Peptidase leucine aminopeptidase

A method by Berger[12] utilized the stereoselective hydrolysis of diastereomeric peptides by leucine aminopeptidase. Z-L-Ala-D-Ala-OH was coupled to an all L-Ala peptide such as l-Ala-L-Ala-ONbz. Epimerization during coupling resulted in the formation of a small amount of L-Ala-L-Ala-L-Ala-L-Ala after deprotection, and since the peptidase has an absolute specificity for the all-L peptide, only the epimerized product was hydrolyzed. Quantification of the degradation products gave the extent of epimerization. These classical procedures, however, are specific to the particular coupling reaction under consideration and the results may not be fully applicable to all systems. Furthermore, they give no direct information about the rate of racemization. 

Ribonuclease Ti is fairly resistant to proteases. The threonine residue at the carboxyl terminal of the enzyme can be removed by carboxy-peptidase A without loss of activity (67). Leucine aminopeptidase does not release amino acids from the amino terminal (68). Ribonuclease Ti is not inactivated by trypsin or chymotrypsin in the presence of 0.2 M phosphate (69), which probably binds the enzyme and protects it from inactivation (67). Treatment of the enzyme with trypsin in the absence of phosphate inactivates it (67). Ribonuclease Tj is hydrolyzed by pepsin with progressive loss of activity (69). 

Various peptide Michael acceptors have been described as a new class of inactivators for cysteine proteases. 5-7 The carbonyl group of the scissile peptide bond in the substrate is replaced by a nucleophile trapping moiety such as a vinylogous structure. An amino acid vinyl sulfone, l-(methylsulfonyl)-4-phenylbut-l-en-3-amine [H2NCH(Bzl)CH=CHS02Me] and a dipeptide derivative, Gly-HNCH(Bzl)CH=CHS02Me have both been prepared as inhibitors of cysteine proteases, leucine aminopeptidase and dipeptidyl peptidase I, respectively.1 5 A series of peptide vinyl sulfones has been synthesized as potent inhibitors for different cysteine proteases. 1A8 ... 

One of the earliest suggestions that total enzymatic hydrolysis was possible came from the studies of Frankel (1916), who showed that over 90 % of the bonds in several proteins could be broken when proteolysis with pepsin, trypsin, and chymotrypsin was followed by prolonged hydrolysis with the erepsin preparation of Cohnheim (1901). The recognition in later years of several peptidases in intestinal exti acts which will specifically act upon bonds that are not susceptible to the endopoptidases (Bcrg-mann, 1942) probably accounts for these obseiwations. The specific peptidases such as prolidase, iminodipeptidase (prolinase), glycylglycine dipeptidase, tripeptidase, and leucine aminopeptidase, whi( h are present in mucosa, attack many of the bonds that resist the action of endopoptidases. 

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... 

Proteasomes rather than cytosolic carboxy-peptidases act to trim the C-terminal amino acids to conform the peptide to the proper size for MHC class-I presentation. Presentation from N-extended precursors is inhibited by acetylation of the terminal a-amino group at the N-terminus [354], which prevents the peptide to be cleaved by aminopeptidases e.g. leucine aminopeptidase) but not by proteasomes or endopeptidases. The TAP system transports peptides to the ER including both mature epitopes and longer precursors. It seems then that the peptides to be presented by MHC class-I can arise from N-extended precursors both in the cytosol and in the endoplasmic reticulum (ER). This assertion has been experimentally confirmed [355,356]. 

This mechanism may best be exemplified by metal-dependent proteases and peptidases such as thermolysin, leucine aminopeptidase, and carboxypeptidase A. Of these, the most systematic study of proton transfer has been conducted with ther-... 

Leucine aminopeptidase R = r-Leucyl Trypsin, trypsiniiine amidase R = a-Benzoyl-L-arginine Peptidase R = Phenyi alanyl... 

Aminopeptidases generally require somewhat longer reaction times (60 to 300 s). Commercially available amino peptidases include aminopeptidase M and leucine aminopeptidase. Again, digestion of the -amyloid peptide fragment DAEFR using aminopeptidase would be expected to yield the molecular-ion species ... 

A recent study, however, has shown that aminopeptidase activity is present on the surface of porcine buccal mucosa, and that various aminopeptidase inhibitors, including amastatin and sodium deoxycholate, reduce the mucosal surface degradation of the aminopeptidase substrate, leucine-enkephalin [149], Since the peptidases are present on the surface of the buccal mucosa, they may act as a significant barrier to the permeability of compounds which are substrates for the enzyme. In addition to proteolytic enzymes, there exist some esterases, oxidases, and reductases originating from buccal epithelial cells, as well as phosphatases and carbohydrases present in saliva [154], all of which may potentially be involved in the metabolism of topically applied compounds. 

There is evidence for the presence of active hydrolytic and proteolytic enzymes within the nasal mucosal, including various aminopeptidases (25). Hussain et al (31) have reported extensive enzymatic hydrolysis of leucine enkephalin, intranasally, which can be inhibited in vitro by various competitive di-and tripeptide fragments. The data on nasal peptidase activity is too incomplete to predict the metabolic consequences for other peptide drugs. 

Aminopeptidases, enzymes that cleave olf the N-terminal amino acid from a peptide chain, are bismetallo peptidases, a class of metallopeptidase that contain two metals ions in the catalytic site (117,118). These can be inhibited by compounds related to bestatin (60)(Fig. 15.28), which contains the iV-tenni-nal a-hydroxy-j3-amino acid residue, sometimes referred to as norstatine. In leucine amino peptidase, chelation occurs between both the amide carbonyl group and the adjacent hydroxyl and the hydroxyl and the N-terminal amino group (119,120). 

Figure 6.1. Diagramatic representation of the peptidases involved in the metabolism of the enkephalins. AP (aminopeptidases), DAP (dipeptidylaminopeptidase), ACE (angiotensin converting enzyme), ENK (enkephalinase) and CBXYP (non-specific carboxypeptidases). R represents methionine in f MetJ-enkephalin or leucine in [Leu]-enkephalin.

C16H24N2O4, Mr 308.38, needles, mp. 233-236 C, [a)o -15.5° (1 M HCl). Peptide antibiotic from Strep-tomyces olivoreticuli with antitumor activity, it is a specific inhibitor of aminopeptidase B, a leucine amino-peptidase. B. activates macrophages and T-lympho-cytes and in vitro increases not only blastogenesis of lymphocytes but also the manifestation of delayed hypersensitivity. It is used clinically in cancer therapy. tM. Annu. Rev. Microbiol. 36, 75 (1982)"J. Org. Chem. 54, 4235 (1989) 55, 2232 (1990) Merck-Index (12.), No. 9973 Tetrahedron Lett. 33,6803 (1992) Ullmann (5.) A2,531. -(CAS 58970-76-6]... 

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