Peptidases metallopeptidase

Like aspartic peptidases, metallopeptidases act by activating a H20 molecule, and they do not form a covalent intermediate with the substrate. Here, the activation of a H20 molecule is mediated by a residue that acts as general base (e.g., Glu, His, Lys, Arg, or Tyr), with a divalent cation (usually Zn2+ but sometimes Co2+ or Mn2+) perhaps also contributing. The major role of the metal cation, however, is to act as an electrophilic catalyst by coordinating the carbonyl (or phosphoryl) O-atom in the substrate and orienting the latter for nucleophilic attack by the HO ion generated from H20 by the general base. 

Peptidases have been classified by the MEROPS system since 1993 [2], which has been available viatheMEROPS database since 1996 [3]. The classification is based on sequence and structural similarities. Because peptidases are often multidomain proteins, only the domain directly involved in catalysis, and which beais the active site residues, is used in comparisons. This domain is known as the peptidase unit. Peptidases with statistically significant peptidase unit sequence similarities are included in the same family. To date 186 families of peptidase have been detected. Examples from 86 of these families are known in humans. A family is named from a letter representing the catalytic type ( A for aspartic, G for glutamic, M for metallo, C for cysteine, S for serine and T for threonine) plus a number. Examples of family names are shown in Table 1. There are 53 families of metallopeptidases (24 in human), 14 of aspartic peptidases (three of which are found in human), 62 of cysteine peptidases (19 in human), 42 of serine peptidases (17 in human), four of threonine peptidases (three in human), one of ghitamicpeptidases and nine families for which the catalytic type is unknown (one in human). It should be noted that within a family not all of the members will be peptidases. Usually non-peptidase homologues are a minority and can be easily detected because not all of the active site residues are conserved. 

It is recommended that a well-characterized peptidase should have a trivial name. Although not rigidly adhered to, there is a different suffix for each catalytic type, metallopeptidases names end with lysin , aspartic peptidases with pepsin , cysteine pqrtidase with ain and serine peptidases with in . 

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. 

Metallopeptidases display an equal or greater catalytic efficiency toward peptides than toward the corresponding esters [84], This behavior is the opposite of that observed with serine peptidases and is unexpected, since the ester bond is chemically more labile than an amide bond. It has been postulated that the difference in catalytic efficiency is due to difference in product release, meaning that this step could be rate-limiting for esters but not for amides [85]. 

After translocation into the mitosome, the N-terminal presequences of mitosomal proteins are cleaved off, most likely by a peptidase that is homologous to the mitochondrial processing peptidase (MPP). The MPP is a matrix-localized metallopeptidase with a zinc binding motif His-X-X-Glu-His that is conserved in the putative mitosomal processing peptidase reported in G. intestinalis (Dolezal et al. 2005). 

Owing to the complementary roles of NEP and APN in enkephalin inactivation, selective inhibitor of only one of the two peptidases gives weak antinoceptive effects even after ICV administration. This led us to propose the concept of mixed inhibitors, that is, compounds able to simultaneously block NEP and APN activities [reviews in 9,20]. This was possible owing to the fact that these two membrane-bound enzymes belong to the superfamily of zinc metallopeptidases. 

The use of specific quenched fluorescent substrates (QFS) provides a rapid and sensitive method to measure peptidase activity, and is readily adaptable to high-throughput screening of potential peptidase inhibitors. In this chapter, we discuss general considerations for the development of QFS assays, and describe in detail an assay protocol for the mammalian metallopeptidase, endothelin-converting enzyme. 

Aminopeptidases, enzymes that cleave olf the N-terminal amino acid from a peptide chain, are bismetallo peptidases, a class of metallopeptidase that contain two metals ions in the catalytic site (117,118). These can be inhibited by compounds related to bestatin (60)(Fig. 15.28), which contains the iV-tenni-nal a-hydroxy-j3-amino acid residue, sometimes referred to as norstatine. In leucine amino peptidase, chelation occurs between both the amide carbonyl group and the adjacent hydroxyl and the hydroxyl and the N-terminal amino group (119,120). 

In the C-terminal domain are five helices in a closed bundle. This characteristic fold is typical of thermolysin-like peptidases. Clan MC contains metallocarbox-ypeptidases which belong to only one family (M14) which is divided into the subfamilies A, B and C. Typical for this clan is that one zinc ion is tetrahedrally coordinated by a water molecule, two histidine and one glutamate residues. Clan MF includes aminopeptidases that require cocatalytic zinc ions for their enzymatic activity. The well-known leucyl aminopeptidase has a two-domain structure bearing the active site in the C-terminal domain. Whereas exopeptidases of clan MG require cocatalytic ions of cobalt or manganese, clan MH contains the third group of metallopeptidases that also require cocatalytic metal ions, but here these are all zinc ions. The third clan in which cocatalytic metal ions are necessary is clan MF with zinc or manganese. Only one catalytic zinc ion is required for peptidases of clans MA, MB, MC, MD and ME. 

Thermolysin (TEN EC 3.4.24.28), a thermostable bacterial protease isolated from Bacillus thermoproteolyticus, has been studied as the prototype of zinc-metallopeptidases at a time where no crystal structure was available for this class of proteases [122]. Crystallographic analysis of a number of TLN/inhibitor complexes has allowed an understanding of the binding mode of these inhibitors and allowed the mechanism of action of this protease to be determined [122]. These seminal studies have greatly inspired the development of NEP inhibitors, given the close stmctural relationship between TEN and NEP [123]. To examine further the structural relationships between these two peptidases, various phosphinic peptides were prepared. One of these compounds (58, Table 1) exhibits a Ki value of 26 nM toward thermolysin and 22 nM toward NEP [124]. 

Many bacteria express a variety of peptidases with a different degree of specificity ranging from nonspecific to specific enzymes. Some of them degraded proteins involved in human innate immunity (for review, see Potempa and Pike 2009). An example is the metallopeptidase aureolysin from Staphylococcus aureus that cleaves and inactivates LL-37 (Sieprawska-Lupa et al. 2004). 

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