Serine hydrolase reactivation

Fig. 3.6. Stereoelectronic control of the cleavage of the tetrahedral intermediate during hydrolysis of a peptide bond by a serine hydrolase. The thin lines represent the reactive groups of the enzyme (serine, imidazole ring of histidine) the thick lines represent the tetrahedral intermediate of the transition state. The full circles are O-atoms open circles are N-atoms. The dotted lines represent H-bonds the thick double arrow indicates an unfavorable dipole-dipole interaction [21]. A (R)-configured N-center B (S)-configured N-center.

The greatly increased nucleophilicity of the catalytic serine distinguishes it from all other serine residues and makes it an ideal candidate for modification via activity-based probes [58]. Of the electrophilic probe types to profile serine hydrolases, the fluorophosphonate (FP)-based probes are the most extensively used and were first introduced by Cravatt and coworkers [38, 39]. FPs have been well-known inhibitors of serine hydrolases for over 80 years and were first applied as chemical weapons as potent acetylcholine esterase inhibitors. As FPs do not resemble a peptide or ester substrate, they are nonselective towards a particular serine hydrolase, thus allowing the entire family to be profiled. FPs also show minimal cross-reactivity with other classes of hydrolases such as cysteine-, metallo-, and aspartylhydrolases [59]. Furthermore, FP-based probes react only with the active serine hydrolase, and not the inactive zymogen, allowing these probes to interact only with functional species within the proteome [59]. Extensive use of this probe family has demonstrated their remarkable selectivity for serine hydrolases and resulted in the identification of over 100 distinct serine hydrolases... 

Recently, Li etal,162 used ABPP competitive profiling to screen libraries of carbamate compounds to identify specific inhibitors for uncharacterized serine hydrolases. The carbamate reactive group reacted irreversibly with the active site serine, which, in turn, blocked subsequent labeling with a fluorescent FP probe. The carbamates were decorated with various side chains in order to find the optimal composition for dedicated target binding. Utilizing this method, a potent and selective inhibitor for a//3-hydrolase domain 6, which may play important functions in nervous system metabolism or signaling, was discovered. 

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. 

For the malonate group to be used for fatty acid synthesis, it must first be transferred from malonyl-CoA to malonyl-ACP by the 32.4-kDa monomeric malonyl-CoA ACP transacy-lase, the product of the fabD gene (Fig. 2). A stable malonyl-serine enzyme intermediate is formed during the course of the FabD reaction, and subsequent nucleophilic attack on this ester by the sulfhydryl of ACP yields malonyl-ACP. The high reactivity of the serine in malonyl-ACP transacylase is due to the active site being composed of a nucleophilic elbow as observed in alpha/beta hydrolases. The serine is hydrogen bonded to His-201 in a fashion similar to serine hydrolases. 

Figure 2.3 Serine protease and hydrolase ABPs. (A) Reaction of a general serine hydrolase probe containing a fluorophosphonate (FP) reactive electrophile. This class of probes has been used extensively to label various classes of serine hydrolases including proteases, esterases, lipases and others. (B) The peptide diphenyl phosphonate (DPP) reacts with the serine nucleophile in the active site of serine proteases. This probe is much less reactive than the FP class of probes but is more selective towards serine proteases over other types of serine hydrolases.(C) The natural product epoxomicin contains a keto-epoxide that selectively reacts with the catalytic N-terminal threonine of the proteasome P-subunit. This reaction results in the formation of a stable six-membered ring. This class of electrophile has been used in probes of the proteasome.

An exceptionally reactive serine residue has been identified in a great number of hydrolase enzymes, e. g., trypsin, subtilisin, elastase, acetylcholine esterase and some lipases. These enzymes appear to hydrolyze their substrates by a mechanism analogous to that of chymotrypsin. Hydrolases such as papain, ficin and bromelain, which are distributed in plants, have a cysteine residue instead of an active serine residue in their active sites. Thus, the transient intermediates are thioesters. 

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... 

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