Active-site serine residue

AEBSF, an irreversible inhibitor of serine proteases, was found to completely inhibit MCA-hydrolysing activity in the concentrated crude culture filtrate at a concentration of 1 mM. We studied AEBSF inhibition of CinnAE at concentrations of 1 and 5 mM AEBSF and found activity was reduced to less than 1 % of that found in the uninhibited sample within 18 h of treatment. These results indicate that CinnAE has an active site serine residue. 

The discovery of the ethylidenecarbapenems, the asparenomycins, as naturally occurring /J-lactamase inactivators in the early 1980s was another striking point in /J-lactamase inhibitor research. The substituted exomethylene function in asparenomycins is a distinctive feature of this class of compounds, which many scientists recognized could be a key factor for /J-lactamase inhibition. The exo cyclic methylene is expected to increase the acylation ability, and form an a,/J-unsaturated ester of the active site serine residue as an acyl-enzyme complex. This ester will be similar in structure to the acyl-enzymes formed from clavulanate and sulfone fragmentation, and will be quite resistant to hydrolytic deacylation. Thus, the exocyclic methylene promotes acylation by the enzyme and subsequently represses deacylation. Based on... 

As a result, the penicillin occupies the active site of the enzyme, and becomes bound via the active-site serine residue. This binding causes irreversible enzyme inhibition, and stops cell-wall biosynthesis. Growing cells are killed due to rupture of the cell membrane and loss of cellular contents. The binding reaction between penicillinbinding proteins and penicillins is chemically analogous to the action of P-lactamases (see Boxes 7.20 and 13.5) however, in the latter case, penicilloic acid is subsequently released from the P-lactamase, and the enzyme can continue to function. Inhibitors of acetylcholinesterase (see Box 7.26) also bind irreversibly to the enzyme through a serine hydroxyl. 

The answer is D. Organophosphates react with the active site serine residue of hydrolases such as acetylcholinesterase and form a stable phosphoester modification of that serine that inactivates the enzyme toward substrate. Inhibition of acetylcholinesterase causes overstimulation of the end organs regulated by those nerves. The symptoms manifested by this patient reflect such neurologic effects resulting from the inhalation or skin absorption of the pesticide diazinon. 

Fidder and coworkers (50) developed a versatile procedure that identifies phosphylated butyrylcholi-nesterase. Adducted butyrylcholinesterase is isolated from plasma by affinity chromatography (procainamide column), digested with pepsin, and a nonapep-tide containing the phosphylated active-site serine residue detected using LC/ESI/MS/MS (quadrupole-TOF hybrid instrument). A C18 150 x 0.3-mm LC column was used, eluted with a gradient of water-acetonitrile-0.2 % formic acid. The method was applied successfully to casualties of sarin poisoning from the Tokyo subway attack (see Chapter 17). 

The action of p-lactam antibiotics is considered to be due to the formation of an acyl enzyme with carboxypeptidases and transpeptidases which are involved in the biosynthesis of bacterial cell walls38). A three-step mechanism involving a stable acyl-enzyme intermediate (El ), a participating active site serine residue, and a very slow decay process (k4.) was proposed [Eq. (9)]59). 

Sulfonyl Fluorides. Sulfonyl fluorides inhibit serine proteases by reacting with the active-site serine residue. Previously we investigated the rates of inhibition of human leukocyte elastase and cathepsin G by a variety of sulfonyl fluorides and found relatively little selectivity or reactivity (38). However, we have discovered recently that the introduction of fluoroacyl groups into the sulfonyl fluoride structure gives considerable reactivity and selectivity for elastase (39). 

Interaction of CarbE with nerve agents follows a kinetic of first order characterized by inhibition of CarbE at the active site serine residue described by a bimolecular rate constant, ki (Maxwell and Brecht, 2001). For noncharged nerve agents (e.g. sarin and soman) the ki of rat serum CarbE was found to be >10 M min whereas cationic substrates (e.g. VX) are converted with poor reactivity (ki < 10" M min ). This specificity is explained by the electrostatic characteristics of the large active site containing only a few cation-II bonding and anionic residues (Maxwell and Brecht, 2001 Satoh and Hosokawa, 2006). 

Most irreversible enzyme inhibitors combine covalently with functional groups at the active sites of enzymes. These inhibitors are usually chemically reactive, and many of them show some specificity in terms of the amino acid groups which they react with. Diisopropyl fluorophosphate (DFP), for example, forms a covalent adduct with active site serine residues, such as in the serine proteases, and in acetylcholinesterase, which explains its toxic effect on animals. Irreversible enzyme inhibition can be used to identify important active site residues. A special case of irreversible enzyme inhibition is the effect of suicide inhibitors, which are generally chemically unreactive compounds that resemble the substrate of the target enzyme and bind at the active site. The process of enzyme turnover begins, but the inhibitor is so... 

Figure 9.10. The Hydrophobic Pocket of Chymotrypsin. The hydrophobic pocket of chymotrypsin is responsible for its substrate specificity. The key amino acids that constitute the binding site are labeled, including the active-site serine residue (boxed). The position of an aromatic ring bound in the pocket is shown in green.

Figure 9.13. The Sj Pockets of Chymotrypsin, Trypsin, and Elastase. Certain residues play key roles in determining the specificity of these enzymes. The side chains of these residues, as well as those of the active-site serine residues, are shown in color.

Figure 3 Design of an ABPP probe. The design is driven by the choice of the active site-directed group (e.g., fluorophosphonate (FP), shown in green) and a fluorescent tag (rhodamine, shown in red) separated by a spacer group. FPs react covalently with the active site serine residue of the serine hydrolase enzyme family (see also Figure 9). B, base. Reproduced by permission of The Royal Society of Chemistry.

These organic phosphates inhibit acetylcholinesterase by reacting with the active-site serine residue to form a stable phosphorylated derivative. They cause respiratory paralysis by blocking synaptic transmission at cholinergic synapses. 

Another series of irreversible inhibitors—alkylcarbamic acid aryl esters, with apparent specificity for FAAH—has also been reported (Kathuria et al. 2003 Tarzia et al. 2003). These inhibitors, which do not bind to CB or CB2 receptors or inhibit MGL or AEA cellular uptake, act by carbamoylation of the active site serine residue. The most potent of the series is URB597 (Kathuria et al. 2003). Of added significance is that these analogs, although difficult to emulsify, are also active as inhibitors of FAAH in vivo, resulting in an elevation of brain AEA content of approximately threefold at a dose of 0.3 mg/kg without an effect on the content of 2-AG (Kathuria et al. 2003). 

A recombinant protein NEST corresponding to human NTE residues 727-1216 has been expressed in Escherichia coli which composes the esterase domain of NTE [34], This polipeptide contains the active site serine residue, Ser-996, as well as two aspartates, Asp-960 and Asp-1086, that are necessary for esterase activity. NEST has cnzymological properties similar to full-length NTE, including inactivation by OP compounds and hydrolysis of membrane lipids [34,44]. The availability of recombinant NTE esterase domains, NEST, has facilitated investigations of the catalytic properties of NTE in vitro. 

Stone, S.R., LeBonniec, B.F. 1997. Inhibitory mechanism of serpins. Identification of steps involving the active-site serine residue of the protease, / Mol Biol 265, 344—362. 

MNPHP is a well-known irreversible inhibitor of lipases that is highly specific for reaction with active-site serine residues. Thus, MNPHP was selected as the inhibitor to determine, by titration, the fraction of catalytic sites that are accessible and active. Since we are concerned with CALB activity in organic media, inhibition was studied in heptane. LC-MS was used to determine the release of p-nitrophenol (pNP) which corresponds with accessible active sites. To ensure that adsorption of pNP by resins was taken into consideration, pNP concentration was corrected as follows. A fixed quantity of enzyme-free resin was incubated overnight in acetonitrile with different concentrations of j NP. Standard curves of pNP adsorption by each resin as a function of pNP concentration were constructed from LC-MS measurements. MNPHP-inhibited immobilized enzymes were used for e-caprolactone ring-opening polymerizations in toluene (70 C). No conversion of monomer was observed in 30 minutes. Hence, MNPHP titration resulted in complete inhibition of CALB activity. 

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