Esterases catalytic triad

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). 

To help the reader gain a better understanding of the three-dimensional structure of the catalytic site of an esterase, Fig. 3.8 presents the 3D structure of human butyrylcholinesterase (EC 3.1.1.8) obtained by homology modeling [42], The overall structure of the enzyme is shown in Fig. 3.8, a, while Fig. 3.8,b shows a closeup of the active site with the catalytic triad highlighted and the close spatial relationship of the Ser-His-Glu residues revealed. 

A sutfonylating agent (abbreviated PMSE) that irreversibly inhibits many serine esterases and serine proteases . Target enzymes usually react with PMSE and related alkylating agents through the activated imidazole group of a histidyl residue that is part of the catalytic triad. 

All of these esterases appear to act by mechanisms closely related to those of proteases. Acetylcholinesterase contains an active site serine that reacts with organophosphorus compounds (Box 12-E) and is part of an Asp-His-Ser catalytic triad which lies in a deep "gorge" as well as an oxyanion hole.637 A surprise is the absence of an essential carboxylate group that might bind the positively charged trimethylammonium... 

Hydroxynitrile lyase (HNL) oxynitrilase esterase catalytic triad Ser-His-Asp Wagner, 1996... 

GXSXG lipase consensus sequence 8 an-kyrin repeats Ca2+-in-dependent catalytic activity, proline-rich consensus motif (PX5PX8HHPX12NX4Q), LH-iPLA 2 (long isoform, 88 kDa) but not SH-iPLA2 (short isoform, 85 kDa) is activated by ATP GDSXV modified consensus sequence of serine esterase family catalytic triad Ser-His-Asp P and y subunits form a heter-otrimer with a 45-kDa noncatalytic subunit Ca2+-indepen-dent catalytic activity 0 Ca2+-independent catalytic activity... 

A similar concept was used in the development of artificial chymotrypsin mimics [54]. The esterase-site was modeled by using the phosphonate template 75 as a stable transition state analogue (Scheme 13.19). The catalytic triad of the active site of chymotrypsin - that is, serine, histidine and aspartic acid (carboxy-late anion) - was mimicked by imidazole, phenolic hydroxy and carboxyl groups, respectively. The catalytically active MIP catalyst 76 was prepared using free radical polymerization, in the presence of the phosphonate template 75, methacrylic acid, ethylene glycol dimethacrylate and AIBN. The template removal conditions had a decisive influence on the efficiency of the polymer-mediated catalysis, and best results were obtained with aqueous Na2CC>3. 

Fig. 12. Ribbon diagram of wild type pN R esterase, looking down into the active site. Loops that are not visible in the electron density are shown with dashed lines, and loops that reorganize most significantly are shown in gold. The catalytic triad is shown in red, and secondary structures are labeled. (Figure from Spiller et al., 1999.)...

The most thermophilic variant of j/NB esterase, 8G8, has only thirteen mutations compared to the wild-type esterase, making it 97% identical to the wild-type esterase sequence, with a root-mean-square deviation of only 0.44 A between the two C backbone structures. As with the 5-6C8 organophile structure, the catalytic triads of 8G8 and wild-type / NB esterase are superimposable. This high sequence and structural identity, in conjunction with the availability of crystal structures for both the wild type and thermophile, affords an interesting opportunity to study the structural basis for thermostability. Thermophile 8G8 is the product of eight generations of directed evolution, screening for retention of activity... 

Clan SC peptidases are a/p hydrolase-fold enzymes that consist of parallel P-strands surrounded by a-helices. The a/p hydrolase-fold provides a versatile catalytic platform that, in addition to achieving proteolytic activity, can either act as an esterase, lipase, dehalogenase, haloperoxidase, lyase, or epoxide hydrolase (18). Six phylogenetically distinct families of clan SC are known, and oifly four of them have known structure. Catalytic amenability of the a/p hydrolase-fold may underlie why clan SC peptidases are the second largest family of serine peptidases in the human genome. Other mechanistic classes need not use the catalytic serine and instead use cysteine or glutamic acid (19). Clan SC peptidases present an identical geometry to the catalytic triad observed in clans PA and SB, yet this constellation is ordered differently in the polypeptide sequence. Substrate selectivity develops from the a-helices that surround the central P-sheet core. Within clan SC, carboxypeptidases from family SIO are unique for their ability to maintain... 

Yet another example of the catalytic triad has been found in carboxypeptidase II from wheat. The structure of this enzyme is not significantly similar to either chymotrypsin or subtilisin (Figure 9.15). This protein is a member of an intriguing family of homologous proteins that includes esterases such as acetylcholine esterase and certain lipases. These enzymes all make use of histidine-activated nucleophiles, but the nucleophiles may be cysteine rather than serine. 

As stated earlier, lipases act at the interface between hydrophobic and hydrophilic regions, a characteristic that distinguishes lipases from esterases. Similar to serine proteases, lipases share the nucleophile-histidine-acidic residue catalytic triad that manifests itself as either a Ser-His-Asp triad or a Ser-His-Glu triad. The enzyme s catalytic site often is buried within the protein structure, surrounded by relatively hydrophobic residues. An a-helical polypeptide structure acts as a cover, making the site inaccessible to solvents and substrates. For the lipase to be active, the a-helical lid structure has to open so that the active site is accessible to the substrate. The phenomenon of interfacial activation is often associated with reorientation of the lid, increasing the hydrophobicity of the surface in the vicinity of the active site and exposing it. The opening of the lid structure may be initiated on interaction with an oiFwater interface. 

The catalytic site in RmL was identified originally from the location of the known lipase/esterase consensus sequence G-X-S-X-G (Brenner, 1988) containing the nucleophilic serine (Ser-144). This amino acid was found to be involved in a hydrogen-bonded constellation also including His-257 and Asp-203. Overall this hydrogen-bonding network is very reminiscent of the catalytic triad of serine proteinases. However, in contrast to proteinases, the triad is concealed under a short helix, the lid, and is therefore inaccessible to solvent. 

The discovery of catalytic triads in lipases and in related esterases, such as acetylcholinesterase (AChE) (Sussman et al., 1991) and cutinase (Martinez et al., 1992), revived interest in this otherwise well-known constellation of amino acids (like GcL, the AChE triad includes a glutamate). It should also be remembered that there are other functionally... 

Laurell and coworkers have identified an alternatively spliced shorter (80 kDa) form of human HSL that is exclusively expressed in WAT and devoid of both esterase and lipase activities, and is presumably generated by skipping of exon 6, which encodes fhe serine residue of the catalytic triad [295]. Subsequenfly, two HSL immunoreactive bands (88 kDa = L form and 84 kDa = S form) in homogenates from human obese adipose tissue were reported [296]. Immunodetection experiments with an antibody specifically recognizing the domain encoded by exon 6 suggested fhat fhe 80 kDa and S forms correspond to the polypeptide translated... 

Karlsson M, Contreras JA, Heilman U, Tornqvist H, Holm C (1997) cDNA cloning, tissue distribution, and identification of the catalytic triad of monoglyceride lipase. Evolutionary relationship to esterases, lysophospholipases, and haloperoxidases. J Biol Chem 272 27218-27223... 

Mechanistic and structural investigations have revealed that while some esterase antibodies promote direct attack by hydroxide on the ester function [ 15], others unexpectedly may also use a covalent mechanism involving a serine-histidine dyad strongly reminiscent of the catalytic triad of serine proteases [16]. 

CE Family 1 is very large and contains members which do not act on carbohydrate-derived substrates. The crystal structure of a CE 1 domain of XynlOB modular enzyme from Clostridium thermocellum has been solved. " The CE 1 domain is a feruloyl esterase which hydrolyses the feruloyl groups attached to some arabinofuranosyl 05 groups in native xylan. (The Xyn lOB protein as a whole consists of two CBM 22 domains, a dockerin domain, and a GH 20 xylanase domain, and forms part of a cellulosome - see Section 5.10.) The enzyme has the common a/p hydrolase fold. Studies of ferulic acid complexes of the inactive alanine mutant of the active site serine revealed the classic catalytic triad, and two main-chain peptide NH bonds are in place to form an oxyanion hole . A remarkable feature is that the enzyme as repeatedly isolated was esterilied on the active site serine by phosphate or sulfate. 

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