Carboxypeptidase Procarboxypeptidase

Procarboxypeptidase A is activated by the removal of a peptide of some 64 residues from the N-terminus by trypsin.153 This zymogen has significant catalytic activity. As well as catalyzing the hydrolysis of small esters and peptides, procarboxypeptidase removes the C-terminal leucine from lysozyme only seven times more slowly than does carboxypeptidase. Also, the zymogen hydrolyzes Bz-Gly-L-Phe with kcsA = 3 s-1 and KM = 2.7 mM, compared with values of 120 s 1 and 1.9 mM for the reaction of the enzyme.154 In contrast to the situation in chymotrypsinogen, the binding site clearly pre-exists in procarboxypeptidase, and the catalytic apparatus must be nearly complete. 

Conejero-Lara, F., J.M. Sanchez-Ruiz, P.L. Mateo, F.J. Burgos, J. Vendrell, and F.X. Aviles. 1991. Differential scanning calorimetric study of carboxypeptidase B, procarboxypeptidase B and its globular activation domain. Eur J Biochem 200 663-670. 

Carboxypeptidases A and B are formed by the hydrolytic action of trypsin upon inactive precursors, procarboxypeptidases. These zymogens are synthesized in the pancreas from which they can be isolated (88). Depending on the preparation method, different forms of carboxypeptidase A are produced, varying in the N-terminal region (89). 

Table 7. Apparent stability constants for metal binding in procarboxypeptidase A, carboxypeptidase A and human carbonic anhydrase B...

Metal binding in procarboxypeptidase A is weaker than in the active enzyme ( 107), Table 7). It was proposed that the bonding involves sulfur and a weaker ligand than N (107). In view of the present concept of the chelating site in carboxypeptidase, further studies of the zymogen are necessary. In that connection, the cobalt complex should be valuable. 

Carboxypeptidase A Procarboxypeptidase A Pancreas Two large fragments, MW 54,000 Trypsin C-terminal amino acids except basics, Pro and Cys... 

Carboxypeptidase B Procarboxypeptidase B Pancreas Uncertain Trypsin C-terminal basic amino acids... 

Chymotrypsinogen, a single polypeptide chain of 245 amino acid residues, is converted to a-chymotrypsin, which has three polypeptide chains linked by two of the five disulfide bonds present in the primary structure of chymotrypsinogen. tt- and S-chymotrypsin also have proteolytic activity. In contrast, the conversion of procarboxypeptidase to carboxypeptidase involves the hydrolytic removal of a single amino acid. 

Figure 15. Conversion of procarboxypeptidase A to carboxypeptidase A by trypsin (151). The subunits are not cyclic polypeptides. ATEEase and HPLAase represents activities of activated subunits I and II on acetyl-1-tyrosine ethyl ester and hippuryl phenyllactic acid, respectively.

Bovine carboxypeptidase A is produced in the pancreas as a zymogen, procarboxypeptidase A, MW = 87,000. The proenzyme is composed of three polypeptide chains (151, schematically shown in Figure 15). On limited digestion with trypsin one or more peptide bonds in subunit II is split resulting in its conversion to an enzyme (ATEEase) having activity on acetyl-L-tyrosine ethyl ester similar to that of chymotrypsin. Continued... 

Fig. 1. Chromatography of bovine pancreatic juice on DEAE-cellulose (anionic proteins) and Amberlite IRC-50 (cationic proteins) (1). RNAase, ribonuclease ChTg-a, chymotrypsinogen A Tg, trypsinogen ProCp-B and Cp-B, procarboxypeptidase B and carboxypeptidase B DNAase, deoxyribonuclease ProCp-A, procarboxypeptidase A.

In 1936, Anson (127) crystallized what is now called carboxypeptidase A from autolyzed bovine pancreas and noted that fresh pancreas did not contain the active enzyme, but an inactive precursor now named procarboxypeptidase A. It has been reported in the preceding sections that pancreatic juices of other species also contain large amounts of procarboxypeptidase A which can be separated by chromatography on DEAE-cellulose at pH 8.0 in a buffer of increasing molarity. 

Cobalt has recently been used as an ESR active substitute in zinc metalloenzymes. Whilst liquid helium temperatures may be needed and theoretical aspects of the spectra are not yet as well understood, cobalt has two important advantages over copper as a metal substitute, namely that many cobalt derivatives show some enzymic activity (e.g. cobalt in carbonic anhydrase, alkaline phosphatase and superoxide dismutase) and that g values and hyperfine splitting are more sensitive to ligand environment, particularly when low spin. ESR data have been reported for cobalt substituted thermolysin, carboxypeptidase A, procarboxypeptidase A and alkaline phosphatase [51]. These are all high spin complexes. Cobalt carbonic anhydrase has been prepared and reacted with cyanide [52]. In... 

It is a proteolytic enzyme, present in the intestine in its inactive form (zymogen), trypsinogen. Trypsinogen is converted into its active form, trypsin, by enteropeptidase, a specialized proteolytic enzyme secreted by intestinal cells. Some free trypsin formed also catalyses the conversion of trypsinogen into trypsin. Trypsin can also convert chymotrypsinogen and procarboxypeptidase into chymotrypsin and carboxypeptidase, respectively. Trypsin has different amino acid specificity when compared with other proteolytic enzymes. Trypsin hydrolyses those peptide bonds whose carboxyl groups are contributed by Lys or Arg residues and if the next residue is not proline. The number of smaller peptides resulting from trypsin action is equal to the total number of Arg and Lys residues in the protein plus one. 

The carboxypeptidases are produced as procarboxypeptidases, which are cleaved to the active form by trypsin. 

The effects of zinc removal from bovine procarboxypeptidase A are similar to those for zinc removal from the carboxypeptidase the difference in activities between the enzyme and the zymogen may be accounted for in part by an altered environment for the catalytically essential glutamate-270 residue. The chloride... 

Replacement of the zinc by cobalt in either bovine procarboxypeptidase or carboxypeptidase resulted in a visible-range CD spectrum, which was inverted and increased in magnitude upon addition in glvcyl-L-tyrosine. Correlations between the optical properties and the catalytic potential of the metalloproteins have been proposed (127). 

The smaller peptides formed by the action of trypsin, chymotrypsin, and elastase are attacked by exopeptidases, which are proteases that cleave one amino acid at a time from the end of the chain. Procarboxypeptidases, zymogens prodnced by the pancreas, are converted by trypsin to the active carboxypeptidases. These exopeptidases remove amino acids from the carboxyl ends of peptide chains. Carboxypeptidase A preferentially releases hydrophobic amino acids, whereas car-boxypeptidase B releases basic amino acids (arginine and lysine). 

Two procarboxypeptidases are found in bovine pancreas. Both of these hexopeptidases are zinc enzymes, and the presence of zinc in the molecule is essential. Indeed, if zinc is eliminated by dialysis, the enzyme loses its activity, but activity may be restored by reintroducing zinc into the molecule. Both enzymes exist in the form of propeptidases which can be activated by splitting a polypeptide chain. The molecular weight of carboxypeptidase A ranges between 94,000 and 96,000, and its isoelectric point is below pH 4.5. 

Carboxypeptidaae. Carboxypeptidase occurs in extracts of pancreas. The pancreas contains an inactive precursor of the enzyme, a zymogen. In the case of carboxypeptidase the precursor has been partially purified. The accumulation of zymogens seems to be characteristic for those proteolytic enzymes that are secreted. In neutral solutions procarboxypeptidase is converted to active carboxypeptidase by the action of other enzymes present in the crude extracts. The mechanism of this activation is not completely known, although it appears that the reaction involves splitting of peptide bonds by other proteolytic enzymes. 

In the examples cited above, both zymogens and enzymes have been crystallized and therefore studied in relatively pure condition. There is evidence, however, that pancreatic carboxypeptidase is secreted as a zymogen, since fresh pancreatic extracts contain little or no carboxypeptidase activity. Upon incubation at 37 or treatment with trypsin the formation of carboxypeptidase occurs. This procarboxypeptidase has been partially purified. 

Pepsin is secreted in the gastric juice as pepsinogen, which is activated by the action of gastric acid, and also by the action of already activated pepsin. In the small intestine, trypsinogen, the precursor of trypsin, is activated by the action of a specific enzyme, enteropeptidase (sometimes known by its obsolete name of enterokinase), which is secreted by the duodenal epithelial cells trypsin can then activate chymotrypsinogen to chymotrypsin, proelastase to elastase, procarboxypeptidase to carboxypeptidase and proaminopeptidase to aminopeptidase. 

Carboxypeptidases may be found in the kidney and spleen, but occur in the digestive juice in particularly large amounts. One carboxypeptidase has been purified to the crystalline stage it has a molecular weight of 34,000 and contains zinc. It is secreted from the pancreas in the form of an inactive precursor (procarboxypeptidase) with a molecular weight of 90,000. 

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