Aspartic hydrolases

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. 

The aspartic hydrolases, metallohydrolases, and calcium-dependent hydrolases, which activate (i.e., render more nucleophilic) a H20 molecule and allow it to attack the substrate. Here, no covalent complex is formed with the enzyme. 

Aspartic endopeptidases (EC 3.4.23) are the best-known aspartic hydrolases and the only ones to be presented here. These enzymes were formerly called acid proteinases because most of them are active at low pH. In con-... 

In the other subdivision, water activation occurs in the first step of the enzymatic cycle. This activation is achieved by a carboxylate group in aspartic hydrolases (Fig. 3.10), Zn2+ and a carboxy group in metallopep-tidases (Fig. 3.12 ), a histidine side chain in calcium-dependent hydrolases (Fig. 3.14), or a Zn2+ in carbonic anhydrase (Fig. 3.15). The substrate, on the other hand, is polarized (activated) by a carboxy group in aspartic hydrolases or by a cation in metallopeptidases and calcium-dependent hydrolases. In this manner, the reactivity of both the water molecule and the substrate is enhanced and fine-tuned to drive formation of a tetrahedral intermediate that will break down to form the hydrolysis products. 

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. 

The NC-IUBMB has introduced a number of changes in the terminology following the proposals made by Barrett, Rawlings and co-workers [7] [8]. The term peptidase should now be used as a synonym for peptide hydrolase and includes all enzymes that hydrolyze peptide bonds. Previously the term peptidases was restricted to exopeptidases . The terms peptidase and protease are now synonymous. For consistency with this nomenclature, the term proteinases has been replaced by endopeptidases . To complete this note on terminology, we remind the reader that the terms cysteine endopeptidases and aspartic endopeptidases were previously called thiol proteinases and acid or carboxyl proteinases , respectively [9],... 

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). 

Another important problem is the development of insects resistant to insecticides. This often arises as a result of increased levels of carboxylesterases which hydrolyze both organophosphates and car-baryl.h/1 A mutation that changed a single active site glycine to aspartate in a carboxylesterase of a blowfly changed the esterase to an organophosphorus hydrolase which protected the fly against insecticides.)... 

Second, consider reaction 6.3.5.4 that has 8 reactants atp + aspartate + glutamine + h2o = amp + ppi + asparagineL + glutamate This reaction can be considered to be made up of three hydrolase reactions atp + h2o = adp + pi... 

ATPase adenosine triphosphate hydrolase Asp aspartic acid... 

GcL contains 544 amino acids in a single chain folded into one domain, making it one of the largest structural domains observed to date in a protein. Like RmL, GcL is an a structure with a central, predominantly parallel jS sheet. There are 11 strands in the central sheet, 3 more in a small additional sheet, and 17 a helices (Fig. 2). The catalytic Ser-217, a part of the G-X-S-X-G pentapeptide, is located at a tight turn between the C terminus of a /3 strand and an N terminus of an a helix, exactly as observed in RmL. The hydroxyl of Ser-217 is hydrogen bonded to the imidazole of His-463, which in turn donates a hydrogen bond to Glu-354. Thus, GcL constitutes the first known example of a serine hydrolase in which the acid residue of the triad is a glutamate and not an aspartate. 

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