Esterase/amidase

The assessment of clearance is complicated by the numerous mechanisms by which compounds may be cleared from the body. These mechanisms include oxidative metabolism, most commonly by CYP enzymes, but also in some cases by other enzymes including but not limited to monoamine oxidases (MAO), flavin-containing monooxygenases (FMO), and aldehyde oxidase [45, 46], Non-oxidative metabolism such as conjugation or hydrolysis may be effected by enzymes such as glucuronyl transferases (UGT), glutathione transferases (GST), amidases, esterases, or ketone reductases, as well as other enzymes [47, 48], In addition to metabolic pathways, parent compound may be excreted directly via passive or active transport processes, most commonly into the urine or bile. 

R R Alkene c=c r R CYP, GST Epoxidation, glutathione adduct formation 0 Amide R-C-NH-R Amidase (esterase)... 

Clostripain (EC 3.4.22.8) an SH-dependent, liyp-sin-like protease (M, 50,000) with endopeptidase and amidase-esterase activity, isolated from culture filtrates of Clostridium histolyticum. Ihe endopeptidase acivity hydrolyses proteins, while the amidase-esterase activity cleaves synthetic amino acid amides and amino acid esters. It attacks only arginyl and lysyl re-adues, and is therefore used to isolate large peptide fragments without prior chemical modification of the sutetrate. 

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. 

Several classes of enzymes have been used to separate stereoisomers of a-H-and a-disubstituted amino acids, eg amidases, nitrilases, hydantoinases, acylases and esterases. 

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. 

Hydrolases Epoxide hydrolase Amidases and esterases Glycosidases... 

Carboxylesterases and amidases catalyze hydrolysis of carboxy esters and carboxy amides to the corresponding carboxylic acids and alcohols or amines. In general those enzymes capable of catalyzing hydrolysis of carboxy esters are also amidases, and vice versa (110). The role of these enzymes in metabolsim of drugs and insecticides has been reviewed (111, 112). In addition to the interest in mammalian metabolism of drugs and environmental chemicals, microbial esterases have been used for enantioselective hydrolyses (113, 114). 

Although hydrolytic enzymes, esterases and amidases, are named after their major substrates, the same enzyme can often hydrolyze esters, thioesters, and amides therefore, the differentiation between esterases and amidases is sometimes artificial. The highest hydrolytic activity is in the liver, but the enzyme pseudocholinesterase is found in the serum. Gut bacteria also contain hydrolytic enzymes. 

Esterolytic antibodies have also been produced by Suga using a different bait and switch strategy (Appendix entry 2.1) (Suga et al., 1994a). A 1,2-aminoalcohol function was designed for generating not only esterases but also amidases. Of three haptens synthesized, [15], [16] and [17], two contained... 

Fig. 9 Three haptens, [15]—[17], containing a 1,2-aminoalcohol functionality were investigated as alternatives for esterase and amidase induction. Of antibodies raised against hapten [15], 50% were shown to catalyse the hydrolysis of ester [18], thereby establishing the necessity for a compact haptenic structure. Hapten [19] along with [16] was employed in a heterologous immunization programme to elicit both a general and acid/base function in the antibody binding site.

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. 

B. Ab, T. Imamura, Characteristics Of Rat Lung Carboxylesterases/Amidases , Proc. West. Pharmacol. Soc. 1985, 28, 319-322 B. Ab, S. Kaur, Mammaban Tissue Acet-ylsabcybc Acid Esterase(s) Identification, Distribution and Discrimination from Other Esterases , J. Pharmacol. Exp. Then 1983, 226, 589 - 594. 

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). 

Metabolic reactions in the liver and the small intestine are well documented [24]. However, only sparse information is available on drug metabolism in the eolon. Drug metabolism in the colon can be brought about by the host enzymes in the epithelial cells or by the microbial enzymes in the gut flora. Metabolie aetivities in the wall of the colon can be attributed to the eytochrome P450, esterases, amidases, and various transferases [25]. Reductive drug metabolism does not appear to be important at this site. 

Esterases and amidases E. coli, P. vulgaris, B.. subtilis, B. mycoides Cleavage of esters or amidases of carboxylic acids... 

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). 

A major class of enzymes that catalyze hydrolytic cleavage reactions. Examples include esterases, phosphatases, sulfatases, nucleases, glycosidases, peptidases, protein-ases, and amidases. 

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. 

Hydrolyses Esters and Amides. The plasma, liver, kidney, and intestines contain a wide variety of nonspecific amidases and esterases. These catalyze the metabolism of esters and amides, ultimately leading to the formation of amines, alcohols, and carboxylic acids. Kinetically, amide hydrolysis is much slower than ester hydrolysis. These hydrolyses may exhibit stereoselectivity. 

It is a cleavage of drug molecule by taking up a molecule of water. The most hydrolytic enzymes are found outside the endoplasmic reticulum, and in higher concentrations in liver, kidney and plasma. The metabolism of an ester by an enzyme esterase results in the formation of an acid and alcohol. The examples are meperidine, procaina-mide, pethidine and lidocaine etc. Meperidine is catalyzed by esterases to be changed into meperidinic acid and procainamide is catalyzed by amidases. 

The findings that, both in ester and amide cleavage, an alkaline-earth metal ion is still catalytically active when complexed with a crown ether, and that a fraction of the binding energy made available by coordinative interactions with the polyether chain can be translated into catalysis, provide the basis for the construction of supramolecular catalysts capable of esterase and amidase activity. 

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