Esterases and Amidases
Esterases and amidases catalyze the addition of a water molecule to an ester, thio-ester or an amide resulting in the formation of the corresponding acid and alcohol or amine. Like EHs and esterases are members of the α/β-hydrolase fold family of enzymes. They share a common structure and catalytic mechanism involving the formation and hydrolysis of a covalent intermediate. Both enzymes add water to the substrate with no additional cofactors needed. While the EHs reaction is irreversible, the action of esterases and amidases is reversible and in some conditions these enzyme could be used for thesynthesis of ester or amides. Furthermore, the esterases and amidases used two substrates (ester and water) to form two products (acid and alcohol or amine).
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Esterases and amidases play an important role in maintaining normal physiology and metabolism, detoxifying various drugs and environmental toxicants in living systems and are increasing important for chemical synthesis in industry.While we are focusing on the role of esterases and amidases in the metabolism of pyrethroid insecticides, we also investigate their role in the metabolism of endocannabinoids, and thus their role in inflammation and analgesia. As indicated in the table at right, we have cloned and super-expressed 15 human esterases and amidases. We are studying their role in the metabolism of numerous compounds, including pesticides, pharmaceutical drugs and endogenous compounds. To study these enzymes, we have developed an array of fluorescent substrates and a variety of chemical inhibitors. |
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Metabolism of pyrethroid insecticides
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Pyrethroids
are the
dominant insecticides used nowdays. Although
regarded as generally safe, human exposure can result in neuropathies.
Long term effects have been suggested; multiple acute symptoms and even
occasional deaths have been reported. The major pyrethroids (left) are
esters, and many are sold as geometrically and/or optically rich
isomers. |
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Cypermethrin for example has 8 optical and geometrical isomers with vastly different biological properties. We have a library of pyrethroids, have extensive expertise in their synthesis and analysis of these chiral esters using GLC-MS, LC-MS and biosensors, and we have prepared numerous immunoassaysbfor pyrethroids and metabolites. We developed a novel class of intensely fluorescent, stable, optically active surrogate substrates for pyrethroid esterase activity. They facilitate high throughput assays, monitoring biochemical purification and biomonitoring using very small samples. |
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The
importance of
esterases has been long recognized in
toxicology.
This is in part because carbamate and organophosphate insecticides
could result in chemical knockouts. In the last few years the
pyrethroid insecticides have emerged as the dominant insecticides and
most are degraded largely by esterases. Thus, we are investigating the
roles of esterases in their metabolism. Clearly genetic or chemical
alterations in esterase could alter the risk not only of insecticides
like pyrethroids but a variety of xenobiotics including common drugs
and prodrugs as well as natural flavors and odors.
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To test the hypothesis that hydrolysis of a single pyrethroid stereoisomer by carboxylesterases correlates to that of the corresponding authentic pyrethroid stereoisomer, we synthesized all the stereoisomers of pyrethroid fluorescent substrates mimicking cypermethrin and fenvalerate. The hydrolysis of such stereoisomers by two purified murine hepatic carboxylesterases and human carboxylesterases (CES1 and CES2) are well correlated with hydrolysis of authentic pyrethroids in both activity and stereo-selectivity. Furthermore, using docking experiments, we explained the selectivity of CES1 for these compounds (figure at left). |
Metabolism of endocannabinoids
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Endocannabinoids are substances produced from within the body which activate cannabinoid receptors (CB). The first such compound identified was arachidonoyl ethanolamide (also reported as anandamide). Anandamide is derived from the essential fatty acid arachidonic acid. It binds to the central (CB1) and, to a lesser extent, peripheral (CB2) cannabinoid receptors, where it acts as a partial agonist. It is found in nearly all tissues in a wide range of animals. Another endocannabinoid, 2-arachidonoyl glycerol, binds to both the CB1 and CB2 receptors with similar affinity, acting as a full agonist at both. 2-AG is present at significantly higher concentrations in the brain than anandamide, and thus it is though to be mostly responsible for endocannabinoid signalling in vivo. It binds primarily to the CB1 receptor, and only weakly to the CB2 receptor. More recently, N-arachidonoyl-dopamine (NADA) was found to preferentially bind to the CB1 receptor. Like anandamide, NADA is also an agonist for the vanilloid receptor subtype 1 (TRPV1), a member of the vanilloid receptor family. |
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Endocannabinoids serve as intercellular 'lipid messengers', signaling molecules that are released from one cell and activate the cannabinoid receptors present on other nearby cells. Although in this intercellular signaling role they are similar to the well-known monoamine neurotransmitters, such as acetylcholine, GABA or dopamine, endocannabinoids differ in numerous ways from them. For instance, they are have retrograde signaling. Furthermore, endocannabinoids are lipophilic molecules that are not very soluble in water. They are not stored in vesicles, and exist as integral constituents of the membrane bilayers that make up cells. They are believed to be synthesized 'on-demand' rather than made and stored for later use.
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The main metabolism pathway of endocannabinoids is through hydrolysis by esterases and amidases, especially by FAAH and MGL. We are using our unique esterase inhibitors to ask the endogenous roles for these enzymes and other esterases in chemical mediation. Already we have shown (at right) that inhibition of 2-acylglycerol lipase (MGL) can stabilize a potent endocannabinoid and reduce the proliferation of human prostate cancer cells. Thus as shown at right, alterations in esterase activity can have profound biological effects through both xenobiotic and endogenous esters. |
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