Aspartic hydrolases mechanism

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 previous chapter offered a broad overview of peptidases and esterases in terms of their classification, localization, and some physiological roles. Mention was made of the classification of hydrolases based on a characteristic functionality in their catalytic site, namely serine hydrolases, cysteine hydrolases, aspartic hydrolases, and metallopeptidases. What was left for the present chapter, however, is a detailed presentation of their catalytic site and mechanisms. As such, this chapter serves as a logical link between the preceding overview and the following chapters, whose focus is on metabolic reactions. 

These three catalytic functionalities are similar in practically all hydrolytic enzymes, but the actual functional groups performing the reactions differ among hydrolases. Based on the structures of their catalytic sites, hydrolases can be divided into five classes, namely serine hydrolases, threonine hydrolases, cysteine hydrolases, aspartic hydrolases, and metallohydrolases, to which the similarly acting calcium-dependent hydrolases can be added. Hydrolases of yet unknown catalytic mechanism also exist. 

Hydrolases possess a common feature for its mechanistic action. Their active site is built around three key amino acid residues, which are histidine (His), serine (Ser), and aspartic acid (Asp) or alternatively glutamic acid (Glu). This site, namely, the catalytic triad, forms a charge-relay network to polarize and activate the nucleophile, allowing the serine hydrolase mechanism to start [19]. This review covers the acylation of alcohols and amines using mainly esters so Figure 9.1 depicts the mechanism for these types of reactions is depicted. 

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). 

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). 

The protein is a 12-stranded anti-parallel p-barrel with amphipathic P-strands traversing the membrane (Fig. 4). The active-site catalytic residues are similar to a classical serine hydrolase triad except that in addition to the serine (Ser-144) and histidine (His-142), there is an asparagine (Asn-156) in place of the expected aspartic acid. Calcium at the active site is predicted to be involved in the reaction mechanism facilitating hydrolysis of the ester. 

Hydrolysis of epoxides, esters, amides, and related structures is an important biotransformation reaction that limits the therapeutic activity of many drugs and generates therapeutically active drugs from prodmg structures. In a few cases, hydrolytic reactions can generate a toxic structure. Epoxide hydrolases and esterases are members of the a/(3 hydrolase-fold family of enzymes (Morisseau and Hammock, 2005 Satoh and Hosokawa, 2006). Although their substrate specificities are radically different (e.g., lipids, peptides, epoxides, esters, amides, haloalkanes), their catalytic mechanisms are similar. All of these enzymes have an active site catalytic triad composed of a nucleophilic serine or cysteine residue (esterases/amidases), or aspartate residue (epoxide hydrolases) to activate the substrate, and histidine residue and glutamate or aspartate residues that act cooperatively in an acid—base reaction to activate a water molecule for the hydrolytic step. 

Borderline-SN2-Type Mechanism. Some enzymes, such as limonene-1,2-epoxide hydrolase, have been shown to operate via a single-step push-pull mechanism [573]. General acid catalysis by a protonated aspartic acid weakens the oxirane to facilitate a simultaneous nucleophilic attack of hydroxyl ion, which is provided by deprotonation of H2O via an aspartate anion. Due to the borderline-SN2-character of this mechanism, the nucleophile preferentially attacks the higher substituted carbon atom bearing the more stabilized 5 -charge. After liberation of the glycol, proton-transfer between both Asp-residues closes the cycle. 

Figure 25.3 shows the relationship of active site of serine hydrolases. The serine hydrolases include serine proteases, lipases, and PHB depolymerases. A common feature of the serine proteases is the presence of a specific amino acid sequence -Gly-Xl-Ser-X2-Gly-. The catalytic mechanism of these enzymes is very similar and the catalytic center consists of a triad of serine, histidine, and aspartate residues [54]. The serine from this sequence attacks the ester bond nucleophilically [55]. Lipases and PHB depolymerases also have a common amino acid sequence around the active site, -Gly-Xl-Ser-X2-Gly-. These serine hydrolases may share a similar mechanism of substrate hydrolysis [21, 56]. In terms of origin of enzymes, it would be wise to consider that the enzyme had wide substrate specificity initially, and then it started to evolve gradually for each specific substrate. In the case of polyester hydrolysis, lipases showed the widest substrate specificity among serine hydrolases for hydrolysis of various polyesters ranging from a-ester bonds to (o-ester bonds. PHB depolymerases would become more specific for microbial PHB that has / -ester bonds, though it could also hydrolyze other polyesters that have -ester and y-ester bonds. Serine proteases such as proteinase K, subtilisin, a-chymotrypsin, elastase, and trypsin hydrolyze only optically active PLLA with a-ester bonds and various proteins with a-amido bonds. 

Peptidases including keratinases are hydrolases able to hydrolyze peptide bonds in proteins and peptides. They are classified using three different approaches (1) the chemical mechanism of catalysis (based on the catalytic amino acid or metal ion at then-active site, represented by serine, cysteine, threonine, aspartic, asparagine, glutamic and metallocatalytic type), (2) the catalytic reaction (this type of classification depends on the selectivity for the bonds that the peptidases will hydrolyze), and (3) the molecular structure and homology. In this latter approach, amino acid... 

Most EHs have a/ 3-hydrolase fold topology and consist of a core and a lid domain [65,66]. The lid domain is mainly a-helical and contains two tyrosine residues that point toward the catalytic triad and cover the core domain. Both tyrosine residues are involved in substrate binding, Uansition-state stabilization, and activation of the epoxide by protonation. The catalytic center is composed of two aspartate and one histidine residue. The first crystal structure of an epoxide hydrolase was solved for the enzyme from Agrobacterium radiobacter ADI (EchA) [67]. The reaction mechanism of EHs is depiaed in Scheme 9.9. First, a nucleophilic attack of the aspartic residue on the epoxide ring of the substrate 31 takes place and a covalently bound ester 32 is formed. This intermediate is subsequently hydrolyzed by a so-called charge relay system (general base catalysis) and the diol 33 is released from the active site. Key reaction parmers are a histidine residue and a water molecule. It is worth mentioning that a limonene epoxide hydrolase discovered by Arand et al. displayed a different crystal structure and catalytic cycle that is discussed elsewhere [68]. 

Classification of this enzyme as a serine, cysteine, aspartic, or metallo-dependent enzyme [50] is somewhat problematic because occasionally inhibitors from the same class led to contradictory results. The observed effects suggest, however, that both metal ions and sulfhydryl groups may play a major role in the hydrolytic mechanism. Therefore, most probably the enzyme is a metallocysteine hydrolase. 

---