Pro-carboxypeptidase

Sato T Miwa T, Asatsu H, et al. Pro-carboxypeptidase R is an acute phase protein in the mouse, whereas carboxypeptidase N is not. J Immunol 2000 165 1053-1058. 

Dietary proteins, with very few exceptions, are not absorbed rather they must be digested into amino acids, or di- and tripeptides. Protein digestion begins in the stomach, where proenzyme pepsinogen is autocatalytically converted to pepsin A. Most proteolysis takes place in the duodenum via enzymes secreted by the pancreas, including trypsinogen, chymotrypsinogen and pro-carboxypeptidase A. These serine and zinc proteases are produced in the form of their respective proenzymes they are both endopeptidase and exopeptidase, and their combined action leads to the production of amino acids, dipeptides and tripeptides. 

Carboxypeptidase is an exopeptidase that specifically hydrolyzes the C-terminal peptide bond and releases the C-terminal amino acid. Two problems are associated with its use the substrate specificity of the enzyme and the continuous action of the enzyme. The continuous action may yield the second, third, and additional residues from some chains even before the terminal residues on every chain are quantitatively released. Thus, it may be difficult to determine which residue is the C terminus. However, monitoring the sequential release of amino acids can often reveal the sequence of several residues at the C terminus. Concerning specificity, carboxypeptidase A releases all C-terminal residues except Lys, Arg, and Pro carboxypeptidase B cleaves C-terminal Arg and Lys residues and carboxypeptidase C hydrolyzes C-terminal Pro residues. Thus, more than one method may be needed to establish the C-terminal amino acid. 

Group II consists of the enkephalins which come from the 267-aniino acid piecuisoi pro-enkephalin A [88402-54-4] (Fig. 2). This proteia contains four copies of Met-enkephalin, one copy of Leu-enkephalin, and the extended peptides Met-enkephalin-Arg -Phe (the last Met-enkephalin sequence ia Fig. 2) and Met-enkephalin-Arg -Gly -Leu (the fourth Met-enkephalin sequence ia Fig. 2) (25,26). AH of these products ate formed by trypsin-like cleavage between pairs of basic residues. The extended enkephalin peptides are further cleaved by carboxypeptidase E (27) to form authentic Met-enkephalin. 

Increased permeability is just one prerequisite in the development of useful peptide prodrugs. Another condition is that efficient bioactivation must follow absorption. Mucosal cell enzymes able to hydrolyze peptides include exopeptidases such as aminopeptidases and carboxypeptidases, endopepti-dases, and dipeptidases such as cytosolic nonspecific dipeptidase (EC 3.4.13.18), Pro-X dipeptidase (prolinase, EC 3.4.13.4), and X-Pro dipeptidase (prolidase, EC 3.4.13.9). For example, L-a-methyldopa-Pro was shown to be a good substrate for both the peptide transporter and prolidase. This dual affinity is not shared by all dipeptide derivatives, and, indeed, dipeptides that lack an N-terminal a-amino group are substrates for the peptide transporter but not for prolidase [29] [33] [34],... 

The decapeptide Hoe 140 (6.94), its D- and/or artificial residues being d-Arg, ((4/ )-hydroxy)Pro, (2-thienyl)Ala, D-[(l,2,3,4-tetrahydroisoquinolin-3-yl)carbonyl] and i.-[(3aS, 7aS)-octahydroindol-2-yl carbonyl, is a potent and long-acting antagonist of bradykinin receptors [219][220]. This compound proved highly resistant to enzymatic degradation. It is not a substrate for kini-nase II and carboxypeptidases, and is only slowly degraded in human plasma. 

ADEPT strategies have been described but only the carboxypeptidase G2 approach has been tested in patients so far. In a phase I clinical trial, patients with non-resectable metastatic or locally recurrent colorectal carcinoma were treated with ADEPT. Carboxypeptidase G2 activity was found in metastatic tumour biopsies. The pro-drug was converted into the active drug but leakage into the bloodstream also occurred [22,144]. 

Mechanisms similar to the one described for carboxypeptidase appear to operate in the hydrolysis of amide and ester bonds catalyzed by a number of pro-... 

Membrane Pro-X Carboxypeptidase P, Zinc, manganese 1,10-Phenanthroline, EDTA,... 

While establishing purifying for aspartic proteinase (aspergillopepsin I, EC 3.4.23.18) [7, 8] of Aspergillus saitoi, which is a food microorganism strain, it was discovered to be a rich source of acid carboxypeptidase (EC 3.4.16.5), which removes acidic, neutral, and basic amino acids as well as proline from the carboxyterminal position at pH around 3 [79, 80], The optimum pH with Z-Tyr-Leu (Z- = bebzyloxycarbonyl-) of the acid carboxypeptidase from A. saitoi was 3.5. The optimum with Z-Glu-Tyr was 3.1, and that with Z-Gly-Pro-Leu-Gly, 3.2. 

Several qualitative conclusions can be drawn from the results. The acid carboxypeptidase splits off neutral and basic amino acids from the substrates and has the ability to hydrolyze the peptide bond, —X-Pro in oligopeptides. 

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

In order to determine the carboxy-terminus of Hez-PBAN,. 200 pmol of purified peptide was digested with carboxypeptidase P. Released amino acids were periodically analyzed as their PTC derivatives. Leu was found to be the C-terminal residue followed by Arg, Pro, Ser, Phe, Tyr, and Lys, respectively, thus confirming the automated Edman degradation data (Table I, run 5). However, none of the data could distinguish between a C-terminal amide or free acid. 

Figure 3 Cysteine protease and subtilisin-like protease pathways for proneuropeptide processing. Distinct cysteine protease and subtilisin-like protease pathways have been demonstrated for pro-neuropeptide processing. Recent studies have identified secretory vesicle cathepsin L as an important processing enzyme for the production of the endogenous enkephalin opioid peptide. Preference of cathepsin L to cleave at the NH2-terminal side of dibasic residue processing sites yields peptide intermediates with NH2-terminal residues, which are removed by Arg/Lys aminopeptidase. The well-established subtilisin-like protease pathway involves several prohormone convertases (PC). PC1/3 and PC2 have been characterized as neuroendocrine processing proteases. The PC enzymes preferentially cleave at the COOH-terminal side of dibasic processing sites, which results in peptide intermediates with basic residue extensions at their COOH-termini that are removed by carboxypeptidase E/H.

Deduce the sequence of amino acids in a peptide from the following information (a) composition = phe4- pro+gIu-l-2 lys (b) treatment with the Edman reagent yielded PTH-glutamale and (c) trypsin, car-boxypeptidase A, and carboxypeptidase B did not release any smaller peptides or amino acids. 

Although any of several combinations of proteases can be used, ideally, one or more non-specific endopeptidases should be used first to convert the protein into many small peptides. These small peptides can then be degraded to amino acids by aminopeptidases and prolidase (hydrolyzes X-Pro bonds). Sometimes, carboxypeptidases are also used. Although leucine aminopeptidase has been used as the amino-peptidase (see Hill and Schmidt 1962), it may be preferable to use aminopeptidase M (Rohm and Haas, supplied by Henley and Co. of N.Y.), since this enzyme removes most residues at acceptable rates. Leucine aminopeptidase removes hydrophobic residues most rapidly, whereas some other residues are removed very slowly. Most procedures should probably include the use of prolidase (Miles) since many aminopeptidases do not cleave X-Pro bonds at appreciable rates. If it is found that proline is not released quantitatively by these procedures, the use of citrus leaf carboxypeptidase C (Rohm and Haas) can be tried after the initial endopeptidase hydrolysis and before the addition of aminopeptidase M and prolidase. Carboxypeptidase C (also yeast carboxypeptidase Y - see Hayashi et al. 1973) hydrolyzes proline bonds (as well as all others), but if proline is at or adjacent to the NH2 terminus of a peptide, it would probably not be released. In all procedures a control consisting of the enzymes only should be run in parallel with the hydrolyzed sample, and corrections should be made for any amino acids found by analysis of the control. suhic / /< > , mi... 

The kinetics of action of carboxypeptidase are very complex. The pH-rate profile for CPA-catalyzed hydrolysis of peptides is bell shaped, with apparent pK values of about 6.5 and 7.5. The former pK value could be attributed to Glu-270. The pH dependency of the kinetics of the CPA-catalyzed enolization of the ketonic substrate (R)-2-benzyl-3-(p-methoxybenzoyl)pro-pionic acid leads to the establishment (with minimum complications from this relatively simple reaction) of a pK value of 6.03 for the Co" and Zn" CPA, which is probably due to Glu-270. The binding of the substrate to both Zn" and Co" CPA appears to depend on an enzyme-bound group with pXa = 7.56 and 8.29 respectively, and on a group with pXa>9. These are attributed to the ionization of Tyr-248 and the bound water molecule. 

A limited amount of information can be obtained by the use of proteolytic enzymes that detach either amino acids or dipeptides sequentially from the C-terminus. They are thus complementary to the aminopeptidases and dipeptidyl aminopeptidases. Two pancreatic enzymes, carboxypeptidases A and B, differ in specificity. The former preferentially liberates C-terminal amino acids with aromatic side chains, somewhat less readily amino acids with alkyl side chains and, more slowly still, other amino acids, but not Pro, Arg, Lys and His. In contrast, carboxypeptidase B releases only C-terminal Arg, Lys and His. Carboxypeptidase Y is much less specific and is capable of removing all amino acids, although Gly and Pro are liberated only slowly. As with aminopeptidases, it is advisable to analyse the hydrolysate at intervals in order to determine the C-terminal sequence of amino acids. An interesting recent development (Carles et al., 1988) uses carboxypeptidase to effect transpeptidation between the protein being sequenced and a tritiated amino acid. The labelled protein is then degraded by various specific methods and then the labelled fragments are isolated by gel electrophoresis and subjected to Edman degradation. 

Dipeptidyl carboxypeptidases remove the C-terminal dipeptide intact and therefore are analogous to the dipeptidyl aminopeptidases such as cathepsin C. One such enzyme, angiotensin-converting enzyme, is important biologically for converting angiotensin I into the hypertensive angiotensin II (see Section 9.3). This enzyme does not hydrolyse bonds of the type X—Pro but will hydrolyse Pro—X bonds. The use of dipeptidyl carboxypeptidases for sequence determination would probably increase if pure enzymes were readily available commercially. 

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