Carboxylesterases substrate specificity

Second, esterases have broad (or even very broad) and overlapping substrate specificities. For example, carboxylesterase (EC 3.1.1.1) also catalyzes reactions characteristic of a number of other hydrolases. The discovery that individual isoenzymes of carboxylesterases may be identical to or closely related to acylglycerol lipase, acylcamitine hydrolase, and palmitoyl-CoA hydrolase (see Sect. 2.4.3) has increased the confusion surrounding esterase classification [59], Many esterases are able to hydrolyze amides, thiolesters,... 

Fourth, most esterases are highly polymorphic enzymes. Many of the purified carboxylesterases are mixtures of isoenzymes that have different substrate specificities [60][61]. For example, the substrate(s) used to isolate pig liver carboxylesterase influences the isoenzyme composition, and, hence, the substrate specificity of the resulting esterase preparation. 

A number of rat liver carboxylesterases identified by their pI values are listed in Table 2.6 [73] five nonspecific carboxylesterases were purified from rat liver and were characterized according to their p/ values [61]. They appeared to be isoenzymes, since they had similar substrate specificities toward phenyl and naphthyl esters and monooleylglycerol. Subsequent studies, however, revealed different specificities with respect to their physiological substrates. The pI 5.2 and 5.6 enzymes were shown to be acylcamitine hydrolases (EC 3.1.1.28), and a p/ 6.0 enzyme an octanoylglycerol lipase. The p/... 

Three isoenzymes of carboxylesterase were purified from rat liver micro-somes and were named RL1, RL2, and RH1. These differ from each other in their response to hormone treatment, inducibility, substrate specificity, and immunological properties [75], It was shown that RL1, RL2, and RH1 resemble hydrolases p/ 6.2/6.4, pI 6.0, and pI 5.6, respectively. Enzyme RL2 was found to be identical to egasyn, a protein with esterase activity found in the endoplasmic reticulum [76], The role of egasyn is to stabilize glucuronidase (EC 3.2.1.31) by noncovalent binding to the microsomal membrane. 

Series of homologous esters have been investigated to try to establish structure-metabolism relationships, however partial and limited the latter may be. This aspect will be discussed again in the context of prodrugs (Chapt. 8). Here, we mention a few representative studies in which model substrates were used. Table 7.2 documents the substrate specificity of a rabbit liver carboxylesterase (ES-1A) toward homologous series of methyl, 4-nitrophenyl, a-naphthyl, /1-naphthyl, and 4-methylumbelliferyl esters [41]. In... 

Table 7.2. Substrate specificity of rabbit hepatic carboxylesterase ES-1A... 

Esters, amides, hydrazides, and carbamates can all be metabolized by hydrolysis. The enzymes, which catalyze these hydrolytic reactions, carboxylesterases and amidases, are usually found in the cytosol, but microsomal esterases and amidases have been described and some are also found in the plasma. The various enzymes have different substrate specificities, but carboxylesterases have amidase activity and amidases have esterase activity. The two apparently different activities may therefore be part of the same overall activity. 

Hojring, N. and Svensmark, O. (1976) Carboxylesterases with different substrate specificity in human brain extracts. Journal of Neurochemistry, 27, 525-528. 

Hydrolysis. Carboxylesterases are frequently one of the major factors in OP resistance. In some insects, for instance the house fly (28), there are highly substrate specific esterases which attack only one or a very few molecules. "Malathionase", the prominent esterase responsible for many cases of malathion resistance, is highly specific for malathion. It cleaves one or both of the ethyl ester groups leaving malathion mono- or diacid (29). This enzyme is a true serine carboxylesterase that is inhibited by malaoxon (28) and does not hydrolyze any of the phosphoester bonds. In Anopheles stephensi from Pakistan, the malathion resistance decreased with adult age, but there was no concommittant decrease in general esterase activity as measured with 1- and 2-naphthylace-tate as model substrates (301. other mosquitoes have a carboxylesterase with broad substrate specificity that is associated with resistance (31-331. As mentioned above, the green peach aphid has a carboxylesterase, E4, with broad substrate specificity that sequesters toxicants (24). 

Pancreatic carboxylester lipase, secreted by the pancreas as an active enzyme without proteolytic activation, displays broad substrate specificity and has therefore received many names in the literature carboxylesterase, bile salt-stimulated (or activated or dependent) lipase (due to its absolute requirement for bile salts to hydrolyze insoluble substrates), carboxylester lipase or hydrolase, cholesterol esterase, lysophospholipase, nonspecific lipase, and monoglyceride lipase. The IUPAC classification of the enzyme has been either EC.3.1.1.1 (carboxylester hydrolase) or EC.3.1.1.13 (cholesterolester hydrolase) (Table 2). 

Chahinian H, Ali YB, AbousaUiam A, Petty S, Mandrich L, Manco G, Canaan S, Sarda L (2005) Substrate specificity and kinetic properties of enzymes belonging to the hormone-sensitive lipase family comparison with non-lipolytic and lipolytic carboxylesterases. Biochim Biophys Acta 1738 29-36... 

Drugs may also undergo hydrolysis by intestinal esterases (hydrolases), more specifically carboxylesterases (EC 3.1.1.1) in the intestinal lumen and at the brush border membrane [58, 59]. It has been shown that intestinal hydrolase activity in humans was closer to that of the rat than the dog or Caco-2 cells [60]. In these studies, six propranolol ester prodrugs and p-nitrophenylacetate were used as substrates, and the hydrolase activity found was ranked in the order human > rat Caco-2 cells > dog for intestinal microsomes. The rank order in hydrolase activity for the intestinal cytosolic fraction was rat > Caco-2 cells = human > dog. The hydrolase activity towards p-nitrophenylacetate and tenofovir disoproxil has also been reported in various intestinal segments from rats, pigs and humans. The enzyme activity in intestinal homogenates was found to be both site-specific (duodenum > jejunum > ileum > colon) and species-dependent (rat > man > Pig)-... 

The in vivo metabolism of a homologous series of alkyl carbamates (7.2, Fig. 7.3) has yielded some informative results [13]. The hydrolysis of these esters liberates carbamic acid (7.3, Fig. 7.3), which breaks down spontaneously to C02 and NH3, allowing the extent of hydrolysis to be determined conveniently and specifically by monitoring C02 production. When such substrates were administered to rats, there was an inverse relationship between side-chain hydroxylation and ester-bond hydrolysis. Thus, for compounds 12 the contribution of hydrolysis to total metabolism (90 - 95% of dose) decreased in the series R=Et (ca. 85-90%), Bu (ca. 60-65%), hexyl (ca. 45 - 50%), and octyl (ca. 30%). Ethyl carbamate (urethane) is of particular toxicological interest, being a well-established carcinogen in experimental animals. In vitro studies of adduct formation have confirmed the competition between oxidative toxification mediated by CYP2E1 and hydrolytic detoxification mediated by carboxylesterases [14]. 

Immobilisation of an Acetobacter aceti strain in calcium alginate resulted in improvement of the operational stability, substrate tolerance and specific activity of the cells and 23 g phenylacetic acid was produced within 9 days of fed-batch cultivation in an airlift bioreactor [133]. Lyophilised mycelia of Aspergillus oryzae and Rhizopus oryzae have been shown to efficiently catalyse ester formation with phenylacetic acid and phenylpropanoic acid and different short-chain alkanols in organic solvent media owing to their carboxylesterase activities [134, 135] (Scheme 23.8). For instance, in n-heptane with 35 mM acid and 70 mM alcohol, the formation of ethyl acetate and propylphenyl acetate was less effective (60 and 65% conversion yield) than if alcohols with increased chain lengths were used (1-butanol 85%, 3-methyl-l-butanol 86%, 1-pentanol 91%, 1-hexanol 100%). This effect was explained by a higher chemical affinity of the longer-chain alcohols, which are more hydrophobic, to the solvent. 

The esterases are involved in the hydrolysis of ester linkages of various types. The products formed are acid and alcohol. These enzymes may hydrolyze triglycerides and include several lipases for instance, phospholipids are hydrolyzed by phospholipases, and cholesterol esters are hydrolyzed by cholesterol esterase. The carboxylesterases are enzymes that hydrolyze triglycerides such as tributyrin. They can be distinguished from lipases because they hydrolyze soluble substrates, whereas lipases only act at the water-lipid interfaces of emulsions. Therefore, any condition that results in increased surface area of the water-lipid interface will increase the activity of the enzyme. This is the reason that lipase activity is much greater in homogenized (not pasteurized) milk than in the non-homogenized product. Most of the lipolytic enzymes are specific for either the acid or the alcohol moiety of the substrate, and, in the case of esters of polyhydric alcohols, there may also be a positional specificity. 

When a chromogenic or fluorogenic enzyme substrate is immobilized at the flber tip, which then is immersed in an enzyme solution, it will be hydrolyzed, which results in an increase in absorbance or fluorescence. This offers the possibility of remote (or invasive) determination of enzyme activities [84]. Specifically, the activity of carboxylesterases was determined. 

The amide linkage is similar to the ester linkage and is hydrolyzed in plants by amidases. There has been speculation, however, that some car-boxylamidases may be carboxylesterases with extended specificity partially purified carboxylesterases from mammalian sources are known to utilize amide substrates. ... 

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