Aspartate Catalytic triads

The metabolic breakdown of triacylglycerols begins with their hydrolysis to yield glycerol plus fatty acids. The reaction is catalyzed by a lipase, whose mechanism of action is shown in Figure 29.2. The active site of the enzyme contains a catalytic triad of aspartic acid, histidine, and serine residues, which act cooperatively to provide the necessary acid and base catalysis for the individual steps. Hydrolysis is accomplished by two sequential nucleophilic acyl substitution reactions, one that covalently binds an acyl group to the side chain -OH of a serine residue on the enzyme and a second that frees the fatty acid from the enzyme. 

Figure 29.2 MECHANISM Mechanism of action of lipase. The active site of the enzyme contains a catalytic triad of aspartic acid, histidine, and serine, which react cooperatively to carry out two nucleophilic acyl substitution reactions. Individual steps are explained in the text.

The elucidation of the X-ray structure of chymotrypsin (Ref. 1) and in a later stage of subtilisin (Ref. 2) revealed an active site with three crucial groups (Fig. 7.1)-the active serine, a neighboring histidine, and a buried aspartic acid. These three residues are frequently called the catalytic triad, and are designated here as Aspc Hisc Serc (where c indicates a catalytic residue). The identification of the location of the active-site groups and intense biochemical studies led to several mechanistic proposals for the action of serine proteases (see, for example, Refs. 1 and 2). However, it appears that without some way of translating the structural information to reaction-potential surfaces it is hard to discriminate between different alternative mechanisms. Thus it is instructive to use the procedure introduced in previous chapters and to examine the feasibility of different... 

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. 

This model clearly shows that the catalytic machinery involves a dyad of histidine and aspartate together with the oxyanion hole. Hence, it does not involve serine, which is the key amino acid in the hydrolytic activity of lipases, and, together with aspartate and histidine, constitutes the active site catalytic triad. This has been confirmed by constructing a mutant in which serine was replaced with alanine (Serl05Ala), and finding that it catalyzes the Michael additions even more efficiently than the wild-type enzyme (an example of induced catalytic promiscuity ) [105]. 

The molecular weight of these enzymes is around 27,000 g/mol. The active site where the catalysis takes place consists of a catalytic triad of Serine-221, Histidine-64, and Aspartate-32 (the numbers indicates the position of the amino acid in the peptide chain). A model of a subtilisin showing the binding cleft and the amino acids of the catalytic triad is illustrated through Figure 1. 

Figure 1. A model of a subtilisin showing the binding cleft and the amino acids of the catalytic triad (Serine-221, Histidine-64, and Aspartate-32)...

The mechanism by which serine peptidases, particularly serine endopep-tidases (EC 3.4.21), hydrolyze peptide bonds in peptides and proteins has been extensively investigated by X-ray crystallography, site-directed mutagenesis, detection of intermediates, chemical modification, H-NMR spectroscopy, and neutron diffraction [2-14], These studies revealed that all serine peptidases possess a catalytic triad, composed of a serine, a histidine, and an aspartate residue, and a so-called oxyanion hole formed by backbone NH groups. 

The mechanism schematized above is a summary of the current knowledge. The role of Asp102 has long been controversial [10], Indeed, the catalytic triad has been depicted as a charge-relay system, meaning that the activation of the serine residue involves a concerted transfer of two protons, i.e., from serine to histidine and then to aspartic acid. More recent studies have shown that aspartic acid remains ionized and serves to stabilize the ionic transition state [6] [14-16],... 

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). 

The first class of DUBs discovered, the ubiquitin C-terminal Aydrolases (UCHs), is a relatively small class vith only four members in humans and one in budding yeast. UCHs are cysteine proteases related to the papain family of cysteine proteases. Most UCHs consist entirely of a catalytic core that has a molecular mass of about 25 kDa, although Bapl and UCH37 have C-terminal extensions [21, 22], All UCHs have a highly conserved catalytic triad consisting of the active-site cysteine, histidine, and aspartate residues that are absolutely required for function [23]. 

The ubiquitin specific processing proteases (referred to as UBPs in yeast and USPs in human and mouse) were the second class of DUBs discovered. Catalytically, the UBPs are very similar to the UCHs in that they also utilize the catalytic triad of an active-site cysteine and a conserved histidine and aspartate. The UBP catalytic core... 

In addition to the lack of sequence homology, ULPs have little structural homology to other DUB classes except in the active site. The structure of ULPl (see Figure 8.2) in complex with the C-terminal aldehyde of yeast SUMO (SMT3) illustrates that, like most other DUBs, ULPs are thiol proteases, utilizing a conserved catalytic triad consisting of an active-site cysteine, histidine, and aspartate [40]. 

Lipases belong to the subclass of a/P-hydrolases and their structure and reaction mechanism are well understood. All lipases possess an identical catalytic triad consisting of an aspartate or glutamate, a histidine, and a nucleophilic serine residue [67], The reaction mechanism of CALB is briefly discussed as a typical example of lipase catalysis (Scheme 7). 

Figure 3-22 Stereoscopic view of a section of the structure of cutinase from the fungus Fusarium solani determined to a resolution of 0.10 nm. The three amino acid residues shown are serine 120 (top), histidine 188, and aspartate 175 (lower left). The structure is presented as a contour map with a "wire mesh" drawn at a "cutoff" level of density equal to 1 a above the average, where a is the root mean square density of the entire map. The side chains of these three residues constitute the "catalytic triad" in the active site of this enzyme (see Chapter 12). At this resolution more than one conformation of a group may often be seen. For example, the gamma oxygen (OG) of S120 is seen in two positions, the major one being toward His 188. When the map is drawn with a lower contour level the N-H proton on His 188 that is hydrogen bonded to Asp 175 can also be seen.410 Courtesy of Christian Cambillau.

Below the active site of aspartate aminotransferase, as shown in Fig. 14-6, is a cluster of three buried histidine side chains in close contact with each other. The imidazole of H143 is hydrogen bonded to the D222 carboxylate, the same carboxylate that forms an ion pair with the coenzyme. This system looks somewhat like the catalytic triad of the serine proteases in reverse. As with the serine proteases, the proton-labeled Hb in Fig. 14-6 can be "seen" by NMR spectroscopy (Fig. 3-30). So can the proton Ha on the PLP ring. These protons... 

The active site of serine proteases is characterized by a catalytic triad of serine, histidine, and aspartate. The mechanism of lipase action can be broken down into (i) adsorption of the lipase to the interface, responsible for the observed interfacial activation (ii) binding of substrate to enzyme (iii) chemical reaction and (iv) release of product(s). 

Serine proteases are characterized by a catalytic triad of Ser, His, and Asp. They work in pairs of Ser-His and His-Asp. Replacement of Asp by a second His revealed the auxiliary nature of aspartate, probably in orienting the histidine with respect to the catalytic serine. All of the catalytic steps are performed by the dyad serine-histidine. 

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. 

Amongst the subnanomolar chymotrypsin inhibitors, modelling of one of the best variants implied a novel inhibitory mechanism for protein serine protease inhibitors, in which two amino acid side chains (arginine and aspartic acid) intrude into the proximity of the catalytic triad of the protease rather than binding in the substrate-binding pockets (see Fig. 4). 

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