Hydrolysis carboxylesterases

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). 

In addition to ester bonds with P (Section 10.2.1, Figures 10.1 and 10.2), some OPs have other ester bonds not involving P, which are readily broken by esteratic hydrolysis to bring about a loss of toxicity. Examples include the two carboxylester bonds of malathion, and the amido bond of dimethoate (Figure 10.2). The two carboxylester bonds of malathion can be cleaved by B-esterase attack, a conversion that provides the basis for the marked selectivity of this compound. Most insects lack an effective carboxylesterase, and for them malathion is highly toxic. Mammals and certain resistant insects, however, possess forms of carboxylesterase that rapidly hydrolyze these bonds, and are accordingly insensitive to malathion toxicity. 

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)-... 

Keywords Carboxylesterase CYP Ester hydrolysis Metabolism Oxidation Pyrethroid... 

Carboxylesterases (CESs) catalyze hydrolysis of pyrethroids. The expression of CESs is ubiquitous in mammals. The highest hydrolase activity is present in liver. 

Crow JA, Borazjani A, Potter PM, Ross MK (2007) Hydrolysis of pyrethroids by human and rat tissues examination of intestinal, liver and serum carboxylesterases. Toxicol Appl Pharmacol 221 1-12... 

Huang H, Fleming CD, Nishi K, Redinbo MR, Hammock BD (2005) Stereoselective hydrolysis of pyrethroid-like fluorescent substrates by human and other mammalian liver carboxylesterases. ChemRes Toxicol 18 1371-1377... 

Yang D, Pearce RE, Wang X, Gaedigk R, Wan YJ, Yan B (2009) Human carboxylesterases HCE1 and HCE2 ontogenic expression, inter-individual variability and differential hydrolysis of oseltamivir, aspirin, deltamethrin and permethrin. Biochem Pharmacol 77 238-247... 

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). 

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 hydrolysis of the amide bond in chloramphenicol (4.26), which liberates dichloroacetic acid (4.27) and the primary amine (4.28), has been shown in bacteria, rodents, and humans [13-15]. In the microsomal fraction of guinea pig liver, moreover, the enzyme responsible for hydrolysis has been identified as one of the B-type carboxylesterase isoenzymes [16]. 

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]. 

Enol esters are distinct from other esters not because of a particular stability or lability toward hydrolases, but due to their hydrolysis releasing a ghost alcohol (an enol), which may immediately tautomerize to the corresponding aldehyde or ketone. A well-studied example is that of vinyl acetate (CH3-C0-0-CH=CH2), a xenobiotic of great industrial importance that, upon hydrolysis, liberates acetic acid (CH3-CO-OH) and acetaldehyde (CH3-CHO), the stable tautomer of vinyl alcohol [25], The results of two studies are compiled in Table 7.1, and demonstrate that vinyl acetate is a very good substrate of carboxylesterase (EC 3.1.1.1) but not of acetylcholinesterase (EC 3.1.1.7) or cholinesterase (EC 3.1.1.8). The presence of carboxylesterase in rat plasma but not in human plasma explains the difference between these two preparations, although the different experimental conditions in the two studies make further interpretation difficult. 

For example, the hydrolysis of phenyl acetate (7.15) by carboxylesterase isozymes was investigated over a broad pH range, allowing many insights into their catalytic mechanisms [30] (see Chapt. 3). This substrate was also used together with various inhibitors to characterize esterases in human and rat tissues [31], Thus, the approximate values of Km (in pM) and Vmax (in pmol min 1 (g tissue)-1) in human tissues were 300 and 60 in liver micro-somes, 200 and 40 in liver cytosol, and 1500 and 250 in plasma, respective-... 

In comprehensive studies, the hydrolysis of some 30 naphthyl esters by human, rat, and mouse liver carboxylesterases was investigated [43], A general trend that was apparent was that the rate of hydrolysis of a- and /3-naphthyl carbonates (7.21, R = alkyl or arylalkyl) catalyzed by human microsomes or rat hydrolases showed a tendency to decrease with increasing lipophilic-ity (range ca. 2 to 5). A similar trend was not seen with naphthyl aryl carbonates nor with a-naphthyl carboxylates. These results tell us that, even with purified enzymes and large series of substrates, it is very difficult indeed to discern sound structure-hydrolysis relationships due to the complexity of the structural and enzymatic factors involved. 

In an homologous series of phthalate diesters (7.24, R = Me, Et, allyl, 2-methoxyethyl, Bu, i-Bu, and PhCH2), hydrolysis by some human and rat liver carboxylesterases was fastest for the diethyl and diallyl esters [50], This suggested a maximal rate of hydrolysis for phthalate esters with a lipophilic-ity (log P in the octanol/water system) of 3 - 4. Interestingly, and compatible with this interpretation, monoesters were not hydrolyzed. However, that re-gioisomeric monoesters are hydrolyzed clearly indicates that factors other than lipophilicity (e.g., steric factors) are also influential. 

A variety of hydrolases catalyze the hydrolysis of acetylsalicylic acid. In humans, high activities have been seen with membrane-bound and cytosolic carboxylesterases (EC 3.1.1.1), plasma cholinesterase (EC 3.1.1.8), and red blood cell arylesterases (EC 3.1.1.2), whereas nonenzymatic hydrolysis appears to contribute to a small percentage of the total salicylic acid formed [76a] [82], A solution of serum albumin also displayed weak hydrolytic activity toward the drug, but, under the conditions of the study, binding to serum albumin decreased chemical hydrolysis at 37° and pH 7.4 from tm 12 1 h when unbound to 27 3 h for the fully bound drug [83], In contrast, binding to serum albumin increased by >50% the rate of carboxylesterase-catalyzed hydrolysis, as seen in buffers containing the hydrolase with or without albumin. It has been postulated that either bound acetylsalicylic acid is more susceptible to enzyme hydrolysis, or the protein directly activates the enzyme. 

In human blood, only 6-acetylmorphine was formed from heroin, with no 3-acetylmorphine or morphine being detected. Four kinetically distinct enzyme activities were seen, namely one in plasma, one in the cytosol of red blood cells, and two in red blood cell membranes [92], In human plasma at 37°, hydrolysis to 6-acetylmorphine occurs with a tm value of some minutes, the enzyme responsible being cholinesterase [90] [93]. These and other results tend to indicate that the formation of morphine from 6-acetylmorphine is due to tissue carboxylesterases, in particular cerebral enzymes [94],... 

Several drugs, in particular neuropharmacological agents, feature a car-boxylate group esterified to an aminoalkyl moiety. As a rule, such lipophilic compounds are good substrates for hydrolases, and their duration of action is influenced by their rate of hydrolysis (see also Sect. 7.3.4). A simple example is that of procaine (7.56), which is rapidly inactivated by hydrolysis [41] [76a], Various hydrolases catalyze the reaction, in particular plasma cholinesterase and cellular carboxylesterases. As often reported, atropine and scopolamine are rapidly hydrolyzed by plasma carboxylesterases in rabbits (with very large differences between individual animals), but are stable in human plasma [1] [75] [76a] [110]. 

Natural (-)-cocaine (7.57, Fig. 7.8), which has the (2/ ,3S)-configuration, is a relatively poor substrate for hepatic carboxylesterases and plasma cholinesterase (EC 3.1.1.8), and also a potent competitive inhibitor of the latter enzyme [116][121], In contrast, the unnatural enantiomer, (+)-(2S,3/ )-cocaine, is a good substrate for carboxylesterases and cholinesterase. Because hydrolysis is a route of detoxification for cocaine and its stereoisomers, such metabolic differences have a major import on their monooxygenase-catalyzed toxification, a reaction of particular effectiveness for (-)-cocaine. 

It, thus, appears that the capacity to catalyze reactions of transesterification and esterification is a characteristic of various hydrolases (Chapt. 3). Apart from the carboxylesterases discussed here, lipoprotein lipase has the capacity to synthesize fatty acid ethyl esters from ethanol and triglycerides, or even fatty acids [127]. Ethanol, 2-chloroethanol, and other primary alcohols serve to esterify endogenous fatty acids and a number of xenobiotic acids [128-130]. In this context, it is interesting to note that the same human liver carboxylesterase was able to catalyze the hydrolysis of cocaine to benzoylecgonine, the transesterification of cocaine, and the ethyl esterification of fatty acids [131]. 

Simple alkyl or aryl thioesters are commonly assayed as substrates of hydrolases, witness the hydrolysis of phenyl thioesters by horse serum carbox-ylesterase [150], For most substrates investigated, e.g., phenyl thioacetate, phenyl thiopropionate, and phenyl thiobutyrate (7.66, R = Me, Et, and Bu, respectively), kcat values were found, which were a few times larger than those of corresponding nitrophenyl esters, whereas the affinities were lower by approximately one order of magnitude. Methyl and phenyl esters of various linear thioacids were also found to be good substrates of mammalian liver carboxylesterases and serum cholinesterases [151]. 

Interesting findings have been reported for the hydrolysis of a- and fi-naphthyl thiocarbonates (7.67, R = Me or Et) by mammalian liver carboxylesterases [43]. In comparison with their carbonate analogues (7.21), the thiocarbonates were more lipophilic by one log P unit and were hydrolyzed at rates modestly to markedly slower. These two findings are apparently unrelated. 

T. L. Huang, A. Szekacs, T. Uematsu, E. Kuwano, A. Parkinson, B. D. Hammock, Hydrolysis of Carbonates, Thiocarbonates, Carbamates, and Carboxylic Esters of a-Naph-thol, /LNaphthol, and p-Nitrophenol by Human, Rat, and Mouse Liver Carboxylesterases , Pharm. Res. 1993, 10, 639 - 648. 

R. Mentlein, W. Butte, Hydrolysis of Phthalate Esters by Purified Rat and Human Liver Carboxylesterases , Biochem. Pharmacol. 1989, 38, 3126-3128. 

R. B. Melchert, C. Goldlin, U. Zweifel, A. A. Welder, U. A. Boelsterli, Differential Toxicity of Cocaine and Its Enantiomers, (+)-Cocaine and (-)- -Cocaine, is Associated with Stereoselective Hydrolysis by Hepatic Carboxylesterases in Cultured Rat Hepatocytes , Chem.-Biol. Interact. 1992, 84, 243-258. 

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