Chymotrypsin, carboxyl ester hydrolysis

Fig. 5. Reaction mechanism for carboxyl ester hydrolysis by chymotrypsin.

Various esterases exist in mammalian tissues, hydrolyzing different types of esters. They have been classified as type A, B, or C on the basis of activity toward phosphate triesters. A-esterases, which include arylesterases, are not inhibited by phosphotriesters and will metabolize them by hydrolysis. Paraoxonase is a type A esterase (an organophosphatase). B-esterases are inhibited by paraoxon and have a serine group in the active site (see chap. 7). Within this group are carboxylesterases, cholinesterases, and arylamidases. C-esterases are also not inhibited by paraoxon, and the preferred substrates are acetyl esters, hence these are acetylesterases. Carboxythioesters are also hydrolyzed by esterases. Other enzymes such as trypsin and chymotrypsin may also hydrolyze certain carboxyl esters. 

As with peptide hydrolysis, several enzyme systems exist that catalyze carboxylic and phosphoric ester hydrolysis without the need for a metal ion. They generally involve a serine residue as the nucleophile in turn, serine may be activated by hydrogen-bond formation—or even proton abstraction—by other acid-base groups in the active site. The reaction proceeds to form an acyl- or phosphory 1-enzyme intermediate, which is then hydrolyzed with readdition of a proton to the serine oxygen. Mechanisms of this type have been proposed for chymotrypsin. In glucose-6-phosphatase the nucleophile has been proposed to be a histidine residue. ... 

Some examples of DKR based on racemization of secondary alkyl amine via Shiff base were shown in Scheme 5.9. Schiffbase formation of a-amino carboxylic esters significantly increases the acidity of the a-proton in comparison to that of the parent amino acid, thus enabling enantiomer-selective hydrolysis and ammonoly-sis through DKR [23]. For example, chymotrypsin catalyses the hydrolysis of a Schiff base of phenylalanine ethyl ester and an aromatic aldehyde (Scheme 5.9, Equation 5.6) [23b]. In this particular case, natural phenylalanine precipitates, leaving the aldehyde and unhydrolysed enantiomeric ester in solution. Addition of l,4-diazabicyclo[2.2.2]octane (DABCO) (as the Bronsted base) allows DKR to take place by promoting racemization of the Schiffbase. Similarly, Novozym 435... 

Lipases and esterases typically show selectivity toward the alcohol or amine part of a carboxylic acid derivative. Favored acyl groups are simple straight chains like acetate or butyrate. In contrast, proteases show higher specificity for the acyl part of a carboxylic acid derivative. Proteases contain a specificity pocket for the acyl group. For example, subtilisins and chymotrypsin favor ester and amides of phenylalanine esters. Another difference between the enzyme classes is that lipases and esterases catalyze hydrolysis of only esters, whereas proteases catalyze hydrolysis of both amides and esters. Several good books on hydrolases in organic synthesis are available [2-4]. 

The structural analysis has been carried out right up to the recognition of molecular Level (i, 2). a-Chymotrypsin is poly(amino acid) consisting of 245 amino adds, having relatively deep grooves. It catalyzes the hydrolysis of carboxylic acid derivatives such as protein, simple amides, esters, etc. The active site is composed of aspartic add, Asp (102). .. histidine, His (57). .. serine, Ser (195), and the distances between Asp. .. His and His... Ser are 2.8 A and 3.0 A, respectively. Electronic structures of these moieties depend on the pH of the reaction system. In the range of pH > 7 at which a-chymotrypsin is active, —COO" of Asp attracts N4 proton in imidazolyl of His, and Nj in the imidazolyl of His attracts the proton in OH of Ser. It is called charge-relay system . 

Peptide-based polymers 62, containing imidazole, carboxyl, and hydroxymethyl functionalities, have been prepared from optically active 50d and tested as mimics of enzymes, such as chymotrypsin, which have the same functionalities (Scheme 41) [70]. These polymers exhibit markedly higher activities than the corresponding low molecular weight compounds in the hydrolysis of nitrophenyl and dinitrophenyl esters. Increased activities were... 

Proteases such as a-chymotrypsin, papain, and subtilisin are also useful biocatalysts for regio-selective or stereoselective hydrolytic biotransformations. For example, dibenzyl esters of aspartic and glutamic acid can be selectively deprotected at the 1-position by subtilisin-catalyzed hydrolysis (Fig. 6). In addition, a-chymotrypsin is used in the kinetic resolution of a-nitro-a-methyl carboxylates, which results in l-configured enantiomers of the unhydrolyzed esters with high optical purity (>95% e.e.). ... 

The hydrolase-catalyzed reactions utilized most for the selective transformation of such substrates are hydrolysis (Schemes 11.1-1, 11.1-2, 11.1-4, 11.1-5 and 11.1-11), acylation (transesterification) (Schemes 11.1-3, 11.1-6 and 11.1-11) and alcoholysis (transesterification) (Schemes 11.1-7,11.1-8 and 11.1-15). Hydrolase-catalyzed esterification of an alcohol with a carboxylic acid, although highly useful in some casesl6Z, has been utilized to a lesser extent. Catalysis of formation and cleavage of the C-O bond of an ester or lactone by pig liver esterase, most lipases, a-chymotrypsin and subtilisin, which are all serine hydrolases, involves the following steps (Scheme... 

Recently, communications by Mashelkar and coworkers [14] described efforts to develop materials that mimic chymotrypsin-like activity. These proteins hydrolyze ester and amide linkages at the carbonyl group of phenylalanine or tyrosine. The active site in the enzyme is comprised of a hydroxyl, carboxyl, and imidazole side chains, respectively. The rate of hydrolysis displayed by chymotrypsin is attributed in part to cooperative effects brought on by the close proximity of the aforementioned functional groups within the active site of the enzyme. 

ABSTRACT. The synthesis and characterization of an artificial acyl-enzyme intermediate of chjrmotrypsin, and an artificial enzyme, chymotryp-sin, are described. They both contain the same three catalytic groups, an imidazolyl group, a hydroxyl group, and a carboxylate ion. In the acyl-enzyme, the three groups are attached to a norbornane backbone, but in the artificial enzyme, the three catalytic groups are attached to a cycloamylose as binder. The artificial acyl-enzyme shows a rate of hydrolysis 154,000 times faster than an ordinary ester and only 18-fold slower than the real acyl-chymotrypsin. The artificial chymotrypsin is over a thousand fold slower than real chymotrypsin, presumably because of impurities in the preparation. 

Alpha-chymotrypsin (Fig. 2) catalyzes the facile hydrolysis of peptide bonds, in particular those adjacent to the carboxyl group of aromatic amino acids (tryptophan, tyrosine, phenylalanine) as well as a variety of esters derived from similar N-acylated amino acids. The enzyme... 

Selective hydrolysis of esters is a well-established procedure for the resolution of chiral carboxylic acids. Enzymes such as hydrolases, lipases and proteases are utilized. Due to their high selectivity for (5)-amino acids, proteases have been widely used in the selective transformations of amino acids and their derivatives. a-Chymotrypsin- a serine protease -catalyzes not only the hydrolysis of amide bonds, but also the cleavage of various esters, including a-alkyl-a-amino acid esters. One application is the synthesis of (5)-a-[ C]-methyltryptophan The anion synthesized by LDA-deprotonation of M-benzylidene tryptophan methyl ester (T) was alkylated with [ CJmethyl iodide to obtain the methyl A-benzylidene derivative 2, which was hydrolyzed under acidic conditions. Subsequent selective cleavage of the ester group with a-chymotrypsin provided the enantiomerically pure (5)-amino acid 3 in 33% radiochemical yield. 

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