Leucine aminopeptidases

Piacenza L., Acosta, D., Basmadjan, I., Dalton, J.P. and Carmona, C. (1999) Vaccination with cathepsin L proteases and with leucine aminopeptidase induces high levels of protection against fascioliasis in sheep. Infection and Immunity 67, 1954-1961. 

Non-corrin cobalt has a number of interesting applications in the chemical industry, for example in the hydroformylation (OXO) reaction between CO, H2 and olefins. A number of non-corrin Co-containing enzymes have been described, including methionine aminopep-tidase, prolidase, nitrile hydratase and glucose isomerase. We describe the best characterized of these, namely the E. coli methionine aminopeptidase, a ubiquitous enzyme, which cleaves N-terminal methionine from newly translated polypeptide chains. The active site of the enzyme (Figure 15.13) contains two Co(II) ions that are coordinated by the side-chain atoms of five amino acid residues. The distance between the two Co2+ is similar to that between the two Zn2+ atoms in leucine aminopeptidase, and indeed the catalytic mechanism of methionine aminopeptidase shares many features with other metalloproteases, in particular leucine aminopeptidases. 

See Section IV.1 for alternative methods of chiral resolution. Partial chemical hydrolysis of proteins and peptides with hot 6 M HC1, followed by enzymatic hydrolysis with pronase, leucine aminopeptidase and peptidyl D-amino acid hydrolase, avoids racemiza-tion of the amino acids281. The problems arising from optical rotation measurements of chiral purity were reviewed. Important considerations are the nonideal dependence of optical rotation on concentration and the effect of chiral impurities282. 

A novel concept of using bioadhesive polymers as enzyme inhibitors has been developed [97]. Included are derivatives of poly acrylic acid, polycarbophil, and car-bomer to protect therapeutically important proteins and peptides from proteolytic activity of enzymes, endopeptidases (trypsin and a-chymotrypsin), exopeptidases (carboxypeptidases A and B), and microsomal and cytosolic leucine aminopeptidase. However, cysteine protease (pyroglutamyl aminopeptidase) is not inhibited by polycarbophil and carbomer [97]. 

LEUCINE 2,3-AMINOMUTASE LEUCINE AMINOPEPTIDASE LEUCINE AMINOTRANSFERASE... 

Enzymes activities are particularly sensitive to the anticoagulant used in collecting the specimen. Heparin inhibits acid phosphatase (W16) and muramidase (Z5). Amylase activity is inhibited by oxalate or citrate (MIO), and lactic dehydrogenase and acid phosphatase lose activity in oxalate (C2). Alkaline phosphatase is stable in oxalate, oxalate-fluoride, or heparin, but 25 mAf citrate inhibits 50% of the activity, and as little as 50 mlf EDTA is completely inhibitory (B19). Leucine aminopeptidase is inhibited by EDTA, as is creatine phosphokinase (F3). Amylase activity has been reported to be only 83% of that in serum when oxalate or citrate-plasma is used (MIO). Heparin plasma appears to have no inhibitory effect. Despite the fact that clotting factor V is not stable in oxalate or EDTA, these are often used as anticoagulants to obtain plasma for prothrombin determinations (Z2, Z4). 

Fig. 36. The three-dimensional structure of the binuclear zinc cluster of leucine aminopeptidase, as determined by X-ray crystallographic methods (Burley etai, 1990 S. K. Burley and W. N. Lipscomb, personal communication).

For the analytical characterization of sulfated tyrosine peptides, spectroscopic methods as well as amino acid analysis and, more recently, mass spectrometry are employed. In Table 2 the spectroscopic data of tyrosine 0-sulfate are compared to those of the related sulfonic acid derivatives as possible byproducts in the chemical sulfation of the tyrosine or tyrosine peptides.[361 In the course of the synthesis of tyrosine 0-sulfate peptides and, particularly in the final deprotection step, desulfation may occur which limits the characterization of the final compounds in terms of quantitative identification of the tyrosine 0-sulfate. This is achieved by amino acid analyses of basic hydrolysates of the sulfated tyrosine peptides or preferably by analyses of the enzymatic hydrolysates with aminopeptidase M or 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. 

Four minor metabolites, psammaplins B-D (503-505) and presammaplin A (506) were isolated from Psammaplysilla purpurea, in addition to psammaplin A (500). Psammaplin B (503) is a thiocyanate bromotyrosine derivative, while psammaplin C (502) is a sulfanamide. Psammaplin D (505) displayed antimicrobial activity and mild tyrosine kinase inhibition [429]. The psammaplins Ai (507) and A2 (508) and aplysinellins A (509) and B (510) were isolated from Aplysinella rhax from both Pohnpei and Palau. These compounds inhibit famesyl protein transferase and leucine aminopeptidase [430]. Another sample of A. rhax from the Great Barrier Reef, Australia contained psammaplin A 11 -sulfate (511) and bisaprasin ll -sulfate (512), both of which inhibited [3H]-l,3-dipropyl-8-cyclopentylxanthine binding to rat brain adenosine Ai receptors [431]. 

Thiourea probably acts in a manner comparable to that of Naddtc and should also be administered after cis-Pt treatment (78). Like Naddtc, thiourea is able to remove platinum from platinated enzymes, such as leucine aminopeptidase (76, 128), y-glutamyltranspeptidase (76,128), and fumarase (129) (Fig. 9), and from Pt-methionine model adducts (Table III) (131). However, thiourea appears to be less useful as an inhibitor of nephrotoxicity it also reacts quite rapidly with platinum-DNA cross-links (56). 

Many aminopeptidases are metalloenzymes.437 Most studied is the cytosolic leucine aminopeptidase which acts rapidly on N-terminal leucine and removes other amino acids more slowly. Each of the subunits of the hexameric enzyme contains two divalent metal ions, one of which must be Zn2+ or Co2+438/439 A methionine aminopeptidase from E. colt contains two Co2+ ions440/441 and a proline-specific aminopeptidase from the same bacterium two Mn2+.442 In all of these enzymes the metal ions are present as dimetal pairs similar to those observed in phosphatases and discussed in Section D,4 and to the Fe-Fe pairs of hemerythrin and other diiron proteins (Fig. 16-20). A hydroxide ion that bridges the metal ions may serve as the nucleophile in the aminopeptidases.438 A bound bicarbonate ion may assist.4383... 

Racemic piperazine-2-carboxylic acid (4-azapipecolic acid, 22) is obtained by hydrogenation of pyrazine carboxylic acid salts/236 For its enantiomeric resolution several methods have been described, e.g. fractional crystallization of its S-camphor-10-sulfonic add salt/236,237 fractional crystallization of l,4-dibenzylpiperazine-2-carboxylic add menthyl ester/238 or selective digestion of racemic piperazine-2-carboxylic add amide by leucine aminopeptidase/239 ... 

Reports relating to the quaternary structure of leucine aminopeptidases have appeared,1297,1298 including a preliminary X-ray study.1299 In the latter work at 2.2 A resolution a P6322 space group is revealed, the dimensions of the unit cell being a = 132 and c = 122 A, The asymmetric unit is a protomer of RMM 54 000. 

Leucine aminopeptidase may be isolated from Aspergillus oryzae.1300 The RMM (37 500) of this enzyme is significantly smaller than that from mammalian sources, but, like the latter, it is a metalloenzyme. 

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