Exopeptidases carboxypeptidases

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]. 

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). 

Exopeptidases Carboxypeptidase A Carboxypeptidase B removes the C-terminal amino acid (not Arg or Lys) removes the C-terminal amino acid (only Arg or Lys)... 

EXAMPLE 5.15 X-ray analysis of crystals of the pancreatic exopeptidase carboxypeptidase A, with a bound pseudo substrate (z. false substrate that is not degraded by the enzyme, i.e., an inhibitor) indicates that the susceptible peptide bond is twisted out of the normal planar configuration that is usually seen with peptide bonds (Chap. 4). This distortion leads to a loss of resonance energy in the bond, and enhances its susceptibility to hydrolytic attack. 

Carboxypeptidases A (EIC 3.4.12.2) and B (EC 3.4.12.3) are pancreatic exopeptidases. Carboxypeptidase A preferentially hydrolyzes terminal peptide bonds in which the amino donor is a hydrophobic amino acid whereas the specificity of carboxypeptidase B is directed toward the... 

Among the best known hydrolytic enzymes of which enough structural and mechanistic information are available are the exopeptidase carboxypeptidase A (see Chapter 6), ribonuclease A (see Chapter 3), and lysozyme. In this chapter we shall examine the chemistry of this last enzyme. 

There are several different types of exopeptidases aminopeptidases, carboxypeptidases, dipeptidyl-peptidases, tripeptidy 1-peptidases, peptidyl-... 

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. 

Metallo proteases Exopeptidase group Peptidyl dipeptidase-A (ACE) Aminopeptidase-M Carboxypeptidase-A... 

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. 

SC 6 Families of endopeptidases (oligopeptidases) and exopeptidases (including lysosomal carboxypeptidase A in family S10) Ser, Asp, His a,/3-Hydrolase... 

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). 

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 in vitro hydrolysis of insulin has been shown to be catalyzed by exopeptidases and endopeptidases. Carboxypeptidase A (EC 3.4.17.1) cleaves the C-terminus of the B-chain (ThrB3°) and that of the A-chain (AsnA21) [145], Leucyl aminopeptidase (EC 3.4.11.1) cleaves the N-terminus of the B-chain (PheB1) and can continue to shorten it. But, leucyl aminopeptidase appears also able to cleave the N-terminus of the A-chain (GlyA1). In addition to these exopeptidases, entire insulin is also cleaved by endopeptidases of the... 

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. 

The exopeptidases attack peptides from their termini. Peptidases that act at the N terminus are known as aminopeptidases, while those that recognize the C terminus are called carboxypeptidases. The dipeptidases only hydrolyze dipeptides. 

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

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-... 

Thermolysin differs from the carboxypeptidases in being an endopeptidase rather than an exopeptidase. This is manifested in the nature of the binding site ... 

Peptidases are often classified as either exopeptidases or endopeptidases, depending on the positional specificity of the bonds they hydrolyze. Exopeptidases act at peptide bonds located at either the N or C terminus of the protein. Those acting at the C terminus are referred to as carboxypeptidases, those acting at the N terminus as aminopeptidases. Endopeptidases, on the other hand, act at peptide bonds internal to the polypeptide chain. 

Specific enzymes, called exopeptidases, that cleave one residue at a time from the end of a polypeptide chain, can be used to provide information on the terminal residues. Aminopeptidases cleave amino acids from the N terminus carboxypeptidases from the C terminus. The released amino acid can then be identified as above by comparison with known standards. 

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