Amidases peptide amidase

This amide, readily formed from an amine and the anhydride or enzymatically using penicillin amidase, is readily cleaved by penicillin acylase (pH 8.1, A -methylpyrrolidone, 65-95% yield). This deprotection procedure works on peptides, phosphorylated peptides, and oligonucleotides, as well as on nonpeptide substrates. The deprotection of racemic phenylacetamides with penicillin acylase can result in enantiomer enrichment of the cleaved amine and the remaining amide. An immobilized form of penicillin G acylase has been developed. ... 

Tretter, V., Altmann, F. and Marz, L. (1991) Peptide-A74-(A-acetyl-P-glucosamiriyl) asparagine amidase F cannot release glycans with fucose attached al-3 to the asparagine-linked A-acetylglucosamin e residue. European Journal of Biochemistry 199, 647-652. 

Cysteine string protein (CSP) Cytochrome b561 Peripheral membrane protein that is paimitoylated on >10 cysteines. May have a role in Ca2+ sensitivity of exocytosis. Electron-transport protein required for intravesicular monooxygenases in subsets of secretory vesicles. Required for dopamine- -hydroxylase and peptide amidase activity. 

Such results are certainly very interesting, but the stability of /3- and y-peptides toward a greater variety of amidases (EC 3.5) and even esterases (EC 3.1) should also be examined in detail. 

In one case, a small peptide with enzyme-like capability has been claimed. On the basis of model building and conformation studies, the peptide Glu-Phe-Ala-Ala-Glu-Glu-Phe-Ala-Ser-Phe was synthesized in the hope that the carboxyl groups in the center of the model would act like the carboxyl groups in lysozyme 17). The kinetic data in this article come from assays of cell wall lysis of M. lysodeikticus, chitin hydrolysis, and dextran hydrolysis. All of these assays are turbidimetric. Although details of the assay procedures were not given, the final equilibrium positions are apparently different for the reaction catalyzed by lysozyme and the reaction catalyzed by the decapeptide. Similar peptide models for proteases were made on the basis of empirical rules for predicting polypeptide conformations. These materials had no amidase activity and esterase activity only slightly better than that of histidine 59, 60). 

An exo-linker according to Fig. 10.1 must contain an enzyme labile group R, which is recognized and attacked by the biocatalyst Possible combinations could be phenylacetamide/penicillin amidase, ester/esterase, monosaccharide/glycosid-ase, phosphate/phosphatase, sulfate/sulfatase and peptides/peptidases [41]. The following systems have been worked out (Tab. 10.2). 

Valina ALB, Mazumder-Shivakumar D, Bruice T C (2004) Probing the Ser-Ser-Lys catalytic triad mechanism of peptide amidase computational studies of the ground state, transition state, and intermediate. Biochemistry 43 15657-15672... 

The use of enzymes and whole cells as catalysts in organic chemistry is described. Emphasis is put on the chemical reactions and the importance of providing enantiopure synthons. In particular kinetics of resolution is in focus. Among the topics covered are enzyme classification, structure and mechanism of action of enzymes. Examples are given on the use of hydrolytic enzymes such as esterases, proteases, lipases, epoxide hydrolases, acylases and amidases both in aqueous and low-water media. Reductions and oxidations are treated both using whole cells and pure enzymes. Moreover, use of enzymes in sngar chemistiy and to prodnce amino acids and peptides are discnssed. 

The optimum yield of a condensation product is obtained at the pH where Ka has a maximum. For peptide synthesis with serine proteases this coincides with the pH where the enzyme kinetic properties have their maxima. For the synthesis of penicillins with penicillin amidase, or esters with serine proteases or esterases, the pH of maximum product yield is much lower than the pH optimum of the enzymes. For penicillin amidase the pH stability is also markedly reduced at pH 4-5. Thus, in these cases, thermodynamically controlled processes for the synthesis of the condensation products are not favorable. When these enzymes are used as catalysts in thermodynamically controlled hydrolysis reactions an increase in pH increases the product yield. Penicilhn hydrolysis is generally carried out at pH about 8.0, where the enzyme has its optimum. At this pH the equiUbrium yield of hydrolysis product is about 97%. It could be further increased by increasing the pH. Due to the limited stability of the enzyme and the product 6-aminopenicillanic acid at pH>8, a higher pH is not used in the biotechnological process. 

When eledoisin-related peptide was used as a substrate, the enzyme quickly released methionine-amide rather than methionine from the carboxy-terminus of-FIGLM-NH2. In this case, the P3 position was occupied by the hydrophobic amino acid isoleucine. Furthermore, when D-amino acid occupied the P3 position of the substrate, as in enkepharin-amide, the enzyme did not liberate a carboxy-terminal methionine-amide but acted only as amidase for this substrate. It can be concluded that the S3-P3 interaction is important in increasing... 

SteUces-Ritter, U., Wyzgol, K., and Kula, M. 1995. Purification and characterization of a newly screened microbial peptide amidase. Applied Microbiology and Biotechnology, 44 393-8. 

A particularly elegant solution for the assay of proteases and acylases is offered by the fluorogenic detection of free amino acids by decomplexation of copper from calcein, which removes its quenching effect This principle has been used for assays of acylases, amidases, and proteases (Scheme 1.13) [52, 53]. For the case of proteases combinatorial assays are particularly in demand for testing multiple peptides in parallel and determining the cleavage specificity [54]. New solutions... 

The additional opportunity to hydrolyze selectively C-terminal peptide amides is of particular interest. For example, a peptide amidase from the flavedo of oranges has broad substrate specificity and accepts Boc-, Trt-, Z-, and Bz-protected and N-terminal unprotected peptide amides (Scheme The C-terminal amides are saponified by this biocatalyst in high yields... 

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