Hydrolysis chymotrypsinogen

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. 

Procarboxypeptidase A is activated by the removal of a peptide of some 64 residues from the N-terminus by trypsin.153 This zymogen has significant catalytic activity. As well as catalyzing the hydrolysis of small esters and peptides, procarboxypeptidase removes the C-terminal leucine from lysozyme only seven times more slowly than does carboxypeptidase. Also, the zymogen hydrolyzes Bz-Gly-L-Phe with kcsA = 3 s-1 and KM = 2.7 mM, compared with values of 120 s 1 and 1.9 mM for the reaction of the enzyme.154 In contrast to the situation in chymotrypsinogen, the binding site clearly pre-exists in procarboxypeptidase, and the catalytic apparatus must be nearly complete. 

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-... 

Hydrolysis of peptides or proteins with acid yields a mixture of free a-amino acids. When completely hydrolyzed, each type of protein yields a characteristic proportion or mixture of the different amino acids. The 20 common amino acids almost never occur in equal amounts in a protein. Some amino acids may occur only once or not at all in a given type of protein others may occur in large numbers. Table 3-3 shows the composition of the amino acid mixtures obtained on complete hydrolysis of bovine cytochrome c and chymotrypsinogen, the inactive precursor of the digestive enzyme chymotrypsin. These two proteins, with very different functions, also differ significantly in the relative numbers of each kind of amino acid they contain. 

Their mode of appearance in the lumen of the intestine is rather complicated and involves activation of trypsinogen secretion by enterokinase. Once trypsin is formed it activates chymotrypsinogen. Pancreatic lipase is also secreted into the lumen with the pancreatic fluid. The digestion process of fatty acids by their lipase-mediated hydrolysis is completed by bile salts, which are also secreted in the duodenum and are crucial for micellization of lipophilic compounds. The micelles formed in the duodenum enable the absorption of hydro-phobic drugs such as steroids. They pose, however, a serious constraint for the stability of drug delivery carriers such as liposomes and emulsions. 

Figure 5. Schematic of the role of limited hydrolysis in the conversion of bovine chymotrypsinogen A to ir-, 8-, and a-chymotrypsins (32)...

Fio. 7. Stepwise formation of the three chains of a-chymotrypsin (A ) (64). ChTg, bovine chymotrypsinogen A. ChT-jr, -i and -a, respectively, ir-chymotrypsin (Ai), S-chymotrypsin (Aj), and -chymotrypsin (Ad. Neo-ChTg, neochymotrypsinogens (degraded and still activatable forms of chymotrypsinogen A). + and —, NHs- and COOH-terminal residues, respectively. T.C. and C.C., hydrolysis catalyzed by trypsin and chymotrypsin, respectively. Asp, asparagine residue. A, B, and C, chains A, B, and C of a-chymotrypsin (Ad. 

The answer is d. (Murray, pp 48-62. Scriver, pp 3 5. Sack, pp 1-3. Wilson, pp 101-120.) Pepsin is secreted in a proenzyme form in the stomach. Unlike the majority of proenzymes, it is not activated by protease hydrolysis. Instead, spontaneous acid hydrolysis at pH 2 or lower converts pepsinogen to pepsin. Hydrochloric acid secreted by the stomach lining creates the acid environment. All the enzymes secreted by the pancreas are activated at the same time upon entrance into the duodenum. This is accomplished by trypsin hydrolysis of the inactive proenzymes trypsino-gen, chymotrypsinogen, procarboxypeptidase, and proelastase. Primer... 

Specific Activities of Proteins. The average specific activities of proteins after y-radiolysis to a dose of about 6 Mrads (except for myoglobin which was irradiated to 23 Mrads), HST labeling and acid hydrolysis were as follows ribonuclease, 0.76 0.21 lysozyme, 0.66 0.21 chymotrypsinogen, 0.76 d= 0.06 insulin, 0.18 0.06 myoglobin 0.46 0.23 CM reduced ribonuclease, 0.96 0.38 CM reduced lysozyme,... 

The low pH of the stomach contents stimulates the secretion of the hormone secretin, as it passes into the small intestine. This hormone is secreted into the bloodstream, and when it reaches the pancreas, it stimulates that organ to secrete bicarbonate into the gut which neutralizes the low pH of the entering stomach contents. Entering amino acids stimulate the secretion by intestinal cells of the specialized enzyme enterokinase. It specificity is directed toward the conversion of the zymogen, trypsinogen, secreted by the pancreas, to the active proteolytic enzyme, trypsin. Trypsin then converts chymotrypsinogen to the active chymotrypsin. These two enzymes reduce polypeptides to small peptides. The further hydrolysis of the small peptides to their constituent amino acids is accomplished by two other enzymes secreted by intestinal cells, carboxypeptidase and aminopeptidase. The mixture of amino acids is then transported across the intestinal cells, enters the blood, and is transported to the liver. 

Trypsinogen, chymotrypsinogen, proelastase, and procarboxypeptidase are all synthesized as single polypeptide chains of around 25-30 kDa. The initial step in the activation is the hydrolysis of a hexapeptide from the N terminus of trypsinogen. This hydrolysis produces trypsin and is catalyzed by enteropeptidase (Fig. 14-8), an enzyme on the membranes of brash border cells of the small intestine. 

Comparison of the structures of bovine trypsinogen and chymotrypsinogen A. Solid arrows show the hydrolysis sites that result in the activation of trypsinogen to trypsin, and chymotrypsinogen to chymotrypsin. Broken arrows are additional hydrolysis sites, leading to formation of a-chymotrypsin. Shaded circles represent amino acids that are Identical or similar in the two proteins. Disulfide bridges are lettered A to G. H represents the histidine residues, and S the serine residue of the active site this serine residue reacts with diisopropylfluorophosphate. Deletions are shown by lines between the circles. To aid comparison, the residue numbers of both structures are based on those of chymotrypsinogen. [B.S.Hartley etal. Nature 207 (1965) 1157-1159]... 

As mentioned above, the hydrolysis by trypsin of a single peptide bond between arginine 15 and isoleucine 16 converts bovine chymotrypsin (245 amino acids) to active a-chymotrypsinogen. Of course, this is followed by the autocatalytic cleavage of other bonds leading to other active chymotrypsins. 

The mechanism of action of a-chymotrypsin is probably understood in more detail than any other enzyme at the present time. Its physiological function is to catalyze the hydrolysis of peptide bonds of protein foods in the mammalian gut. It is secreted in the pancreas as an inactive zymogen precursor, chymotrypsinogen, having a single polypeptide chain of245 amino... 

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