Amidases and Proteases

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... 

As discussed above, OPs are enzyme inhibitors, most obviously antiesterases and the most important esterase OPs inhibit is acetylcholinesterase. However, there is increasing evidence that some toxic effects are not mediated by inhibition of acetylcholinesterase. In fact, OPs bind to a variety of enzymes including esterases other than acetylcholinesterase e.g. carboxylesterase, long chain fatty acid hydrolase), serine peptidases, amidases and proteases and others, often modulating them (see review by Lockridge and Schopfer and section 10.3.2.1). ... 

Ester/amide hydrolysis or synthesis Hydrolases (lipases, esterases, amidases and proteases)... 

Chirazymes. These are commercially available enzymes e.g. lipases, esterases, that can be used for the preparation of a variety of optically active carboxylic acids, alcohols and amines. They can cause regio and stereospecific hydrolysis and do not require cofactors. Some can be used also for esterification or transesterification in neat organic solvents. The proteases, amidases and oxidases are obtained from bacteria or fungi, whereas esterases are from pig liver and thermophilic bacteria. For preparative work the enzymes are covalently bound to a carrier and do not therefore contaminate the reaction products. Chirazymes are available form Roche Molecular Biochemicals and are used without further purification. 

Enantioselective acylation of amine and hydrolysis of amide are widely studied. These reactions are catalyzed by acylases, amidases and lipases. Some examples are shown in Figure 21.22 Aspartame, artificial sweetener, is synthesized by a protease, thermolysin (Figure 21(a)).22a In this reaction, the L-enantiomer of racemic phenylalanine methyl ester reacted specifically with the a-carboxyl group of N-protected L-aspartate. Both the separation of the enantiomers of the phenylalanine and the protection of the y-carboxyl group of the L-aspartate were unnecessary, which simplified the synthesis. 

The serine hydrolase family is one of the largest and most diverse classes of enzymes. They include proteases, peptidases, lipases, esterases, and amidases and play important roles in numerous physiological and pathological process including inflammation [53], angiogenesis [54], cancer [55], and diabetes [56]. This enzyme family catalyzes the hydrolysis of ester, thioester, and amide bonds in a variety of protein and nonprotein substrates. This hydrolysis chemistry is accomplished by the activation of a conserved serine residue, which then attacks the substrate carbonyl. The resulting covalent adduct is then cleaved by a water molecule, restoring the serine to its active state [57] (Scheme 1). 

Enzymes such as proteases (122), subtilisin (123), acylases, peptidases, amidases, and lipases (124) are reported to catalyze amide bond formation with, in some cases, enantiospeciflcity of over 99%. Despite limited enzyme-substrate compatibility, specific conditions have been developed to reverse their natural reactivity, which is in favor of the hydrolysis. For example, Kyotorphin (Tyr-Arg) (125), a potent analgesic, was produced on an industrial scale using a-chymotrypsin, a peptidase isolated from bovine pancreas. 

Specific peptidase and protease systems which involve Mn(II) include thrombin limited-proteolysis of prothrombin [122], insulin protease [123], enkephalin-degrading amino-peptidase [124], camosinase [125,129], ki-ninase [127], and trypsin activation [128]. A metalIo(Mn)-protease is involved in the processing of mitochondrial precursor proteins [130]. Several aminopeptidases are also specifically manganese-dependent, namely Leu-aminopeptidase [131] and prolidase or C-terminal proline dipeptidase [132-135]. Other systems that hydrolyze linear and cyclic G-N bonds include various amino-acylases, deacetylases, amidases and methylene-... 

This is the most common application of biocatalysis in organic synthesis and represents the majority of published examples. Enzymes that catalyze acyl transfer reactions of esters and amides are widely distributed in nature and belong to the lipase/esterase and protease/amidase families, respectively. They play key roles in the metabolism of lipids and proteins and the choice of names, lipase versus esterase, is subject to debate. Normally, acyl ttansfer occurs almost exclusively to water, resulting in hydrolysis. This is particularly valuable for amide hydrolysis that normally requires forcing conditions and strong acid or... 

Historically, the most popular enzymes used for chemical synthesis are lipases, esterases, proteases, acylases and amidases, among others. Recently, a number of recombinant biocatalysts have been discovered and isolated, significantly expanding the toolbox for biotransformations. In this section, the focus will be on these new enzymes. 

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). 

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. 

Hydrolases that form an acyl-enzyme intermediate, such as some proteases and amidases, can be effectively used in this approach. On the other hand, this method is not applicable to metallo- and carboxyproteases. 

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