Peptidases reactions

Figure C2.2.2 Peptidase reaction hydrolysis of peptide bond.

In carboxypeptidases and thermolysins, the Zn + ligands are two histidines and one Glu C02, separated by a different number of amino-acid spacers. The Zn +-H20 activated by Glu C02 is involved in catalysis. Human leucotriene A4 hydrolase, Clostridium botulinum neuro-toxin A, and VanX D-Ala-D-Ala carboxypeptidase belong to the thermolysin family, although they do not necessarily catalyze peptidase reactions. 

Additionally, the nitrocellulose surface provides an opportunity to carry out enzymatic and chemical reactions directly on the sample foil. These are discussed below, and in more detail later in Chapter 10, and include carboxy and amino-peptidase reactions that can be used to produce peptide ladder mixtures for determining amino acid sequence or the location of post-translational modifications. Following the on-foil reaction, the enzyme can be washed off prior to PDMS analysis of the resulting products. 

Cleavage of a peptide bond is an example of a nucleophilic attack. The nucleophile in the reaction is either an activated water molecule or part of the side-chain of an amino acid, and peptidases are described as having either a water nucleophile or a protein nucleophile. Peptidases with a water nucleophile either utilize one or two metal ions as ligands for the water molecule, in which case the peptidase generally acts... 

Other interesting examples of proteases that exhibit promiscuous behavior are proline dipeptidase from Alteromonas sp. JD6.5, whose original activity is to cleave a dipeptide bond with a prolyl residue at the carboxy terminus [121, 122] and aminopeptidase P (AMPP) from E. coli, which is a prohne-specific peptidase that catalyzes the hydrolysis of N-terminal peptide bonds containing a proline residue [123, 124]. Both enzymes exhibit phosphotriesterase activity. This means that they are capable of catalyzing the reaction that does not exist in nature. It is of particular importance, since they can hydrolyze unnatural substrates - triesters of phosphoric acid and diesters of phosphonic acids - such as organophosphorus pesticides or organophosphoms warfare agents (Scheme 5.25) [125]. 

The antibiotic activity of certain (3-lactams depends largely on their interaction with two different groups of bacterial enzymes. (3-Lactams, like the penicillins and cephalosporins, inhibit the DD-peptidases/transpeptidases that are responsible for the final step of bacterial cell wall biosynthesis.63 Unfortunately, they are themselves destroyed by the [3-lactamases,64 which thereby provide much of the resistance to these antibiotics. Class A, C, and D [3-lactamases and DD-peptidases all have a conserved serine residue in the active site whose hydroxyl group is the primary nucleophile that attacks the substrate carbonyl. Catalysis in both cases involves a double-displacement reaction with the transient formation of an acyl-enzyme intermediate. The major distinction between [3-lactamases and their evolutionary parents the DD-peptidase residues is the lifetime of the acyl-enzyme it is short in (3-lactamases and long in the DD-peptidases.65-67... 

The coupling of solute transport in the GI lumen with solute lumenal metabolism (homogeneous reaction) and membrane metabolism (heterogeneous reaction) has been discussed by Sinko et al. [54] and is more generally treated in Cussler s text [55], At the cellular level, solute metabolism can occur at the mucosal membrane, in the enterocyte cytosol, and in the endoplasmic reticulum (or microsomal compartment). For peptide drugs, the extent of hydrolysis by lumenal and membrane-bound peptidases reduces drug availability for intestinal absorption [56], Preferential hydrolysis (metabolic specificity) has been targeted for reconversion... 

There were also less concrete considerations. In the early 1950s glycogenolysis was still believed to be completely reversible. UTP dependency and the glycogen synthase reactions had not yet been discovered nor had phosphofructokinase been shown to act irreversibly. The mechanism of protein synthesis was still a mystery. Laboratories studying proteolysis had shown that the peptide bond could be resynthesized by peptidases, although under very restricted conditions. Reversibility seemed to be an accepted property of the major metabolic pathways. 

One of the general principles of the Nomenclature Committee is that enzymes should be classified and named according to the reaction they catalyze. However, the overlapping specificities of and great similarities in the action of different peptidases render naming solely on the basis of function impossible [10]. For example, some enzymes can act as both endo- and exopeptidases. Thus, cathepsin H (EC 3.4.22.16) is not only an endopeptidase but also acts as an aminopeptidase (EC 3.4.11), and cathepsin B (EC 3.4.22.1) acts as an endopeptidase as well as a peptidyl-dipeptidase (EC 3.4.15). The actual classification of peptidases is, therefore, a compromise based not only on the reaction catalyzed but also on the chemical nature of the catalytic site, on physiological function, and on historical priority. 

The evolutionary classification has a rational basis, since, to date, the catalytic mechanisms for most peptidases have been established, and the elucidation of their amino acid sequences is progressing rapidly. This classification has the major advantage of fitting well with the catalytic types, but allows no prediction about the types of reaction being catalyzed. For example, some families contain endo- and exopeptidases, e.g., SB-S8, SC-S9 and CA-Cl. Other families exhibit a single type of specificity, e.g., all families in clan MB are endopeptidases, family MC-M14 is almost exclusively composed of carboxypeptidases, and family MF-M17 is composed of aminopeptidases. Furthermore, the same enzyme specificity can sometimes be found in more than one family, e.g., D-Ala-D-Ala carboxypeptidases are found in four different families (SE-S11, SE-S12, SE-S13, and MD-M15). 

Fig. 2.3. Schematic representation of consecutive zymogen activation reactions (cascade). Following initiation by a physiological signal ( ), the zymogens X, Y, and Z are sequentially activated. The resulting peptidases (Xa and Ya) are inactivated by specific inhibitors (Ix and Iy) to limit their action (modified from [20a]).

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. 

It is interesting to note that serine peptidases can, under special conditions in vitro, catalyze the reverse reaction, namely the formation of a peptide bond (Fig. 3.4). The overall mechanism of peptide-bond synthesis by peptidases is represented by the reverse sequence f-a in Fig. 3.3. The nucleophilic amino group of an amino acid residue competes with H20 and reacts with the acyl-enzyme intermediate to form a new peptide bond (Steps d-c in Fig. 3.3). This mechanism is not relevant to the in vivo biosynthesis of proteins but has proved useful for preparative peptide synthesis in vitro [17]. An interesting application of the peptidase-catalyzed peptide synthesis is the enzymatic conversion of porcine insulin to human insulin [18][19]. 

There are a few reported cases of esterases that catalyze not only hydrolysis but also the reverse reaction of ester formation, in analogy with the global reaction described for serine peptidases (Fig. 3.4). Thus, cholesterol esterase can catalyze the esterification of oleic acid with cholesterol and, more importantly in our context, that of fatty acids with haloethanols [54], Esterification and transesterification reactions are also mediated by carboxyleste-rases, as discussed in greater detail in Sect. 7.4. 

Bradykinin (Fig. 6.34) is a vasoactive nonapeptide that is hydrolyzed by a variety of peptidases. Its N-terminus is susceptible to cleavage, but only by aminopeptidase P (X-Pro aminopeptidase, EC 3.4.11.9). Dipeptidyl-pepti-dase IV can then cleave the N-terminus dipeptide of bradykinin-(2-9). However, most cleavage reactions have been found to occur at or close to the C-terminus, with angiotensin-converting enzyme (ACE, peptidyl-dipeptidase A, EC 3.4.15.1) playing an important role. In fact, aminopeptidase P and ACE accounted for ca. 30 and 70%, respectively, of total bradykininase activity in the isolated perfused rat heart [164], As shown in Fig. 6.34, ACE... 

Fig. 6.34. Major peptidases that act on bradykinin. Descending arrows represent primary cleavage reactions, whereas ascending arrows indicate secondary reactions, i.e., cleavage sites...

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