Chymotrypsinogen, activation

Reflect and Apply Explain why cleavage of the bond between arginine 15 and isoleucine 16 of chymotrypsinogen activates the zymogen. 

Chymotrypsin is formed in the pancreas, just as is trypsin, in the form of inactive chymotrypsinogen. Activation is performed by trypsin. The process runs through a series of intermediates which are also enzymically active. As shown in the diagram on page 149, the activation amounts to a double scission of a cyclic peptide, eliminating two dipeptides in the process. 

The polypeptide chain of chymotrypsinogen, the inactive precursor of chymotrypsin, comprises 245 amino acids. During activation of chymotrypsinogen residues 14-15 and 147-148 are excised. The remaining three polypeptide chains are held together by disulfide bridges to form the active chymotrypsin molecule. 

Figure 11.7 Schematic diagram of the structure of chymotrypsin, which is folded into two antiparallel p domains. The six p strands of each domain are red, the side chains of the catalytic triad are dark blue, and the disulfide bridges that join the three polypeptide chains are marked in violet. Chain A (green, residues 1-13) is linked to chain B (blue, residues 16-146) by a disulfide bridge between Cys 1 and Cys 122. Chain B is in turn linked to chain C (yellow, residues 149-245) by a disulfide bridge between Cys 136 and Cys 201. Dotted lines indicate residues 14-15 and 147-148 in the inactive precursor, chmotrypsinogen. These residues are excised during the conversion of chymotrypsinogen to the active enzyme chymotrypsin.

FIGURE 16.16 Comparison of the amino acid sequences of chymotrypsinogen, trypsino-gen, and elastase. Each circle represents one amino acid. Nmnbering is based on the sequence of chymotrypsinogen. Filled circles indicate residues that are identical in all three proteins. Disnlfide bonds are indicated in yellow. The positions of the three catalytically important active-site residues (His, Asp °-, and Ser ) are indicated. 

Active a-chymotrypsin is produced from chymotrypsinogen, an inactive precursor, as shown in the color figure on page 530. 

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. 

Chymotrypsin has also been utilized to promote debridement, as well as the reduction of soft tissue inflammation. It is also used in some opthalmic procedures, particularly in facilitating cataract extraction. It is prepared by activation of its zymogen, chymotrypsinogen, which is extracted from bovine pancreatic tissue. 

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-... 

Fig. Z14. The activation of chymotrypsin via proteolytic cleavage, a) Chymotrypsinogen is transformed into the active forms of chymotrypsin n and a by trypsin and autoproteolysis, b) The N-terminal isoleucine residue Ile6 is particularly important for the activity of chymotrypsin. The positively charge NH2 group of llel6 interacts electrostatically with Aspl94 and stabilizes an active conformation of the catalytic center. After Stryer Biochemistry , with permission.

For some enzymes, an inactive precursor called a zymogen is cleaved to form the active enzyme. Many proteolytic enzymes (proteases) of the stomach and pancreas are regulated in this way. Chymotrypsin and trypsin are initially synthesized as chymotrypsinogen and trypsinogen (Fig. 6-33). Specific cleavage causes conformational changes that expose the enzyme active site. Because this type of activation is irreversible, other... 

Proteolytic Processing Many proteins are initially synthesized as large, inactive precursor polypeptides that are proteolytically trimmed to form their smaller, active forms. Examples include proinsulin, some viral proteins, and proteases such as chymotrypsinogen and trypsino-gen (see Fig. 6-33). 

Chymotrypsinogen consists of a single 245-residue chain. The amino acid residues in chymotrypsin, trypsin, and elastase are usually all numbered according to their position in this zymogen. Inactive proenzymes are formed as precursors to enzymes of many different classes and are activated in a variety of ways. A part of the polypeptide chain of the proenzymes is often folded over the active site, interacting in a nonsubstrate-like fashion and blocking the site.197a... 

Support for this concept is provided by H NMR studies which have identified a downfield resonance of the hydrogen-bonded proton in this pair at 18 ppm in chymotrypsinogen and chymotrypsin at low pH and at 14.9-15.5 ppm at high pH values.246 247 Similar resonances are seen in the a-lytic protease,248 in sub-tilisin,249 in adducts of serine proteases with boronic acids250 251 or peptidyl trifluoromethyl ketones,252 in alkylated derivative of the active site histidine,253 and in molecular complexes that mimic the Asp-His pair in the active sites of serine proteases.254... 

Chymotrypsinogen and related proenzymes have extremely low catalytic activity even though a major part of the substrate binding site as well as the catalytic triad system are already in place. However, the oxyanion hole is created during activation of the proenzyme by a subtle conformational change197,262,271 that involves the chain segment containing Gly 193 (Fig. 12-12). This is further evidence of the importance of this part of the active site structure. 

Some of the serine proteases are stored in the pancreas as inactive precursors that may be activated by proteolysis. Trypsinogen, for example, is converted to trypsin by the removal of the N-terminal hexapeptide on the cleavage of the bond between Lys-6 and Ile-7 by enterokinase. Chymotrypsinogen is activated by the tryptic cleavage of the bond between Arg-15 and He-16. (In this case, further proteolysis by the chymotrypsin that is released during the activation leads to the different forms of the enzyme—Figure 16.5.)... 

The inactive precursors are called trypsinogen, pepsinogen, chymotrypsino-gen, and procarboxypeptidase. These precursors are converted to the active enzymes by hydrolytic cleavage of a few specific peptide bonds under the influence of other enzymes (trypsin, for example, converts chymotrypsinogen to chymotrypsin). The digestive enzymes do not appear to self-destruct, probably because they are so constructed that it is sterically impossible to fit a part of one enzyme molecule into the active site of another. In this connection, it is significant that chymotrypsin attacks denatured proteins more rapidly than natural proteins with their compact structures of precisely folded chains. 

Schematic diagrams of the amino acid sequences of chymotrypsin, trypsin, and elastase. Each circle represents one amino acid. Amino acid residues that are identical in all three proteins are in solid color. The three proteins are of different lengths but have been aligned to maximize the correspondence of the amino acid sequences. All of the sequences are numbered according to the sequence in chymotrypsin. Long connections between nonadjacent residues represent disulfide bonds. Locations of the catalytically important histidine, aspartate, and serine residues are marked. The links that are cleaved to transform the inactive zymogens to the active enzymes are indicated by parenthesis marks. After chymotrypsinogen is cut between residues 15 and 16 by trypsin and is thus transformed into an active protease, it proceeds to digest itself at the additional sites that are indicated these secondary cuts have only minor effects on the enzymes s catalytic activity. (Illustration copyright by Irving Geis. Reprinted by permission.)...

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