Esterases and Other Hydrolases

Many xenobiotics, both synthetic and natnrally occuring, are lipophilic esters. They can be degraded to water-soluble acids and bases by hydrolytic attack. Two important examples of esteratic hydrolysis in ecotoxicology now follow  

Enzymes catalyzing the hydrolysis of esters are termed esterases. They belong to a larger group of enzymes termed hydrolases, which can cleave a variety of chemical bonds by hydrolytic attack. In the classification of hydrolases of the International Union of Biochemistry (lUB), the following categories are recognized  

Organic Pollutants An Ecotoxicological Perspective, Second Edition 

FIGU RE 2.10 Plasma A-esterase activities of birds and mammals. Activities were originally measured as nanomoles product per milliliter of serum per minute, but they have been converted to relative activities (male rat = 1) and plotted on a log scale. Each point represents a mean value for a single species. Substrates , paraoxon , pirimiphos-methyl oxon. Vertical hues indicate limits of detection, and all points plotted to the left of them are for species in which no activity was detected. (Activities in the male rat were 61 4 and 2020 130 for paraoxon and pirimiphos-methyl oxon, respectively.) (From Walker 1994a in Hodgson and Levi 1994.) 

Cholinesterases are another group of B-esterases. The two main types are acetylcholinesterase (EC 3.1.1.7) and unspecific or butyrylcholinesterase (EC 3.1.1.8). Acetylcholinesterase (AChE) is found in the postsynaptic membrane of cholinergic 

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Ester hydrolysis, esterification or acylation, and transesterification reactions play an important role in nature and organic synthesis including the synthesis of natural products [1, 2], Applications of hydrolytic or esterification reactions range from laboratory syntheses (e.g. with the acyl moiety as key intermediate or as protecting group) to industrial scale production of bulk chemicals, biodiesel or food (e.g. ester units as building blocks for polymerisation and polycondensation reactions, transesterification of triglycerides) [3, 4]. Transesterification reactions in nature include for example enzymatic reactions of lipases, esterases and other hydrolases that rely on the catalytic triad of serine proteases, which will be discussed in the first part of this section [5-8]. 

Since MGL is a serine hydrolase, its sensitivity to many of the available serine hydrolase inhibitors has been explored (Table 3). The results support the hypothesis that MGL can be inhibited by compounds that interact with its reactive serine. On the other hand, the potencies of the inhibitors are quite variable in some cases, this likely reflects differences in assay methodology (i.e., substrate concentration, pH, form of the enzyme). However, in a few cases, the same assay conditions revealed very different inhibitory potencies (e.g., compare the platelet and macrophage membrane studies by Di Marzo et al. 1999). In any event, studies of these compounds are not likely to yield selective inhibitors of MGL. All of these compounds are inhibitors of FAAH (see above) and many are also inhibitors of PLA2, diacylglycerol lipase, and acetylcholine esterase, among other hydrolases. By analogy to the development of the URB series of FAAH inhibitors (Kathuria et al. 2003), it is likely that selective inhibitors of MGL will come from other synthetic avenues. 

Phase 1 metabolizing enzymes include the CYP and FMO enzymes, esterases, and epoxide hydrolases, among others. These enzymes and the reactions they catalyze have been described in detail previously (Hodgson and Goldstein, 2001 Parkinson. 2001). The CYP and FMO families are perhap.s the best characterized because they are capable of catalyzing a wide range of reactions with broad substrate specificity. These enzymes are located in the endoplasmic reticulum of the cell and have been studied in many organs and tissues. 

The microsomal fraction consists mainly of vesicles (microsomes) derived from the endoplasmic reticulum (smooth and rough). It contains cytochrome P450 and NADPH/cytochrome P450 reductase (collectively the microsomal monooxygenase system), carboxylesterases, A-esterases, epoxide hydrolases, glucuronyl transferases, and other enzymes that metabolize xenobiotics. The 105,000 g supernatant contains soluble enzymes such as glutathione-5-trans-ferases, sulfotransferases, and certain esterases. The 11,000 g supernatant contains all of the types of enzyme listed earlier. 

Second, esterases have broad (or even very broad) and overlapping substrate specificities. For example, carboxylesterase (EC 3.1.1.1) also catalyzes reactions characteristic of a number of other hydrolases. The discovery that individual isoenzymes of carboxylesterases may be identical to or closely related to acylglycerol lipase, acylcamitine hydrolase, and palmitoyl-CoA hydrolase (see Sect. 2.4.3) has increased the confusion surrounding esterase classification [59], Many esterases are able to hydrolyze amides, thiolesters,... 

Peptide hydrolases (peptidases or proteases, i.e., enzymes hydrolyzing peptide bonds in peptides and proteins, see Chapt. 2) have received particular attention among hydrolases. As already described in Chapt. 2, peptidases are divided into exopeptidases (EC 3.4.11 -19), which cleave one or a few amino acids from the N- or C-terminus, and endopeptidas-es (proteinases, EC 3.4.21-99), which act internally in polypeptide chains [2], The presentation of enzymatic mechanisms of hydrolysis in the following sections will begin with peptidases and continue with other hydrolases such as esterases. 

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). 

Whereas several areas of biocatalysis - in particular the use of easy-to-use hydrolases, such as proteases, esterases and lipases - are sufficiently well research to be applied in every standard laboratory, other types of enzymes are still waiting to be discovered with respect to their applicability in organic-chemistry transformations on a preparative scale. This latter point is stressed in this volume, which concentrates on the newcomer-enzymes which show great synthetic potential. 

Although the hydrolysis of esters with lipases and esterases represents the most common process to obtain chiral intermediates for the synthesis of pharmaceuticals, proteases and other hydrolytic enzymes such as epoxide hydrolases and nitrilases have also been used for this purpose. We show here a few representative examples of the action of these biocatalysts that have been recently published. 

The substrate specificity of many esterases is not high (19) and the same is true of some proteases (amide-hydrolyzing enzymes), such as a-chymo-trypsin (12, 20). Amides may also serve as substrates for some esterases (21). Since esterases and proteases are widespread, hydrolysis of ester or amide linkages often accompanies other transformations by intact organisms. Soluble hydrolases are often present in supernatant fractions of mammalian microsomal preparations, and hydrolytic reactions may also occur when extracts of this type are used. Glycosidases, which catalyze the hydrolysis of... 

Alkaline phosphatase, acid phosphatase, 5 -nucleotidase, monoacyl hydrolase, ribonuclease, type 1 phosphodiesterase, adenosine triphosphatase, adenyl cyclase, glycosyl transferase, esterases and disaccharidase have been biochemically or cytochemically demonstrated in the tegument of various cestodes (152, 210, 250, 374, 491, 620, 624-626, 651, 718, 763, 776, 898). Several of these enzymes - phosphatases, 5 -nucleotidase and phosphodiesterase - probably have a digestive and/or absorptive function but the role of the others is uncertain. 

Enzymes that hydrolyze lysophospholipids are found in nearly all tissues and organisms. They seem to be non-specific esterases of the serine-histidine type (25) and hardly deserve the name lysophospholipase because they also hydrolyze esters other than phospholipids. They should probably be considered together with such enzymes as cholesterol esterases and monoglyceride lipases as amphiphilic carboxyl ester hydrolases. These non-specific esterases have a preference for amphiphilic (hydrophilic-lipophilic) substrates. Such an enzyme may perhaps hydrolyze lysophospholipis, monoglycerides, diglycerides, and cholesterol esters. 

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