Procarboxypeptidase activation

The proteases are secreted as inactive zymogens the active site of the enzyme is masked by a small region of its peptide chain, which is removed by hydrolysis of a specific peptide bond. Pepsinogen is activated to pepsin by gastric acid and by activated pepsin (autocatalysis). In the small intestine, trypsinogen, the precursor of trypsin, is activated by enteropeptidase, which is secreted by the duodenal epithelial cells trypsin can then activate chymotrypsinogen to chymotrypsin, proelas-tase to elastase, procarboxypeptidase to carboxypepti-dase, and proaminopeptidase to aminopeptidase. 

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. 

The inactive precursors are called trypsinogen, pepsinogen, chymotrypsino-gen, and procarboxypeptidase. These precursors are converted to the active enzymes by hydrolytic cleavage of a few specific peptide bonds under the influence of other enzymes (trypsin, for example, converts chymotrypsinogen to chymotrypsin). The digestive enzymes do not appear to self-destruct, probably because they are so constructed that it is sterically impossible to fit a part of one enzyme molecule into the active site of another. In this connection, it is significant that chymotrypsin attacks denatured proteins more rapidly than natural proteins with their compact structures of precisely folded chains. 

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. 

Tan AK, Eaton DL. Activation and characterization of procarboxypeptidase B from human plasma. Biochemistry 1995 34 581 1-5816. 

The trypsin family of proteases plays a role in acute and chronic pancreatitis, as well as leads to its ultimate destruction [4, 105]. In pancreatitis, active exocrine enzymes are prematurely released inside the pancreatic duct. Various factors can contribute to the development of acute pancreatitis. Trypsinogen, chymotrypsinogen, procarboxypeptidase, and proelastase are inactive proforms of proteolytic enzymes produced by the pancreatic acinar cells. Following secretion these enzymes are activated in a cascade that converts trypsinogen to trypsin in the duodenum and/or small intestine. 

In the duodenum, the pancreatic zymogens, trypsinogen, chymotrypsinogen, proelastase and procarboxypeptidase are converted into active enzymes by enteropeptidase and trypsin, as shown in Fig. 15-6. The activation of all the zymogens involves cleavage of peptide bonds and removal of peptides, enabling conformational changes and formation of a functional active site. 

Trypsinogen, chymotrypsinogen, proelastase, and procarboxypeptidase are all synthesized as single polypeptide chains with an Mr around 25,000-30,000. The initial step in the activation is the... 

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... 

Zymogens have been considered to be inactive precursors of enzymes and the activation process to involve the generation of a catalytic or substrate binding site or both 44). Recently, Behnke and Vallee (50) found that the spectral properties of cobalt-substituted procarboxypeptidase A closely resemble those of the cobalt enzyme. Since these spectra were believed to be peculiar to enzymatically active proteins (5i), they investigated the intrinsic catalytic activity of the cobalt zymogen. Remarkably, with certain substrates, cobalt procarboxypeptidase was found to have as much activity, and in some cases even more than the native enzyme. These observations, as well as those of others (52), have questioned the entire concept of zymogens as inactive enzyme precursors. 

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 enzyme is prepared from the exudate of frozen sliced beef pancreas. The precursor, procarboxypeptidase, is activated at 37° C. pH 7.8 for 1 hour a crystalline enzyme is obtained by fractionation (Green and Neurath, 1954). Sevenfold recrystallization yields an enzyme which has been found essentially monodispersed by electrophoresis at pH 8.5, but a minor, faster moving component appears at pH 9.3 (Putnam and Neurath, 1946). The molecular weight has been calculated to be 34,300 (Putnam and Neurath,... 

Pancreatic juice contains the proenzymes trypsinogen, chymotrypsinogen, procarboxypeptidases, and proelas-tase. All are activated by trypsin in the intestinal lumen. Enteropeptidase located in the brush border of the jejunal mucosa converts trypsinogen to trypsin. A trypsin inhibitor in pancreatic juice protects against indiscriminate autodigestion from intraductal activation of trypsinogen. Other enzymes of pancreatic juice and their substrates are listed below. 

Chyme contains potent secretagogues for various endocrine cells in the intestinal mucosa. CCK and secretin cause release of an alkaline pancreatic juice containing trypsinogen, chymotrypsinogen, proelastase, and procarboxypeptidases A and B. Activation begins with that of trypsinogen to trypsin by enteropeptidase (previously called enterokinase) present in the brush-border membranes of the duodenum. 

The answer is d. (Murray, pp 48-62. Scriver, pp 3 5. Sack, pp 1-3. Wilson, pp 101-120.) Pepsin is secreted in a proenzyme form in the stomach. Unlike the majority of proenzymes, it is not activated by protease hydrolysis. Instead, spontaneous acid hydrolysis at pH 2 or lower converts pepsinogen to pepsin. Hydrochloric acid secreted by the stomach lining creates the acid environment. All the enzymes secreted by the pancreas are activated at the same time upon entrance into the duodenum. This is accomplished by trypsin hydrolysis of the inactive proenzymes trypsino-gen, chymotrypsinogen, procarboxypeptidase, and proelastase. Primer... 

V. Villegas, J. C. Martinez, F. X. Aviles et al. Structure of the transition state in the folding process of human procarboxypeptidase A2 activation domain. Journal of Molecular Biology, 283 (1998), 1027 F. Chiti, N. Taddei, P. M. White et al. Mutational analysis of acylphosphatase suggests the importance of topology and contact order in protein folding. Nature Structural Biology, 6 (1999), 1005. 

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... 

The zymogen trypsinogen is cleaved to form trypsin by enteropeptidase (a protease, formerly called enterokinase) secreted by the brush-border cells of the small intestine. Trypsin catalyzes the cleavages that convert chymotrypsinogen to the active enzyme chymotrypsin, proelastase to elastase, and the procarboxypeptidases to the car-boxypeptidases. Thus, trypsin plays a central role in digestion because it both cleaves dietary proteins and activates other digestive proteases produced by the panaeas. 

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). 

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