Malathion esterase

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. 

Getzin and Rosefield" have extracted from soil and partially purified an arylesterase which hydrolysed the insecticide malathion to its monoacid. Under the conditions of assay the reaction proceeded with zero order kinetics. Purification of the extracted enzymes included precipitation of co-extracted humic compounds, resulting in a 9.3 fold increase in specific activity. Following further treatment with ammonium sulphate and separation by ion-exchange chromatography, the specific activity of the partially-purified enzyme had increased to 22 times that of the crude soil extract, with an overall recovery of activity of 32%. The enzyme was optimally active at pH 7.0. Measurements of initial velocities of reaction for different substrate concentrations showed that the reaction conformed to Michaelis-Menten kinetics a Km value for the malathion esterase was calculated to be 2.12 x 10 % for the two enzyme levels tested. 

The malathion esterase activity of soil was destroyed by autoclaving. When the purified enzyme was added to an inoculated, autoclaved soil or to a soil of naturally-low esterase activity, and then immediately extracted with alkali, 50-65% of the added activity was recovered in neutral buffer extracts (Tris-HCl, pH 7.0) of the soils, indicative of a rapid adsorption of the added esterase to soil colloids. Further, the adsorbed enzyme remained in a biologically - stable state since no losses of activity occurred.during an eight-week incubation of the amended non-sterile soils. 

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. 

Metabolites that are less reactive than suicide inhibitors may impact more distant enzymes, within the same cell, adjacent cells, or even in other tissues and organs, far removed from the original site of primary metabolism. For example, organopho-sphates (OPs), an ingredient in many pesticides, are metabolized by hepatic CYPs to intermediates, which, when transported to the nervous system, inhibit esterases that are critical for neural function. Acetylcholinesterase (AChE) catalyzes the hydrolysis of the ester bond in the neurotransmitter, acetylcholine, allowing choline to be recycled by the presynaptic neurons. If AChE is not effectively hydrolyzed by AChE in this manner, it builds up in the synapse and causes hyperexcitation of the postsynaptic receptors. The metabolites of certain insecticides, such as the phos-phorothionates (e.g., parathion and malathion) inhibit AChE-mediated hydrolysis. Phosphorothionates contain a sulfur atom that is double-bonded to the central phosphorus. However, in a CYP-catalyzed desulfuration reaction, the S atom is... 

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). 

Hydrolytic reactions. There are numerous different esterases responsible for the hydrolysis of esters and amides, and they occur in most species. However, the activity may vary considerably between species. For example, the insecticide malathion owes its selective toxicity to this difference. In mammals, the major route of metabolism is hydrolysis to the dicarboxylic acid, whereas in insects it is oxidation to malaoxon (Fig. 5.12). Malaoxon is a very potent cholinesterase inhibitor, and its insecticidal action is probably due to this property. The hydrolysis product has a low mammalian toxicity (see chap. 7). 

The hydrolysis of esters by esterases and of amides by amidases constitutes one of the most common enzymatic reactions of xenobiotics in humans and other animal species. Because both the number of enzymes involved in hydrolytic attack and the number of substrates for them is large, it is not surprising to observe interspecific differences in the disposition of xenobiotics due to variations in these enzymes. In mammals the presence of carboxylesterase that hydrolyzes malathion but is generally absent in insects explains the remarkable selectivity of this insecticide. As with esters, wide differences exist between species in the rates of hydrolysis of various amides in vivo. Fluoracetamide is less toxic to mice than to the American cockroach. This is explained by the faster release of the toxic fluoroacetate in insects as compared with mice. The insecticide dimethoate is susceptible to the attack of both esterases and amidases, yielding nontoxic products. In the rat and mouse, both reactions occur, whereas sheep liver contains only the amidases and that of guinea pig only the esterase. The relative rates of these degradative enzymes in insects are very low as compared with those of mammals, however, and this correlates well with the high selectivity of dimethoate. 

The inhibition by other organophosphate compounds of the carboxylesterase which hydrolyzes malathion is a further example of xenobiotic interaction resulting from irreversible inhibition because, in this case, the enzyme is phosphorylated by the inhibitor. A second type of inhibition involving organophosphorus insecticides involves those containing the P=S moiety. During CYP activation to the esterase-inhibiting oxon, reactive sulfur is released that inhibits CYP isoforms by an irreversible interaction with the heme iron. As a result, these chemicals are inhibitors of the metabolism of other xenobiotics, such as carbaryl and fipronil, and are potent inhibitors of the metabolism of steroid hormones such as testosterone and estradiol. 

Talcott, R.E., Mallipudi, N.M., Umetsu, N., and Fukuto, T.R., Inactivation of esterases by impurities isolated from technical malathion, Toxicol. Appl. Pharmacol., 49,107,1979. 

There are two types of esterases that are important in metabolizing insecticides, namely, carboxylesterases and phosphatases (also called phosphorotriester hydrolases or phosphotriesterases). Carboxylesterases, which are B-esterases, play significant roles in degrading organophosphates, carbamates, pyrethroids, and some juvenoids in insects. The best example is malathion hydrolysis, which yields both a- and (i-monoacids and ethanol (Figure 8.10). 

Figure 9.17. Organophosphate inhibitors of acetylcholinesterase. a The catalytic mechanism, shown here for diiso-propylfluorophosphate(DFP).b Stmcturesof soman and tabun. Like DFP, these were developed during world war II as nerve gases , c Stractures of the insecticides parathion and malathion, and of paraoxon, which is the achve metabolite of parathion. (Malathion likewise requires conversion to malaoxon.) The arrow above the malathione stmcture indicates the esterase cleavage sites in its leaving group esterase cleavage occurs in human plasma and renders the molecule non-toxic.

Decreased renal excretion of penicillin when coadministered with probenecid, potentiating its therapeutic effect Ops (profenofos, sulprofos, DEF) potentiate the toxicity of fenvalerate and malathion by inhibiting esterase, which detoxifies many pyrethroid insecticides and malathion... 

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). 

Mechanism of interaction of A- and B-esterases with OPC is similar. B-esterases initially form Michaelis complex with an OPC inhibitor producing phosphorylated or inhibited enzyme that either reactivates very slowly or does not reactivate at all (Figure 3) [1], However, after forming Michaelis complex with OPC A-esterases perform intensive and permanent hydrolysis of OPC and their catalytic activity and turnover rate are very high. It was already shown that carboxylesterases, as a typical B-esterase, can hydrolyze carboxylic esters that serve as functional groups in OPC such as malathion thus performing detoxication of the compound [26, 27]. 

Metabolism of the local anaesthetic procaine provides an example of esterase action, as shown in figure 4.42. This hydrolysis may be carried out by both a plasma esterase and a microsomal enzyme. The insecticide malathion is metabolized by a carboxyl esterase in mammals, rather than undergoing oxidative desulphuration as in insects (figure 5,10). 

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. 

After acute effects subside, OPs produce a condition called the intermediate syndrome mediated through the enzyme neurotoxic esterase (Annau 1992). Symptoms consist of muscle weakness developing days after initial symptoms. Organophosphate-induced delayed neurotoxicity, a second delayed OP effect, develops weeks after exposure (Davis and Richardson 1980 Ecobichon 1996 Willems et al. 1984). Another condition described as wasting away results from toxic by-products generated during synthesis of OP insecticides, especially malathion (Chambers 1992). 

Advantage of species differences in metabolism was taken with the synthesis of malathion which is attacked by esterases in mammals and excreted rapidly as the diacid before conversion of the innocuous thio-phosphate to the toxic phosphate. Insects have very low levels of esterases, and metabolism to the lethal oxo metabolite can occur unimpeded (Figure 20). 

Mendo/a, C. E. (1976). Toxicity and effects of malathion on esterases of suckling albino rats, To.xicol. Appl. Pharmacol. 35, 229-238. 

Choline electrodes coupled with acetylcholine esterase have been described as sensor for several acetylcholine esterase inhibitors NaF, butoxycarboxime, trichlorfon, dimethoate (25), malathion(2i) paraoxon, aldicarb (2i, 22). All these studies used model toxic compounds and consisted of preliminary laboratory investigations aimed at environmental pesticide control. 

Azinphos-ethyl, carbophenothion, diazinon, ethion, malathion, mevinphos, parathion, and OP pesticides were determined in fruits and vegetables by beef liver esterases and S-bromoindoxy 1 acetate. Their TLC separation was carried out on silica gel plates developed in acetone-hexane (1 4) (163). 

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