Hydrolases serine hydrolase

Organophosphorus reagents that inhibit hydrolases (serine hydrolases) diethyl p-nitrophenyl phosphate (paraoxon) diisopropyl phosphofluoridate (DFP) dimethyl(dichlorovinyl) phosphate (dichlovos) O -methylphosphoramidothioate 3-hydroxy-N-methyl-cis-crotonamide dimethylphosphate tetraethyl pyrophosphate (TEPP)... 

Cholinesterases (ChEs), polymorphic carboxyles-terases of broad substrate specificity, terminate neurotransmission at cholinergic synapses and neuromuscular junctions (NMJs). Being sensitive to inhibition by organophosphate (OP) poisons, ChEs belong to the serine hydrolases (B type). ChEs share 65% amino acid sequence homology and have similar molecular forms and active centre structures [1]. Substrate and inhibitor specificities classify ChEs into two subtypes ... 

Threonine peptidases (and some cysteine and serine peptidases) have only one active site residue, which is the N-terminus of the mature protein. Such a peptidase is known as an N-terminal nucleophile hydrolase or Ntn-hydrolase. The amino group of the N-terminal residue performs the role of the general base. The catalytic subunits of the proteasome are examples of Ntn-hydrolases. 

The lipase (PAL) used in these studies is a hydrolase having the usual catalytic triad composed of aspartate, histidine, and serine [42] (Figure 2.6). Stereoselectivity is determined in the first step, which involves the formation of the oxyanion. Unfortunately, X-ray structural characterization of the (S)- and (J )-selective mutants are not available. However, consideration of the crystal structure of the WT lipase [42] is in itself illuminating. Surprisingly, it turned out that many of the mutants have amino acid exchanges remote from the active site [8,22,40]. 

The mechanism for the lipase-catalyzed reaction of an acid derivative with a nucleophile (alcohol, amine, or thiol) is known as a serine hydrolase mechanism (Scheme 7.2). The active site of the enzyme is constituted by a catalytic triad (serine, aspartic, and histidine residues). The serine residue accepts the acyl group of the ester, leading to an acyl-enzyme activated intermediate. This acyl-enzyme intermediate reacts with the nucleophile, an amine or ammonia in this case, to yield the final amide product and leading to the free biocatalyst, which can enter again into the catalytic cycle. A histidine residue, activated by an aspartate side chain, is responsible for the proton transference necessary for the catalysis. Another important factor is that the oxyanion hole, formed by different residues, is able to stabilize the negatively charged oxygen present in both the transition state and the tetrahedral intermediate. 

The crystal structure of the HNL isolated from S. bicolor (SbHNL) was determined in a complex with the inhibitor benzoic acid." The folding pattern of SbHNL is similar to that of wheat serine carboxypeptidase (CP-WII)" and alcohol dehydrogenase." A unique two-amino acid deletion in SbHNL, however, is forcing the putative active site residues away from the hydrolase binding site toward a small hydrophobic cleft, thereby defining a completely different active site architecture where the triad of a carboxypeptidase is missing. 

All the enzymes discussed above belong to the class of dimetalloenzymes. In this context, it should be mentioned that serine-type hydrolases are irreversibly inhibited by organophosphorus esters, among them highly toxic chemical warfare agents. However, in some cases, for example of human butyrylcholi-noesterase, the inhibited enzyme could be reactivated by proper mutations." Moreover, such mutahons were found to confer phosphotriesterase activity in this... 

Mutagenesis of known enzyme towards a desired activity will be the fastest developing direction. The use of mutants of simple serine-hydrolases, which exhibit the phosphotriesterase activity (in contrast to the native enzymes, which are irreversibly inhibited under such conditions), clearly shows that practically any kind of substrates can be enzymatically transformed. The... 

Fischer F, S Kunne, S Fetzner (1999) Bacterial 2,4-dioxygenases new members of the a/p hydrolase-fold superfamily of enzymes functionally related to serine hydrolases. J Bacterial 181 5725-5733. 

Hernaez MJ, E Andujar, JJ Rios, SR Kaschabek, W Reineke, E Santero (2000) Identification of a serine hydrolase which cleaves the alicyclic ring of tetralin. / Bacfeno/ 182 5488-5453. 

Seah SYK, G Labbe, S Nerdinger, M Johnson, V Snieckus, LD Eltis (2000) Identification of a serine hydrolase as a key determinant in the microbial degradation of polychlorinated biphenyls. J Biol Chem 275 15701-15708. 

Tarzia et al. [69, 70] have recently reported the FAAH inhibitory activity of a series of alkylcarbamic acid aryl esters. The starting point for their studies was the known serine hydrolase inhibitor carbamyl (51) that had no activity at FAAH. Replacement of the small methyl group of carbamyl (51) with more lipophilic groups and, in particular, bulky lipophilic groups resulted in increased affinity at FAAH (Table 6.6). Exploration of replacements of the naphthyl moiety revealed that replacement with a biphenyl group resulted in improved affinity and in particular, the 3-biphenylyl group proved effective... 

The mechanism of serine (3-lactamases is similar to that of a general serine hydrolase. Figure 8.14 illustrates the reaction of a serine (3-lac(amasc with another type of (3-lactam antibiotic, a cephalosporin. The active-site serine functions as an attacking nucleophile, forming a covalent bond between the serine side chain oxygen... 

The power of the pooled GST fusion protein approach will increase as new biochemical reagents and assays become available. The development of chemical probes for biological processes, termed chemical biology, is a rapidly advancing field. For example, the chemical synthesis of an active site directed probe for identification of members of the serine hydrolase enzyme family has recently been described (Liu et al., 1999). The activity of the probe is based on the potent and irreversible inhibition of serine hydrolases by fluorophosphate (FP) derivatives such as diisopropyl fluorophosphate. The probe consists of a biotinylated long-chain fluorophosphonate, called FP-biotin (Liu et al., 1999). The FP-biotin was tested on crude tissue extracts from various organs of the rat. These experiments showed that the reagent can react with numerous serine hydrolases in crude extracts and can detect enzymes at subnanomolar... 

Remarkably, Brassica napus pollen was reported to have a 22 kDa cutinase that cross-reacted with antibodies prepared against F. solani f. pisi cutinase [134]. Although a 22 kDa and a 42 kDa protein that catalyzed hydrolysis of p-nitrophenyl butyrate were found in this pollen, only the former catalyzed cutin hydrolysis. Immunofluorescence microscopic examination suggested that the 22 kDa protein was located in the intine. Since the nature of the catalytic mechanism of this enzyme has not been elucidated, it is not clear whether this represents a serine hydrolase indicating that plants may have serine and thiol cutinases. The role of the pollen enzyme in controlling compatibility remains to be established. 

Most poly(HA) depolymerases are inhibited by reducing agents, e.g., dithio-erythritol (DTT), which indicates the presence of essential disulfide bonds, and by serine hydrolase inhibitors such as diisopropyl-fluoryl phosphate (DFP) or acylsulfonyl derivates. The latter compounds covalently bind to the active site serine of serine hydrolases and irreversibly inhibit enzyme activity [48]. 

A competitive version of ABPP identifies the target(s) and assesses the selectivity of an enzyme inhibitor in biological systems by gauging how well the inhibitor slows the enzyme s reaction with an ABP. For example, fluorophosphonate ABP 3 was used to profile the selectivity of fatty acid amide hydrolase (FAAH) inhibitors within the serine hydrolase superfamily [27] (FAAH hydrolyzes endocannabinoids such as anandamide). Serine hydrolases that exhibited reduced labeling by the probe in the presence of inhibitor were scored as targets of the inhibitor. Urea FAAH inhibitors exemplified by PF-3845 (5) that covalently modify the active-site serine nucleophile of FAAH were found to be exquisitely selective for FAAH in brain and liver... 

Protein dephosphorylation is catalyzed by phospho-hydrolases called protein phosphatases. While the number of protein tyrosine kinases is roughly comparable to the number of protein tyrosine phosphatases, protein serine-threonine kinases vastly outnumber the protein serine-threonine phosphatases, of which about 25 different species are known to exist. This relative under-representation may be accounted for by the alternative diversification... 

Serine hydrolase s active-site motif (GESAG)... 

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