Hydrolases Hydrolytic Enzymes

Many different enzymes possess hydrolase activity. For example, ALDH shows esterase activity. P450 enzymes also seem to have apparent hydrolase activity at times, although in the case of these enzymes the hydrolysis occurs via oxidative dealkylation by alpha oxidation next to an amide or an ester.

Overall reaction: R4COXR2 + H2O ! RiCOOH + HXR2 where X = O or N

Reaction: The following "catalytic triad" is important for the reaction:

• A nucleophilic residue (serine or cysteine for esterases and ami-dases and aspartic acid for expoxide hydrolase) that attacks the ester/thioester substrate and forms an acyl-enzyme intermediate.

• A water molecule that is coordinated by histidine and glutamic acid/aspartic acid then hydrolyzes the acyl enzyme to recycle the enzyme and liberate the hydrolysis product carboxylic acid.

Chemically, electron-deficient substrates (i.e., electron-withdrawing groups adjacent to an amide bond) result in more-labile amides susceptible to hydrolysis.

Classification based on organophosphates:

A-Esterases (also known as paraoxonases) hydrolyze organo-phosphates such as paraoxon. These enzymes have a free thiol (from cysteine) in their active site, which is critical for its functional activity. Inhibited by p-chloromercurobenzoate.

B-Esterases are inhibited by organophosphates and carbamate insecticides. Enzymes in this class include carboxylesterases and cholinesterases (acetylcholinesterase and butyrylcholinester-ase).

C-Esterases include esterases that are not inhibited by organo-phosphates or do not hydrolyze organophosphates.

Subcellular location: Mainly in the ER, cytosol, and lysosomes, lesser amounts in monocytes and macrophages. Reaction: Hydrolysis of esters and amides. Isoforms: hCEi (180 kDa; optimum pH 6.5) and hCE2 (60 kDa; optimum pH 7.5-8) are the two CE isoforms in humans. hCEi is primarily expressed in the liver, with lesser amounts in the intestine, kidney, lung, testes, and heart. hCE2 is primarily expressed in the intestine and to a lesser extent in the liver.

Substrates: Aromatic and aliphatic esters; hCE2 converts CPT-11 to SN-38.

Inhibitors: Organophosphates and 4-benzyl-piperidine-1-carboxylic acid 4-nitrophenyl ester.

Generally, rodents have a considerably greater concentration of CEs than do humans.

Cellular location: Lysosomes, lumen of the ER (same side as uridine diphosphate glucuronosyltransferase (UGT)), and gut bacteria.

1,4-Saccharolactone is typically used to inhibit P-glucuronidase activity in in vitro incubations. Oleson and Court argue that addition of this inhibitor does not result in improved detection of glucuronide metabolites, and in some cases, it results in UGT inhibition (Oleson and Court 2008).

The two classes of EH that are important for xenobiotic metabolism are microsomal EH (mEH; prefers cis epoxide substrates; optimum pH = 9) and soluble EH (sEH; prefers trans epoxide substrates; optimum pH = 7.4) (Morisseau and Hammock 2005).

Organ distribution: Both types are present in all tissues, with the highest level in the liver.

Reactions: Hydrolysis of arene and alkene epoxides to polar diols.

The hydrolysis of epoxides plays a key role in detoxification (for example, the hydrolysis of epoxide derivatives of polycyclic aromatic hydrocarbons such as benzo[a]pyrene 4,5-oxide).

mEH substrates: Benzo[a]pyrene 4,5-oxide, cis-stilbene oxide, and styrene oxide.

mEH inhibitors: 1,1,1 -Trichloropropene-2,3-oxide, divalent heavy metals (Hg2+and Zn2+), and cyclopropyl oxiranes.

sEH substrate: Trans-stilbene oxide.

sEH inhibitors: Chalcone oxides, trans-3-phenylglycidols, Cd2+ and Cu2+. Arylesterases/Paraoxonases (PONs; EC

Organ distribution: PON1 is synthesized in the liver and excreted to plasma and plays an important role in lipid metabolism (van Himbergen et al. 2006); PON2 is ubiquitous; PON3 is found in the liver, GI tract, kidney, lung, and brain.

Substrates: Aromatic esters phenyl acetate (PON1); p-nitrobu-tyrate (PON3 > PON1 = PON2), paraoxon (PON1), and aromatic lactones (Draganov et al. 2005).

Inhibitors: Sulfanilamide and Hg2+. Pseudocholinesterase/Butyrylcholinesterase (BuChE; EC Organ distribution: Widely distributed, high concentration in plasma.

Substrates: Basic compounds containing esters such as succi-nylcholine, mivacurium, procaine, and cocaine.

Inhibitors: Organophosphates and physostigmine. BuChE was the first most widely studied gene for genetic polymorphisms.

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