Prolyl aminopeptidases

Aminopeptidase P (EC 3.4.11.9 prolyl aminopeptidase) is located in the plasma membrane and is a zinc-metalloproteinase. Notable neuropeptide substrates include bradykinin, substance P, neuropeptide Y, peptide YY and enterostatin. Inhibitors include apstatin. 

Prolyl aminopeptidases (PAP) are exopeptidases that hydrolytically cleave off an N-terminal Pro from peptides. The enzymes belong to the a/p hydrolase fold proteins. A PAP from Streptomyces thermoluteus carrying the active site nucleophile mutation S144C was used as a catalyst for the synthesis of proline-containing peptides. Dipeptide synthesis was obtained with an amino acid methyl or benzyl ester as the acyl donor and prolyl-OBz as the nucleophile [21]. Under alkaline conditions, cycli-zation and polymerization of prolyl-OBz was observed. 

Yamamoto, Y., Usuki, H., Iwabuchi, M., Hatanaka, T. (2010). Prolyl aminopeptidase from Streptomyces thermoluteus subsp. fuscus strain NBRC14270 and synthesis of proline-containing peptides by its S144C variant. Appl. Environ. Microbiol., 76,6180-6185. 

Other interesting examples of proteases that exhibit promiscuous behavior are proline dipeptidase from Alteromonas sp. JD6.5, whose original activity is to cleave a dipeptide bond with a prolyl residue at the carboxy terminus [121, 122] and aminopeptidase P (AMPP) from E. coli, which is a prohne-specific peptidase that catalyzes the hydrolysis of N-terminal peptide bonds containing a proline residue [123, 124]. Both enzymes exhibit phosphotriesterase activity. This means that they are capable of catalyzing the reaction that does not exist in nature. It is of particular importance, since they can hydrolyze unnatural substrates - triesters of phosphoric acid and diesters of phosphonic acids - such as organophosphorus pesticides or organophosphoms warfare agents (Scheme 5.25) [125]. 

At the C-terminus, prolyl oligopeptidase (postproline endopeptidase, EC 3.4.21.26) cleaves the C-terminal dipeptide [178]. The resulting oxytocin-(1-7) is also a substrate for aminopeptidase. Furthermore, neprilysin (EC 3.4.24.11) can also play a role in oxytocin degradation, although it seems to act with less efficiency than the two other enzymes. 

This enzyme [EC 3.4.11.9] (also known as Xaa-Pro aminopeptidase, X-Pro aminopeptidase, proline amino-peptidase, and aminoacylproline aminopeptidase) catalyzes the hydrolysis of a peptide bond at the iV-terminus of a peptide provided that the iV-terminal amino acyl residue is linked to a prolyl residue by that peptide bond. The enzyme will also act on dipeptides and tripeptides with that same restriction. Either manganese or cobalt is needed as a cofactor. This enzyme appears to be a membrane-bound system in both mammalian and bacterial cells. The protein belongs to the peptidase family M24B. 

This zinc-dependent enzyme [EC 3.4.11.1], also referred to as cytosol aminopeptidase, leucyl aminopeptidase, and peptidase S, catalyzes the hydrolysis of a terminal peptide bond such that there is a release of an N-terminal amino acid, Xaa-Xbb-, in which Xaa is preferably a leucyl residue, but may be other aminoacyl residues including prolyl (although not arginyl or lysyl). Xbb may be prolyl. In addition, amino acid amides and methyl esters are also readily hydrolyzed, but the rates with arylamides are exceedingly slow. The enzyme is activated by heavy metal ions. 

This methodology was employed in a short synthesis of bestatin 26 (Scheme 2.11) [24] which acts as a potent inhibitor of aminopeptidase and prolyl endopepti-dase. 

Peptidases such as aminopeptidases and diamino-peptidases are at lower concentrations in the colon than in the small intestine. However, it has been shown that prolyl endoprotease and collagenase activities are five to six times higher in the colon than in the small intestine. TRH is more readily hydrolyzed to deaminated TRH in colonic homogenates compared with small intestine and rectum. Therefore, peptide drugs that are substrates for prolyl endoprotease and collagenase will likely be degraded in the colon and may not be suitable for colon delivery. 

Bockelmann, W., Fobker, M., and Teuber, M. (1991). Purification and characterization of the X-prolyl-dipeptidyl-aminopeptidase from Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus acidophilus. Ini Dairy J. 1, 51-66... 

Khalid, N. M., and Marth, E. H. (1990d). Purification and partial characterization of a prolyl-dipeptidyl aminopeptidase from Lactobacillus helveticus CNRZ 32. Appl. Environ. Microbiol. 56, 381-388. 

Meyer, J., and Jordi, R. (1987). Purification and characterization of X-prolyl-dipeptidyl-aminopeptidase from Lactobacillus lactis and from Streptococcus thermophilus. J. Dairy Sci. 70, 738-745. 

Miyakawa, H., Hashimoto, I., Nakamura, T., Ishibashi, N., Shimamura, S., and Igoshi, K. (1994). Purification and characterization of an X-prolyl dipeptidyl aminopeptidase from Lactobacillus helveticus LHE-511. Milchwissenschaft 49, 670-673. 

Bordallo, C., Schwencke, J., and Suarez-Renduelles, M. P., 1984. Localization of the thermosensitive X-prolyl-dipeptidyl aminopeptidase in the vacuolar membrane of Saccharomyces cerevisiae, FEES Lett., 173 199. 

S., 1981. A new X-prolyl-dipeptidyl aminopeptidase from yeast associated with a particulate fraction, FEES Lett., 131 296. 

Kato, T Nagatsu, T Kimura, T Sakakibara, S. Fluorescence assay of X-prolyl dipeptidyl-aminopeptidase activity with a new... 

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