The Chymotrypsin Enzyme

A digestive enzyme called chymotrypsin catalyzes the breakdown of peptide bonds in the ileum and duodenum to produce polypeptides and amino acids that the body can use. It is produced as chymotrypsinogen, an inactive form, by the acinar cells in the pancreas. Chymotrypsin is a digestive aid and anti-inflammatory agent used in pharmaceuticals. Its presence in the stool is also employed in a person's fecal diagnostic to ascertain how well the pancreas is working. Treatment of pancreatic diseases requires testing for fecal chymotrypsin. There are studies in progress that prove chymotrypsin aids in the cure of cancer in chemotherapy. In the early 1900s, a scientist by the name Vernon suggested that preparation of pancreatic enzymes could produce an enzyme activator of its own enzyme. He did a milk-clotting experiment discovered that there existed at least two enzymes in the milk, but one enzyme was more prominent than the other (Buchholz, Kasche, & Bornscheuer, 2012). Later on, in 1938, Kvnitz managed to separate the different types of chymotrypsin (α, β, and γ) through a test experiment. The denaturation and folding of chymotrypsin has been researched on of late under varying concentrations with nanoparticle and also molecules of Peg were conjugated. (Worthington, 1993).

Chymotrypsinogen is a proteolytic inactive enzyme belonging to the family of serine proteases and contains one polypeptide chain made up of 245 types of amino acid. It is stored on the edge of the acinar cell membrane granules. Inactive chymotrypsinogen is activated into chymotrypsin by trypsin enzyme during digestion (Nelson & Cox, 2008). α-Chymotrypsin is the most active form of active enzyme of zymogen, in form of Chymotrypsinogen A.

This research paper focuses on the enzyme chymotrypsin, its genetic composition, protein structure, functioning mechanism and how the enzyme is regulated.

Genetics/Evolution

Enzyme chymotrypsin is found in the digestive tract of mammals in the duodenum. It is released from the pancreas as chymotrypsinogen through the pancreatic juice and is activated by trypsin in the small intestines for the digestion process. It is produced by all organisms which possess a pancreas such as mammals, reptiles, birds, etc. An enzyme that resembles trypsin is found in the sea anemone and Pacific dogfish, and the cocoonase enzyme used by the pupa of the silkworm to digest its cocoon is closely related as previous tests have shown (Whitaker, 1994).

Chymotrypsin Structure

Chymotrypsin is a protease consisting of 245 amino acid residues by composition. The molecule is made up of three peptide chains: a  γ chain of 97 residues, α chain of 13 residues, and a β chain of 131 residues.



Figure 1: Structure of chymotrypsin enzyme. Reprinted from Digestive Enzymes. (p. 40), by Bland, Jeffrey, 1993, New Canaan, CT: Keats Publishing, Inc. Copyright [1993] by Bland, Jeffrey. Reprinted with permission.

As shown above (Figure 1), chymotrypsin is made up of three polypeptide chains that are connected via bonds. Examples of the important amino acids include asp, histidine, and serine on chains B, A, and C respectively. When folding is done, these amino acids are moved nearer to the space. When active sites close down, the enzyme is activated. Since asp is negatively charged, it takes away hydrogen ions from the histidine amino acid thereby forming a hydrogen bond between histidine and asp amino acids. Consequently a hydrogen ion is attracted from the ser amino acid, which is a hydroxyl, and recovers the lost hydrogen ion which Asp gave to histidine. This results in a negative charge on ser using the residue from histidine. The process then repeats in the systematic order. The coordination of these three amino acid in time lead to their name ‘catalytic triad’ simply because they function together symbiotically.

Chymotrypsin acts only on specific to aromatic residues available. A site for chymotrypsin to act on specifically is created by a large space. This large space of the enzyme shape acts as the site where aromatic amino acid residues that are hydrophobic bind to residue linings. This causes the peptide bonds to come merge because ser residue is attacked in the binding process. Chymotrypsin becomes inactive when the substrate binding is hindered (Blow, 1976).

Chymotrypsin Function mechanism

During the binding process, polypeptide, which is the substrate, nestles in a hydrophobic space in found on the enzyme thus making the peptide bond prone to attack. One proton is taken from serine to form an alkoxide ion by histidine and therefore serine reacts with the polypeptide. In chymotrypsin, the COH- R-group of asp102 amino acid forms a hydrogen bond with the carbon chain group of histidine 57. During the process, the hydrogen bond is compressed and electrons move to the adjacent nitrogen atom making the his57 to have a high ph value. The histidine 57 removes some of the protons from ser195 which turns it into a strong nucleophile which is able to act on the polypeptide (Gumport & Stryer, 1989).

The Oxygen ion hole makes the tetrahedral intermediate stable. It is formed by linking the peptide NH groups with the hydrogen bond and to the oxyanion. Oxygen becomes negatively charged in the process.

The negative charge is unstable and therefore the charge on the substrate collapses the tetrahedral intermediate. Another double bond is reformed with the carbon that breaks the substrate bond between the amino acid group and carbon. When the oxyanion hole becomes neutral, the bond is broken because one proton from Histidine binds to nitrogen to avoid binding on carbon. After stabilization, the acyl-enzyme is formed.The amine part is removed from the enzyme when the body metabolizes and binds to serine. At this stage, the first product is formed.



Figure 2: Diagrammatic representation of the working mechanism of chymotrypsin enzyme. Reprinted from Biochemistry, 6th edition (p. 80), by JM Berg, JL Tymockzo, L Stryer, 2010, New York, NY: Mackerlin Press. Copyright 2010 by the Association for Memory Research. Reprinted with permission.

The nitrogen terminus is replaced by a water molecule from metabolism. Histidine then removes hydrogen protons from the water creating a hydroxide anion. This causes carbon to be removed from the carboxyl part. The bond is thus disintegrated. Hydrogen ions are removed from the water molecule by acid-base catalysis, producing a nucleophilic negative hydroxide ion. Removal of hydroxide on the R-COOH link of the acyl-enzyme gives rise to another tetrahedral intermediate. The second product is, a carboxylate anion is formed after the disintegration of the tetrahedral intermediate and ser195 is displaced. The hydrogen ion from Histidine is taken back to Serine. The carboxylic acid is released, and the enzyme is made to speed up the next reaction while still in active state (Chang, 2005).

Chymotrypsin Regulation

Enzymes perform many useful processes that are necessary for life, but without limitation, they can cause damage.  Enzyme inhibitors are used as a form of limiting the enzyme once it is activated in the small intestines.  Some organisms have a delicate balance between enzymes and their corresponding inhibitors for easy control of the activation and intracellular catabolism of proteins. Blood in Animals is the medium for carrying the enzyme inhibitors and glycoproteins. This ensures that the inhibitors reach every part of the body quickly in time for deactivation. The protease inhibitors form very stable complexes by using a reactive peptide site bond which acts as the substrate for different types of proteases, and this eliminates protease from the blood. There are different types of protease inhibitors. They all differ in their structures but share the same reactive peptide bond.  Some inhibitors only control trypsin, while others hydrolyse both chymotrypsin and trypsin using different inhibitory domains of the enzyme (Klein, 2012).

            Enzyme inhibitors especially protease are commonly found in plant seeds such as legumes. Inhibitors can have an adverse impact on other animals as well; some inhibitors found in soya beans have been discovered to cause enlargement of the pancreas in certain animal species. Some protease inhibitors also provide nutrition to the organism such as the Bowman-Birk inhibitor found in soybean plant prevents tumorigenesis and nephrotoxicity. 

That is why zymogen has to undergo proteolytic cleavage to form the active enzyme. This prevents the enzyme from attacking other substrates thus making it substrate specific. At first, chymotrypsin, which is the active form, is also synthesized as chymotrypsinogen which is inactive, to avoid attacking other substrates.

Diseases/Mutations/Treatment.

Chymotrypsin has been known to treat inflammation and reduce swelling on the body such as tissue injuries, sprains, hematomas, and muscle cramps. It is also applied in the treatment of arthritis and other autoimmune diseases such as lupus. Ulcers can also be controlled by the enzyme through the production of its inhibitors. Chymotrypsin has also been known to play a role in the treatment of cancer through chemotherapy, although the treatment method is still in the research stages.

Pancreatitis is known to be a mutation of the CTRC gene which replaces the chymotrypsin enzyme. Chymotrypsin is then attacked by trypsin and the digestive protease. The absence of chymotrypsin in the digestion of proteins leads to chronic pancreatitis (Hur, 2014).

Conclusion

In summary, Chymotrypsin is a digestive enzyme that catalyzes the hydrolysis of peptide bonds in the ileum and duodenum to polypeptides and amino acids. It is secreted by the acinar cells of the pancreas in an inactive state known as chymotrypsinogen. It is found the digestive tract of mammals in the duodenum. It is released from the pancreas as chymotrypsinogen through the pancreatic juice and is activated by trypsin in the small intestines for the digestion process. Chymotrypsin is a protease consisting of 245 amino acid residues by composition. The molecule is made up of three peptide chains: an α chain, a β chain, and a γ chain of 97 with a total of 245 residues. At first, chymotrypsin, which is the active form, is synthesized as chymotrypsinogen which is inactive, to avoid attacking other substrates. It is used in medicine to cure inflammation and cancer treatment via chemotherapy.



































References

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