziemlahski, 2001). Zinc plays a role in protein and carbohydrate metabolism and is a component of over 60 metaloenzymes, including alkaline phosphatase, pancreatic carboxypeptidases A and B, alcoholic and lactic dehydrogenases, carbonate anhydrase, and proteases. It also forms bonds with nucleic acids -which is very important for their functioning (Prasad, 1983 Valee and Falchuk, 1993). 

Human proinsulin has been synthesized in homogeneous solution from 11 protected fragments using azide coupling.1151 The difficulties with insoluble intermediates were sufficiently overcome to allow the 86-residue peptide to be synthesized. The product was de-protected, converted into the 5-sulfonate, and then reduced and reoxidized to form the three disulfide bonds. The product was extensively purified and analyzed, and shown to be pure proinsulin. This product could then be converted into insulin by the use of endopeptidases I and II from pancreatic (3-cell granules, together with carboxypeptidase H, which removed the four basic residues 31, 32, 64, and 65, and split out C-peptide.1 6 ... 

II, DJ-I, HSC 54, carboxypeptidase A I, carboxypeptidase A2, and neuropolypeptide h3 decreased (36). Table 3 shows the summary of the proteins whose expression was different between pancreatic cancer tissues and non-cancerous tissues. 

Many secreted proteins, as well as smaller peptide hormones, are acted upon in the endoplasmic reticulum by tryptases and other serine proteases. They often cut between pairs of basic residues such as KK, KR, or RR.214-216 A substilisin-like protease cleaves adjacent to methionine.217 Other classes of proteases (e.g., zinc-dependent carboxypeptidases) also participate in this processing. Serine carboxypeptidases are involved in processing human prohormones.218 Among the serine carboxypeptidases of known structure is one from wheat219 and carboxypeptidase Y, a vacuolar enzyme from yeast.220 Like the pancreatic metallocarboxypeptidases discussed in Section 4, these enzymes remove one amino acid at a time, a property that has made carboxypeptidases valuable reagents for determination of amino acid sequences. Carboxypeptidases may also be used for modification of proteins by removal of one or a few amino acids from the ends. 

Thiosulfate cyanide sulfurtransferase symmetry in 78 TTiiouridine 234 Three-dimensional structures of aconitase 689 adenylate kinase 655 aldehyde oxido-reductase 891 D-amino acid oxidase 791 a-amylase, pancreatic 607 aspartate aminotransferase 57,135 catalytic intermediates 752 aspartate carbamyltransferase 348 aspartate chemoreceptor 562 bacteriophage P22 66 cadherin 408 calmodulin 317 carbonic acid anhydrase I 679 carboxypeptidase A 64 catalase 853 cholera toxin 333, 546 chymotrypsin 611 citrate synthase 702, 703 cutinase 134 cyclosporin 488 cytochrome c 847 cytochrome c peroxidase 849 dihydrofolate reductase 807 DNA 214, 223,228,229, 241 DNA complex... 

There are five distinct families of zinc proteases, classified by the nature of the zinc binding site. These families, and their variously proposed mechanisms, have recently been reviewed in depth.143 The most studied member is the digestive enzyme bovine pancreatic carboxypeptidase A, which is a metalloenzyme containing one atom of zinc bound to its single polypeptide chain of 307 amino acids and Mr 34 472. It is an exopeptidase, which catalyzes the hydrolysis of C-terminal amino acids from polypeptide substrates, and is specific for the large hydrophobic amino acids such as phenylalanine. The closely related carboxypeptidase B catalyzes the hydrolysis of C-terminal lysine and arginine residues. The two en-... 

The pancreatic carboxypeptidases are not homologous with thermolysin (and the G protease), but their active sites have evolved convergently to having striking similarities. 

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. 

In Table II are shown the results from kinetic studies with commercially available gastric and pancreatic enzymes. Trypsin was strongly inhibited, at least at a low concentration of casein as substrate. The hydrolysis of benzoyl arginine ethyl ester (BAEE) by trypsin was non-competitively inhibited, giving a 30% reduction of Vmax at 0.5 mg/ml of the LMW fraction. Carboxypepti-dase A, and to a lesser extent carboxypeptidase B, were non-competitively inhibited as well. Pepsin and chymotrypsin were not affected by the conditions used in these assays. 

P9. Pezzilli, R., Morselli-Labate, A. M., Barbieri, A. R., and Plate, L., Clinical usefulness of the serum carboxypeptidase B activation peptide in acute pancreatitis. JOP 1, 58-68 (2000). 

Competitive inhibition of the carboxypeptidase from A. saitoi by small substrates was found with hydrocinnamic acid, indole-3-propionic acid, and 4-phenylbutyric acid [80], The K for hydrocinnamic acid inhibition was 4 x 10 4 M. Diisopropylfluorophosphate (DFP) and tosyl-L-phenylalanylchloromethane (TPCK) were also powerful inhibitors of the carboxypeptidase from A. oryzae (80). />-Chloromercuribenzoate (PCMB) and iodoacetic acid were also powerful inhibitors of the carboxypeptidase from A. saitoi, while the inhibitors of DFP, TPCK, PCMB, and iodoacetic acid on the carboxypeptidase from A. saitoi were less than that of A. oryzae [80], As the carboxypeptidase activity of A. saitoi has no effect when used with ethylenediaminetetraacetate (EDTA) and o-phenanthroline, the enzyme is a different type of carboxypeptidase from those of the pancreatic sources, carboxypeptidase A and carboxypeptidase B [80],... 

The specificity of the acid carboxypeptidase displays the features typical of all pancreatic carboxypeptidases, hydrolysis of the specific substrate R-X-Y between X and Y (R = peptide residue, Z-, Bz-, Ac-). The amino acid in position Y must have a free carboxyl group dipeptides (having free amino group) are not hydrolyzed. The enzyme hydrolyzes most of the a-amino substituted peptides. The carboxypeptidase was inactive on a number of small amides tried at pH 3.0. A peculiarity of its specificity, however, was its inability to hydrolyze the peptide bond of tripeptides tried in the Table 11. 

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. 

The vertebrate pancreatic carboxypeptidases are one such family of digestive enzymes. Within the family, two broad substrate preferences are known. Enzymes with carboxypeptidase A (CPA) activity cleave hydro-phobic and aromatic residues from the carboxyl terminus of peptides and proteins, whereas those with carboxypeptidase B (CPB) activity prefer substrates with arginine and lysine residues at the C terminus.3 Several duplicates within the family have CPA-like activity but display a range of substrate preferences.5,6... 

Alignment of five completely sequenced members of the pancreatic digestive enzyme carboxypeptidase family1 is shown in Fig. 1, along with hepatopancreatic carboxypeptidase B from crayfish.8 Only the amino acid... 

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