Carboxylesterases esterase activity

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. 

Carboxylesterases (EC 3.1.1.1) can be detected in most mammalian tissues. Besides organs with high carboxylesterase activity such as liver, kidney, and small intestine, esterase activity is present, e.g., in the brain, nasal mucosa, lung, testicle, and saliva. Compared to rat plasma, human plasma contains little carboxylesterase, its esterase activity being essentially due to cholinesterase [61][73][79][89-91],... 

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. 

Esterase activity is important in both the detoxication of organophosphates and the toxicity caused by them. Thus brain acetylcholinesterase is inhibited by organophosphates such as paraoxon and malaoxon, their oxidized metabolites (see above). This leads to toxic effects. Malathion, a widely used insecticide, is metabolized mostly by carboxylesterase in mammals, and this is a route of detoxication. However, an isomer, isomalathion, formed from malathion when solutions are inappropriately stored, is a potent inhibitor of the carboxylesterase. The consequence is that such contaminated malathion becomes highly toxic to humans because detoxication is inhibited and oxidation becomes important. This led to the poisoning of 2800 workers in Pakistan and the death of 5 (see chap. 5 for metabolism and chap. 7 for more details). 

Although carboxylesterases and amidases were thought to be different, no purified carboxylesterase has been found that does not have amidase activity toward the corresponding amide. Similarly enzymes purified on the basis of their amidase activity have been found to have esterase activity. Thus these two activities are now regarded as different manifestations of the same activity, specificity depending on the nature of R, R and R groups and, to a lesser extent, on the atom (O, S, or N) adjacent to the carboxyl group. 

Lipases are enzymes that hydrolyse triglycerides in fats and phospholipases, as the name indicates, hydrolyse phospholipids. Lipases remove long-chain fatty acids from triglycerides, and they are also frequently described as having esterase activity. There are also specific esterases described in the GI tract, for example, carboxylesterase that is secreted by the pancreas. These enzymes are included in the discussion because their activity may be relevant to the use of macromolecular materials in novel formulations, particularly for oral peptide and nucleic acid delivery. 

Carboxylesterase activity is elevated in mastitic milk and colostrum (Fitz-Gerald et al., 1981) and may correspond to that of the reported lipases from somatic cells (Gaffney and Harper, 1965 Azzara and Dimick, 1985a) and colostrum (Driessen, 1976), respectively. The retinyl esterase activity that co-purifies with, but can be separated from, LPL may also be due to a carboxylesterase (Goldberg et al., 1986). It is of interest that the BSSL in human milk that has been shown to be identical with pancreatic carboxylesterase, has retinyl esterase activity (O Connor and Cleverly, 1989). 

The term carboxylesterase refers to a wide variety of enzymes with both esterase and amidase activity. They cleave carboxylesters, carboxylamides, and car-boxylthioesters, producing a carboxylic acid and an alcohol or phenol (Figure 8), amine, or mercaptan, respectively. There are many different esterases, some of which are important for the hydrolysis and detoxication of toxic organophosphate esters. In general, esterases are present in almost all mammalian tissues, occur as multiple isozymes, and are concentrated in the liver. The esterase activity present in plasma is normally due to the release of these enzymes from liver. 

Cocaine metabolism and disposition following acute ethanol administration were studied in the rat to determine if the in vitro effects of ethanol on cocaine methyl esterase and ethyl transferase activities had significance in vivo (Zachman et al. 1993). The rat was used as it possesses both ethyl transferase and methyl esterase activities, is frequently employed for behavioral and toxicity studies of cocaine, and the size provides sufficient tissue for anal 4 ical work. This study was designed to address three questions. First, do significant concentrations of cocaethylene form and accumulate in tissues with controlled coadministrations of cocaine and alcohol Second, does ethanol administration significantly diminish the hydrolysis of cocaine to benzoylecgonine and methanol, as occurs in vitro when cocaine and ethanol are coincubated with purified human liver carboxylesterase (Brzezinski et al. 1994 Dean et al. 1991) Third, does ethanol inhibition of cocaine methyl ester hydrolysis increase the N-oxidative metabolism of cocaine, as noted when rodents are pretreated with nonspecific esterase inhibitors (Thompson etal. 1979) ... 

Malathionase (ME) For measuring the inhibition of malathion esterase activity, general carboxylesterase from porcine liver (Sigma) was used at a final concentration of 16 jig protein/mL in 0.1M Tris HC1 buffer (pH-7.5). The procedure involves an indirect determination of the malathionase activity by coupling the hydrolysis of malathion to the reduction of a tetrazolium dye (42). An acetone solution of malathion was used as substrate to a final concentration of 3x10 4M. 

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). 

A novel biomarker of OP insecticide exposure, egasyn, is an accessory protein of glucuronidase (BG) in the liver micro-somes (Swank and Paigen, 1973). Mcdda et al. (1987) reported that egasyn has esterase activity and it is one of the carboxylesterase (CarbE) isozymes. Later, Hosokawa and coworkers (1987, 1990) purified three CarbE isozymes (RLl, RL2, and RHl) from rat liver microsomes, and ega,syn was identified as RL2 isozyme. In 1998, Satoh and Hosokawa proposed a new das.sificalion of CarbE isozymes based on the molecular homology of the individual esterase isozymes. 

Mo.st OP pesticides react preferentially with BuChE. In contrast, most OP nerve agents react preferentially with AChE. Carboxylesterase is also highly reactive with most OPs. This complicate.s the comparison of studies on rodents with studie,s on monkeys and humans because rodenhs have very high concentrations of carboxylesterase in plasma, whereas monkeys and humans have no carboxylesterase in plasma. On the other hand, albumin has esterase activity (Means and Wu, 1979), and this is sometimes mistaken for carboxylesterase activity in human plasma. 

Carboxylesterase activity of soil extracts was demonstrated in a sensitive assay based on the formation of a fluorescent product, 7-hydroxy 4-methyl umbellipherone from the hydrolysis of the butyl ester of 7-hydroxy 4-methylcoumarin. EDTA, Cu and competitively inhibited esterase activities, Fe inhibited non-competitively. Activities increased after addition of Ca ", Mg " ", Na" " or... 

Sigolaeva, L.V., Dubacheva, G.V., Porus, M.V., et al., 2013. Layer-by-layer tyrosinase biosensor for assay of carboxylesterase and neuropathy target esterase activities in blood. Anal. Methods 5, 3872-3879. 

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)-... 

Organophosphates phosphorylate the OH group of the catalytic serine at the active site of B-esterases (see Sect. 3.3). The rate of dephosphorylation of the enzyme is very slow, thus, the organophosphate acts as a mechanism-based inactivator. B-Esterases are classified as carboxylesterases (EC 3.1.1.1). 

The intracellular localization of carboxylesterases is predominantly microsomal, the esterases being localized in the endoplasmic reticulum [73] [79] [93], They are either free in the lumen or loosely bound to the inner aspect of the membrane. The carboxylesterases in liver mitochondria are essentially identical to those of the microsomal fraction. In contrast, carboxylesterases of liver lysosomes are different, their isoelectric point being in the acidic range. Carboxylesterase activity is also found in the cytosolic fraction of liver and kidney. It has been suggested that cytosolic carboxylesterases are mere contaminants of the microsomal enzymes, but there is evidence that soluble esterases do not necessarily originate from the endoplasmic reticulum [94], In guinea pig liver, a specific cytosolic esterase has been identified that is capable of hydrolyzing acetylsalicylate and that differs from the microsomal enzyme. Also, microsomal and cytosolic enzymes have different electrophoretic properties [77]. Cytosolic and microsomal esterases in rat small intestinal mucosa are clearly different enzymes, since they hydrolyze rac-oxazepam acetate with opposite enantioselectivity [95], Consequently, studies of hydrolysis in hepatocytes reflect more closely the in vivo hepatic hydrolysis than subcellular fractions, since cytosolic and microsomal esterases can act in parallel. 

The physiological functions of carboxylesterases are still partly obscure but these enzymes are probably essential, since their genetic codes have been preserved throughout evolution [84] [96], There is some evidence that microsomal carboxylesterases play an important role in lipid metabolism in the endoplasmic reticulum. Indeed, they are able to hydrolyze acylcamitines, pal-mitoyl-CoA, and mono- and diacylglycerols [74a] [77] [97]. It has been speculated that these hydrolytic activities may facilitate the transfer of fatty acids across the endoplasmic reticulum and/or prevent the accumulation of mem-branolytic natural detergents such as carnitine esters and lysophospholipids. Plasma esterases are possibly also involved in fat absorption. In the rat, an increase in dietary fats was associated with a pronounced increase in the activity of ESI. In the mouse, the infusion of lipids into the duodenum decreased ESI levels in both lymph and serum, whereas an increase in ES2 levels was observed. In the lymph, the levels of ES2 paralleled triglyceride concentrations [92] [98],... 

Other serine hydrolases such as cholinesterases, carboxylesterases, lipases, and fl-lactamases of classes A, C, and D have a hydrolytic mechanism similar to that of serine peptidases [25-27], The catalytic mechanism also involves an acylation and a deacylation step at a serine residue in the active center (see Fig. 3.3). All serine hydrolases have in common that they are inhibited by covalent attachment of diisopropyl phosphorofluoridate (3.2) to the catalytic serine residue. The catalytic site of esterases and lipases has been less extensively investigated than that of serine peptidases, but much evidence has accumulated that they also contain a catalytic triad composed of serine, histidine, and aspartate or glutamate (Table 3.1). 

Thioesters play a paramount biochemical role in the metabolism of fatty acids and lipids. Indeed, fatty acyl-coenzyme A thioesters are pivotal in fatty acid anabolism and catabolism, in protein acylation, and in the synthesis of triacylglycerols, phospholipids and cholesterol esters [145], It is in these reactions that the peculiar reactivity of thioesters is of such significance. Many hydrolases, and mainly mitochondrial thiolester hydrolases (EC 3.1.2), are able to cleave thioesters. In addition, cholinesterases and carboxylesterases show some activity, but this is not a constant property of these enzymes since, for example, carboxylesterases from human monocytes were found to be inactive toward some endogenous thioesters [35] [146], In contrast, allococaine benzoyl thioester was found to be a good substrate of pig liver esterase, human and mouse butyrylcholinesterase, and mouse acetylcholinesterase [147],... 

R. A. Dean, J. Zhang, M. R. Brzezinski, W. F. Bosron, Tissue Distribution of Cocaine Methyl Esterase and Ethyl Transferase Activities Correlation with Carboxylesterase Protein , J. Pharmacol. Exp. Ther. 1995, 275, 965 - 971. 

Another therapeutic class to be briefly discussed is that of the lipid-lowering agents known as fibrates, e.g., clofibrate and fenofibrate (8.5). Here also, the acidic metabolite is the active form clofibrate (an ethyl ester) is rapidly hydrolyzed to clofibric acid by liver carboxylesterases and blood esterases [11], Human metabolic studies of fenofibrate (8.5), the isopropyl ester of fenofibric acid, showed incomplete absorption after oral administration, while hydrolysis of the absorbed fraction was quantitative [12], This was followed by other reactions of biotransformation, mainly glucuronidation of the carboxylic acid group. 

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. 

---