Enzyme specificity pepsin

Figure 4.5. Schematic representation of enzyme specific cieavage of immunogiobuiin G (igG) by pepsin and papain. Treatment of igG with pepsin produces two unique fragments. Fab, with two antigen binding sites and Fj without binding sites. Treatment of igG with papain generates two Fab and one F fragments.

Specificity Each of these enzymes has a different specificity for the amino acid R-groups adjacent to the susceptible peptide bond (Figure 19.5). For example, trypsin cleaves only when Ihe carbonyl group of the peptide bond is contributed by arginine or lysine. These enzymes, like pepsin described above, are synthesized and secreted as inactive zymogens. 

The specificity of pepsin has been investigated using synthetic substrates and proteins of known sequence as substrate. From these studies, the following conclusions have been reached. Pepsin attacks peptide linkages involving L-amino acids. The enzyme is specific for both components of the peptide bonds. The attack of the enzyme on the polypeptide chain is facilitated by the presence of aromatic residues in the chain. For example, in the insulin molecule, highly susceptible bonds are those between Leu 13 and Tyr 14 in the A chain, or Phe 24-Phe 25, and Phe 25-Tyr 26 in the B chain. However, the enzyme specificity is complex, and in insulin the Leu 11-Val 12 peptide bond in the B chain proves to be susceptible to the enzyme, and numerous other points of the molecule are the site of a slower attack by the protease [31]. 

Numerous enzymes also destroy the hypertensive action, such as tyrosinase, intestinal aminopolypeptidases. Plentl and Page (471) drew various conclusions concerning the structure of the peptide, from data on the controlled hydrolysis by four crystalline enzymes, namely pepsin, chymotrypsin, trypsin, carboxypeptidase, all of which destroy the physiological activity. Thrombin (149) and thromboplastin (150), but not prothrombin, also destroy the activity. An enzyme present in plasma, hypertensinase, has been described which destroys the hypertensin action in the animal. This enzyme acts under anaerobic as well as aerobic conditions, and the presence of octyl alcohol and cyanide do not affect its behavior its pH optimum is between 7.8 and 8.5. Hypertensinase is also present in red blood cells, with a pH optimum differing from the plasma enzyme. These enzymes, whose specificity and identity are not yet well established, are probably aminopolypeptidases (151). 

Mammals, fungi, and higher plants produce a family of proteolytic enzymes known as aspartic proteases. These enzymes are active at acidic (or sometimes neutral) pH, and each possesses two aspartic acid residues at the active site. Aspartic proteases carry out a variety of functions (Table 16.3), including digestion pepsin and ehymosin), lysosomal protein degradation eathepsin D and E), and regulation of blood pressure renin is an aspartic protease involved in the production of an otensin, a hormone that stimulates smooth muscle contraction and reduces excretion of salts and fluid). The aspartic proteases display a variety of substrate specificities, but normally they are most active in the cleavage of peptide bonds between two hydrophobic amino acid residues. The preferred substrates of pepsin, for example, contain aromatic residues on both sides of the peptide bond to be cleaved. 

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. 

The proteases are secreted as inactive zymogens the active site of the enzyme is masked by a small region of its peptide chain, which is removed by hydrolysis of a specific peptide bond. Pepsinogen is activated to pepsin by gastric acid and by activated pepsin (autocatalysis). In the small intestine, trypsinogen, the precursor of trypsin, is activated by enteropeptidase, which is secreted by the duodenal epithelial cells trypsin can then activate chymotrypsinogen to chymotrypsin, proelas-tase to elastase, procarboxypeptidase to carboxypepti-dase, and proaminopeptidase to aminopeptidase. 

Soya Proteins. Early attempts to make albumen substitutes from soya protein also ran into problems. A bean flavour tended to appear in the finished product. A solution to these problems has been found. Whipping agents based on enzyme modified soy proteins are now available. The advantage of enzymatic modification is that by appropriate choice of enzymes the protein can be modified in a very controlled way. Chemical treatment would be far less specific. In making these materials the manufacturer has control of the substrate and the enzyme, allowing the final product to be almost made to order. The substrates used are oil-free soy flakes or flour or soy protein concentrate or isolate. The enzymes to use are chosen from a combination of pepsin, papain, ficin, trypsin or bacterial proteases. The substrate will be treated with one or more enzymes under carefully controlled conditions. The finished product is then spray dried. 

The Daily Industiy. The first step in cheese manufacture is the coagulation of milk. Coagulation can be divided into two distinct phases, enzymatic and the non-enzymatic. In the primary enzymatic phase a proteol ic enzyme such as chymosin (rennet), or less effectively, pepsin, carries out an extremely specific and limited proteolysis, cleaving a phenylalanine-methionine bond of /c-casein, making the casein micelle metastabie. In the second, non-enzymatic phase, the... 

This lysosomal endopeptidase [EC 3.4.23.5] is similar to pepsin A, except that the specificity is narrower and will not hydrolyze the Gln" —His peptide bond in the B chain of insulin. The enzyme is a member of the peptidase family Al. 

Microbial proteinases can be classified by mechanism of action. Hartley (1960) divided them into four groups serine proteinases, thio proteinases, metalloproteinases, and acid proteinases. Morihara (1974) classified enzymes within these groups according to substrate specificity. Enzymes which split peptide substrates at the carboxyl side of specific amino acids are called carboxyendopeptidases, and those which split peptide substrates at the amino side of specific amino acids are called aminoendopeptidases. Acid proteinases, such as rennin and pepsin, split either side of specific aromatic or hydrophobic amino acid residues. The action of proteolytic enzymes on milk proteins has been reviewed by Visser (1981). 

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