Serine hydrolase-catalyzed ester hydrolysis

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). 

Hydrolase enzymes were initially chosen as target proteins to catalyze and amplify the optimal constituents from dynamic systems. Among the six enzyme classes, hydrolases (EC 3) are one of the most commonly used, both in bulk industrial processes as well as for laboratory scale reactions [1]. These enzymes do not require any cofactors to perform the reactions, and a large variety is commercially available. Hydrolases furthermore catalyze reactions for a broad range of substrates, e.g., hydrolysis of esters, amides, thiolesters, etc., often accompanied with high stereoselectivity. An example of hydrolases is the family of serine hydrolases, which employs... 

Pig Liver Esterase (PLE). This is the more used car-boxylesterase (carboxylic-ester hydrolase, EC 3.1.1.1, CAS 9016-18-6) which physiologically catalyzes the hydrolysis of carboxylic acid esters to the free acid anion and alcohol. PLE is a serine hydrolase which has been widely used for the preparation of chiral synthons and these applications have been fully reviewed. An active-site model for interpreting and predicting the specificity of the enzyme has been published. In the pioneering studies of the enzyme applications field, PLE was used for the chiral synthesis of mevalonolactone. Prochiral 3-substituted glutaric acid diesters... 

Many pesticides are esters or amides that can be activated or inactivated by hydrolysis. The enzymes that catalyze the hydrolysis of pesticides that are esters or amides are esterases and amidases. These enzymes have the amino acid serine or cysteine in the active site. The catalytic process involves a transient acylation of the OH or SH group in serin or cystein. The organo-phosphorus and carbamate insecticides acylate OH groups irreversibly and thus inhibit a number of hydrolases, although many phosphorylated or carbamoylated esterases are deacylated very quickly, and so serve as hydrolytic enzymes for these compounds. An enzyme called arylesterase splits paraoxon into 4-nitrophenol and diethyl-phosphate. This enzyme has cysteine in the active site and is inhibited by mercury(ll) salts. Arylesterase is present in human plasma and is important to reduce the toxicity of paraoxon that nevertheless is very toxic. A paraoxon-splitting enzyme is also abundant in earthworms and probably contributes to paraoxon s low earthworm toxicity. Malathion has low mammalian toxicity because a carboxyl esterase that can use malathion as a substrate is abundant in the mammalian liver. It is not present in insects, and this is the reason for the favorable selectivity index of this pesticide. 

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... 

The DhiA enzyme functions as a monomer ( 35 kDa) and is composed of two domains a main domain and a cap domain (Figure 2(a)). The main domain consists of a mostly parallel eight-stranded /3-sheet connected by ct-helices on both sides of the sheet. The cap domain is composed of five ct-helices with intervening loops. The active site is an occluded hydrophobic cavity located at the interface of the two domains. The overall fold of the main domain is the hallmark of the o //3-hydrolase fold superfamily of enzymes, to which lipases, esterases, carboxypeptidases, and acetylcholinesterases also belong. These superfamily members catalyze the hydrolysis of ester and amide bonds via a two-step nucleophilic substitution mechanism similar to that of serine proteases. 

Lipases are serine hydrolases (EC 3.1.1.3) that catalyze the hydrolysis of ester bonds in long-chain triacylglycerols with a concomitant release of fatty acids and glycerol (Jaeger et al. 1999 Sharma et al. 2001). The action mechanism of lipases is shown inO Fig. 7.2.0 figure 7.3 represents a structure of a lipase from Pseudomonas aeruginosa (Nardini and Dijkstra 1999). 

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. 

Other enzymes widely used in organic syntheses are lipases. Lipases are serine hydrolases that catalyze the hydrolysis of lipids to fatty acids. An example of their use is in the resolution of racemic methyl 4-benzyloxy-2-(hydroxymethyl)[l- C]butyrate (8), an intermediate for the carbon-13 labeling of the antiviral agents penciclovir and famciclovir. From a limited number of candidates Candida cylindracea lipase turned out to be the enzyme of choice. At optimal reaction conditions (pH 5-7, 35 °C) no nonenzymatic hydrolysis was detectable. Due to insufficient enzymatic stereospecificity, however, the hydrolysis provided the unreacted dextrorotary ester 2 at an optical purity of 94% e.e. and the hydrolyzed levorotary acid 10 at an optical purity of 70% e.e. 

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