Carboxypeptidases pancreatic

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). 

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 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. 

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... 

This discussion of the metalloexopeptidases has focused on the general role of these enzymes in the conversion of dietary proteins into amino acids. In particular, the apparent synergistic relationship which the pancreatic carboxypeptidases have with the major endopeptidases, trypsin, chymotrypsin, and pepsin, in order to facilitate formation of essential amino acids has been stressed. The chemical characteristics, metalloenzyme nature, and mechanistic details of a representative of each class of exopeptidase have been presented. Leucine aminopeptidase from bovine lens was shown to be subject to an unusual type of metal ion activation which may be representative of a more general situation. Carboxypeptidase A of bovine pancreas was discussed in terms of its three-dimensional structure, the implications of x-ray crystallography to mecha ... 

The discovery of teprotide led to a search for new, specific, orally active ACE inhibitors. Ondetti et al. (172) proposed a hypothetical model of the active site of ACE, based on analogy with pancreatic carboxypeptidase A, and used it to predict and design compounds that would occupy the carboxy-terminal binding site of the enzyme. Carboxyalkanoyland mer-captoalkanoyl derivatives of proline were found to act as potent, specific inhibitors of ACE and 2-D-methyl-3-mercaptopropanoyl-L-proline (131) (captopril) was developed and launched in 1981 as an orally active treatment for patients with severe or advanced hypertension. Captopril, modeled on the biologically active peptides found in the venom of the pit viper, made an important contribution to the understanding of hypertension and paved the... 

Two important zinc metalloenzymes in protein digestion are the pancreatic carboxypeptidase A and B. A loss of activity of the pancreatic carboxyeptidase A in zinc deficiency is a consistent finding (91), According to some investigators, within two days of institution of zinc-deficient diet in rats, the enzyme lost 24% of its activity, and within three days of dietary zinc repletion, the activity of pancreatic carboxypeptidases is restored to normal levels (91). These results indicate that the level of food intake has no influence and that a decreased activity of carboxypeptidase A in the pancreas was related specifically to a dietary lack of zinc. As in the case of the alkaline phosphatase, the amount of carboxypeptidase A apoenzyme present appears to be diminished in zinc-deficient pancreas. 

The most studied member of zinc proteases is the digestive enzyme bovine pancreatic carboxypeptidase A (CPA) which is a metalloenzyme containing one atom of zinc bound to its single polypeptide side chain of 307 amino acids with a molecular weight of 34 kD. It is an exopeptidase, which catalyses the hydrolysis of C-terminal amino... 

Enzyme Models .—Two general mechanisms have been proposed for hydrolytic reactions catalysed by bovine pancreatic carboxypeptidase A the first involves formation of an anhydride intermediate and the second involves the residue Glu-270 as a general base. Work on model systems and the enzyme indicates that the general base mechanism is the correct one and a consistent mechanism (Scheme 4) has been proposed in which both zinc and Arg-145 interact with the substrate. 

Hendriks et al. [106] modeled the structure of the enzymatically active subunit of human plasma carboxypeptidase N based on the structure of bovine pancreatic carboxypeptidase A. The aim of the study was twofold improved understanding of the way the molecule functions and understanding the... 

Wintersberger2 isolated from bovine pancreatic carboxypeptidase a peptide containing a thiol group involved in the binding of zinc. He first removed the zinc by exposure to a chelating agent (1,10-phenanthroline) or by denaturation, and then labeled the reactive sulfhydryl group by reaction with DDPM and determined the amino acid sequence. 

Pancreatic carboxypeptidases are also secreted by the pancreas and act in the small bowel as exopeptidases, which trim amino acids one at a time from an end of a polypeptide. Carboxypeptidase A has a pref-... 

Carboxypeptidase A is a metalloenzyme—an enzyme that contains a tightly bound metal ion. The metal ion in carboxypeptidase A is Zn. Carboxypeptidase A is one of several hundred enzymes known to contain zinc. In bovine pancreatic carboxypeptidase A, Zn is bound to the enzyme at its active site by forming a complex with Glu 72, His 196, and His 69, as well as with a water molecule (Figure 24.5). (The source of the enzyme is specified because, although carboxypeptidase As from different sources follow the same mechanism, they have slightly different primary stmctures.)... 

Cushman, Ondetti, and coworkers (17,18,19) used SQ 20,881 and other peptide analogues to provide an enhanced understanding of the enzymatic properties of ACE. Using knowledge of substrate-binding specificities and the fact that ACE has properties similar to those of pancreatic carboxypeptidases, these researchers developed a hypothetical model of the enzyme active site. Carboxypeptidase A, like ACE, is a zinc-containing exopeptidase. The binding of a substrate to carboxypeptidase A involves three major interactions (Fig. 28.5A). 

Removal of contaminating proteins (which bind) during calmodulin purification Purification of pancreatic carboxypeptidase Purification of crayfish carboxypeptidase Purification of wheat-germ aspartate trans-carbamoylase... 

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