Enzymes exopeptidases

Proteolytic enzymes - hydrolyse proteins selectively, either on terminal groups (exopeptidases) or internal linkages (endopeptidases), eg... 

An exopeptidase that sequentially releases an amino from the C-terminus of a protein or peptide. Carbox-ypeptidases are classified in Enzyme Nomenclature according to catalytic type and are included in subsubclasses 3.4.16-3.4.18. 

An exopeptidase that can only degrade a dipeptide. Examples are carnosine dipeptidase I (MEROPS M20.006), which degrades carnosine (beta-Ala-His), and membrane dipeptidase (MEROPS Ml9.001), which is important in the catabolism of glutathione, degrading the dipeptides Cys-Gly. Dipeptidases are included in Enzyme Nomenclature sub-subclass 3.4.13. 

An exopeptidase that sequentially releases a dipeptide from the N-terminus of a protein or peptide. Dipeptidy 1-peptidases are included in Enzyme Nomenclature subsubclass 3.4.14 along with tripeptidyl-peptidases. 

An exopeptidase that does not cleave standard peptide bonds. An example is pyroglutamyl-peptidase I (MEROPS C15.010), which releases an N-terminal pyroglutamyl from hormones such as thyrotropinreleasing hormone and luteinizing hormone. Omega peptidases are included in Enzyme Nomenclature subsubclass 3.4.19. 

There are two main classes of proteolytic digestive enzymes (proteases), with different specificities for the amino acids forming the peptide bond to be hydrolyzed. Endopeptidases hydrolyze peptide bonds between specific amino acids throughout the molecule. They are the first enzymes to act, yielding a larger number of smaller fragments, eg, pepsin in the gastric juice and trypsin, chymotrypsin, and elastase secreted into the small intestine by the pancreas. Exopeptidases catalyze the hydrolysis of peptide bonds, one at a time, fi"om the ends of polypeptides. Carboxypeptidases, secreted in the pancreatic juice, release amino acids from rhe free carboxyl terminal, and aminopeptidases, secreted by the intestinal mucosal cells, release amino acids from the amino terminal. Dipeptides, which are not substrates for exopeptidases, are hydrolyzed in the brush border of intestinal mucosal cells by dipeptidases. 

As mentioned earlier, by far the largest number of zinc enzymes are involved in hydrolytic reactions, frequently associated with peptide bond cleavage. Carboxypeptidases and ther-molysins are, respectively, exopeptidases, which remove amino acids from the carboxyl terminus of proteins, and endopeptidases, which cleave peptide bonds in the interior of a polypeptide chain. However, they both have almost identical active sites (Figure 12.4) with two His and one Glu ligands to the Zn2+. It appears that the Glu residue can be bound in a mono- or bi-dentate manner. The two classes of enzymes are expected to follow similar reaction mechanisms. 

The NC-IUBMB classifies peptidases (EC 3.4) into exopeptidases (EC 3.4.11-19), which remove one or a few amino acids, and endopeptidases (proteinases, EC 3.4.21-99), which catalyze the cleavage of peptide bonds away from either end of the polypeptide chain (Fig. 2.1). Exopeptidases are further subdivided into enzymes that carry out hydrolysis at the N-terminus or the C-terminus (Figs. 2.1 and 2.2). Thus, aminopeptidases (EC 3.4.11) cleave a single amino acid from the N-terminus [3] those removing a dipep-... 

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],... 

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). 

Peptide hydrolases (peptidases or proteases, i.e., enzymes hydrolyzing peptide bonds in peptides and proteins, see Chapt. 2) have received particular attention among hydrolases. As already described in Chapt. 2, peptidases are divided into exopeptidases (EC 3.4.11 -19), which cleave one or a few amino acids from the N- or C-terminus, and endopeptidas-es (proteinases, EC 3.4.21-99), which act internally in polypeptide chains [2], The presentation of enzymatic mechanisms of hydrolysis in the following sections will begin with peptidases and continue with other hydrolases such as esterases. 

The metal ion is held in place by amino acid residues, generally His, Glu, Asp, or Lys. In many metallopeptidases, which may be exopeptidases or en-dopeptidases, only one zinc ion is required. In all Co2+ or Mn2+-dependent, and in some Zn2+-dependent metallopeptidases, two metal ions are present and act cocatalytically these enzymes are exopeptidases [2][73][74],... 

Increased permeability is just one prerequisite in the development of useful peptide prodrugs. Another condition is that efficient bioactivation must follow absorption. Mucosal cell enzymes able to hydrolyze peptides include exopeptidases such as aminopeptidases and carboxypeptidases, endopepti-dases, and dipeptidases such as cytosolic nonspecific dipeptidase (EC 3.4.13.18), Pro-X dipeptidase (prolinase, EC 3.4.13.4), and X-Pro dipeptidase (prolidase, EC 3.4.13.9). For example, L-a-methyldopa-Pro was shown to be a good substrate for both the peptide transporter and prolidase. This dual affinity is not shared by all dipeptide derivatives, and, indeed, dipeptides that lack an N-terminal a-amino group are substrates for the peptide transporter but not for prolidase [29] [33] [34],... 

The synthesis of active centers is not a small problem. The enzyme car-boxypeptidase A is a pancreatic exopeptidase that catalyzes the sequential release of amino acids from the C terminus of polypeptide chains as shown in reaction (1). Much work has been done on the structure 31). Although the... 

A novel concept of using bioadhesive polymers as enzyme inhibitors has been developed [97]. Included are derivatives of poly acrylic acid, polycarbophil, and car-bomer to protect therapeutically important proteins and peptides from proteolytic activity of enzymes, endopeptidases (trypsin and a-chymotrypsin), exopeptidases (carboxypeptidases A and B), and microsomal and cytosolic leucine aminopeptidase. However, cysteine protease (pyroglutamyl aminopeptidase) is not inhibited by polycarbophil and carbomer [97]. 

Protein digestion occurs in two stages endopeptidases catalyse the hydrolysis of peptide bonds within the protein molecule to form peptides, and the peptides are hydrolysed to form the amino acids by exopeptidases and dipeptidases. Enteropeptidase initiates pro-enzyme activation in the small intestine by catalysing the conversion of trypsinogen into trypsin. Trypsin is able to achieve further activation of trypsinogen, i.e. an autocatalytic process, and also activates chymotrypsinogen and pro-elastase, by the selective hydro-... 

These proteolytic enzymes are all endopeptidases, which hydrolyse links in the middle of polypeptide chains. The products of the action of these proteolytic enzymes are a series of peptides of various sizes. These are degraded further by the action of several peptidases (exopeptidases) that remove terminal amino acids. Carboxypeptidases hydrolyse amino acids sequentially from the carboxyl end of peptides. They are secreted by the pancreas in proenzyme form and are each activated by the hydrolysis of one peptide bond, catalysed by trypsin. Aminopeptidases, which are secreted by the absorptive cells of the small intestine, hydrolyse amino acids sequentially from the amino end of peptides. In addition, dipeptidases, which are structurally associated with the glycocalyx of the entero-cytes, hydrolyse dipeptides into their component amino acids. 

In addition, renal tubular cells contain various proteases for the degradation of proteins and oligopeptides. These enzymes are located predominantly in the lysosomes and micro-somes of these cells, but some have been reported on the brush-border membranes [16]. Degradative enzymes include various endopeptidases, exopeptidases and esterases [17]. 

The proteolytic enzymes are classified into endopeptidases and exopeptidases, according to their site of attack in the substrate molecule. The endopeptidases or proteinases cleave peptide bonds inside peptide chains. They recognize and bind to short sections of the substrate s sequence, and then hydrolyze bonds between particular amino acid residues in a relatively specific way (see p. 94). The proteinases are classified according to their reaction mechanism. In serine proteinases, for example (see C), a serine residue in the enzyme is important for catalysis, while in cysteine proteinases, it is a cysteine residue, and so on. 

Zinc proteases carboxypeptidase A and thermolysin have been extensively studied in solution and in the crystal (for reviews, see Matthews, 1988 Christianson and Lipscomb, 1989). Both carboxypeptidase A and thermolysin hydrolyze the amide bond of polypeptide substrates, and each enzyme displays specificity toward substrates with large hydrophobic Pi side chains such as phenylalanine or leucine. The exopeptidase carboxypeptidase A has a molecular weight of about 35K and the structure of the native enzyme has been determined at 1.54 A resolution (Rees et ai, 1983). Residues in the active site which are important for catalysis are Glu-270, Arg-127, (liganded by His-69, His-196, and Glu-72 in bidentate fashion), and the zinc-bound water molecule (Fig. 30). 

Several different proteases can attack a single protein at enzyme-selective amino-acid sequences. Proteases can be divided into two categories. Endopeptidases are enzymes that cleave peptide bonds between specific, nonterminal amino acids. There are endopeptidases specific for just about every amino acid. Exopeptidases are enzymes that cleave terminal peptide bonds at either the C-terminus or N-terminus. 

The metalloproteases include both exopeptidases (e.g., angiotensin-converting enzyme, aminopeptidase-M, and carboxypeptidase-A) and endopeptidases (e.g.,... 

We have chosen to discuss enzyme modification of proteins in terms of changes in various functional properties. Another approach might have been to consider specific substrates for protease action such as meat and milk, legumes and cereals, and the novel sources of food protein such as leaves and microorganisms ( ). Alternatively, the proteases themselves provide categories for discussion, among which are their source (animals, plants, microorganisms), their type (serine-, sulfhydryl-, and metalloenzymes), and their specificity (endo- and exopeptidases, aromatic, aliphatic, or basic residue bond specificity). See Yamamoto (2) for a review of proteolytic enzymes important to functionality. 

There are five distinct families of zinc proteases, classified by the nature of the zinc binding site. These families, and their variously proposed mechanisms, have recently been reviewed in depth.143 The most studied member is the digestive enzyme bovine pancreatic carboxypeptidase A, which is a metalloenzyme containing one atom of zinc bound to its single polypeptide chain of 307 amino acids and Mr 34 472. It is an exopeptidase, which catalyzes the hydrolysis of C-terminal amino acids from polypeptide substrates, and is specific for the large hydrophobic amino acids such as phenylalanine. The closely related carboxypeptidase B catalyzes the hydrolysis of C-terminal lysine and arginine residues. The two en-... 

The International Union of Biochemistry and Molecular Biology recommends that the term peptidase be used synonymously with the term peptide hydrolase (IUBMB, 1992). Thus, in this unit the term peptidase is used in reference to any enzyme that catalyzes the hydrolysis of peptide bonds, without distinguishing between exo- and endopeptidase activities. Peptidases may be assayed using native or modified proteins, peptides, or synthetic substrates. In this unit, the focus is on assays based on the hydrolysis of common, commercially available, protein substrates. Thus, the assays are not intended to be selective for a given peptidase they are designed to provide estimates of overall peptidase activity. Other units in this publication focus on synthetic or model substrates, which can be designed for the measurement of specific endo- and/or exopeptidase activities. 

Liver flukes also possess cathepsin C and LAP exopeptidases that are orthologous to the schistosome enzymes. These exopeptidases most likely complete the digestive process to yield free dipeptides and amino acids, respectively, from peptides generated by endoprote-olytic cysteine protease activity on host proteins. Both cathepsin C and LAP have been immunolocalized to gastrodermal cells (Carmona et al., 1994 Acosta et al., 1998 J.P. Dalton, unpublished data). 

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