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Digestive enzymes play a very important role in providing the energy and proteins that the body needs to survive from the food that is ingested. One important digestive enzyme is pepsin. This enzyme survives the acidic pH of the stomach and is not denatured by it as some enzymes might. Pepsin has a precursor, pepsinogen, which is the inactive form of pepsin. Pepsinogen is structurally different than pepsin because it has an extra peptide chain. Pepsin is in charge of breaking down polypeptides into smaller peptide units in the stomach. Pepsin breaks down proteins through a carbonyl nucleophilic attack that is initiated by the two aspartic acids in the active site of pepsin.
The digestive system is where the digestion of macromolecules and the absorption of their components occur. Throughout the parts of the digestive system, the pH fluctuates to accommodate for the enzymes that are released. The stomach, a part of the digestive system, has a pH between 1 and 3, which is very low compared to the body’s normal pH (VanPutte, Cinnamon L, 2017). The low pH of the stomach is obtained through the production of hydrochloric acid by the parietal cells within the stomach mucosa (VanPutte, Cinnamon L, 2017). The low pH of the stomach that is conducted by HCl has many important functions, most importantly providing a suitable environment for pepsin, which has an optimal pH of 3 or less (VanPutte, Cinnamon L, 2017). Pepsin is a proteolytic enzyme and aspartic proteinase, which is involved in the breakdown of ingested proteins into smaller peptide units (Fujinaga, M,1995).
After the discovery of pepsin in 1836, it was used to treat digestive disorders, which increased the in-depth study of pepsin (Pepsin). Although pepsin was one of the first enzymes to be discovered, the amino acid sequence was later put together by Tang and other scientist in 1973, and Moravek and Kostka in 1974, after the crystallization of pepsin by John H. Northrop in 1930 (“Pepsin”).
Pepsin is not directly released from the stomach. Pepsinogen, the inactive form of pepsin, is produced by the chief cells in the gastric glands and then converted to pepsin. It is crucial that pepsin is released as pepsinogen, the inactive form, because it prevents the digestion of the proteins on the inner lining of the digestive tract (Heda, Rajiv, 2018).
When pepsinogen is released in the lumen of the stomach, HCl and previously converted pepsin catalyze the transformation from pepsinogen into pepsin (Herriott, R M, 1962) (VanPutte, Cinnamon L, 2017). Pepsin, when formed, participates in the cleavage of covalent bonds in the protein, forming smaller polypeptide chain units that can be further broken down by other enzymes in the digestive tract (VanPutte, Cinnamon L, 2017).
The structure of human pepsin follows closely to the structure of porcine pepsin which has been experimented with and studied very well (Fujinaga, M,1995), as seen in Figure 1. Porcine structure will be discussed in this paper.
Pepsin consists of a single peptide chain that is comprised of 327 residues and folds into a globular-shaped peptide (Herriott, R M, 1962) (Sepulveda, P, 1975). “Porcine pepsin has 4 basic residues, and 42 acidic residue (Pepsin).” There are two catalytic residues in the active site within pepsin, Asp 32 and Asp 215 (Kageyama, T, 2002).
The secondary structure of pepsin is made mostly of beta-sheets that are held together by hydrogen bonds (Kageyama, T, 2002), as seen in Figure 2. “There are ten alpha helices encompassing forty-six residues and thirty-two beta strands encompassing one hundred forty-four residues” (Gutierrez, Megan). The hydrogen bonds are the intermolecular bonds that stabilize the secondary structure of the pepsin peptide. The backbone of the active site is comprised of a six-standard antiparallel beta-sheets (Fujinaga, M,1995). The backbone consists of “residues Val I-Leu 6, Asp 149-Val 184, and Gln 308-Ala 326” (Fujinaga, M,1995). Most of these amino acids have hydrophobic side chains which make the active site hydrophobic and negatively charged because of the Asp. “Two antiparallel beta strands on the surface of the protein containing residues Leu-71 through Gly-82 form a loose, flexible flap” (Gutierrez, Megan ). This flap is positioned at that location to facilitate the movement of substrate into and out of the enzyme and to protect the active site from any other side reactions that might occur (Gutierrez, Megan ).Figure 2: Secondary Structure of Pepsin (Gutierrez, Megan).
Since pepsin is a one peptide chain, it cannot have a quaternary structure. It only has a tertiary structure. “The tertiary structure of pepsin shows two lobes with similar folds and it has been suggested that the gene has arisen from an ancient duplication and fusion event “(Rawlings, Neil D, and Alex Bateman, 2009). The single peptide chain folds into a globular-shaped peptide as seen in Figure 3. Also seen in Figure 3 is the active site which is circled and consists of two catalytic residues, Asp 32 and Asp 215, where the substrate binds and is broken down by pepsin. Figure 3: Tertiary Structure of Porcine Pepsin (Kageyama, T, 2002).
Pepsinogen is the zymogen, or the inactive form of pepsin, which is converted to pepsin under the acidic conditions of the stomach as discussed before. The difference between pepsinogen and pepsin is that pepsinogen has an extra peptide chain containing 47 amino acid residues (Josh, and Congress, 2014), as seen in Figure 4. The 47 extra amino acids are cleaved to transform pepsinogen to pepsin (Josh, and Congress, 2014). The conversion of pepsinogen to pepsin is an autocatalytic reaction, which means that pepsinogen has the ability to cleave itself and catalyzes the conversion of the pepsinogen to pepsin through intramolecular reactions (Herriott, R M, 1962). The conversion occurs at a pH under 5 (Herriott, R M, 1962). As seen in Figure 5, “The intramolecular activation of pepsinogen is known to involve several steps. The central helical part of the propeptide undergoes local conformational denaturation in acid.” (Barrett, Alan J., et al, 2004). As seen in Figure 5, the central helical part of pepsinogen that was denatured binds to the active site as a substrate on the pepsinogen that it was denatured from. Then, cleavage occurs first at residues 16 and 17 which form an Ile-Leu bond. The cleavage of the first 16 amino acid residues out the extra 47 on pepsinogen is important because it helps in the denaturation of the reminder of the extra 47 amino acid residues (Barrett, Alan J., et al, 2004). According to Alan Barrett and other writers, “This [step] leads to biomolecular cleavage of the reminder of the propeptide from pepsin. The detailed steps in biomolecular activation of pepsinogen are still unclear. Under certain activation conditions, the release of entire propeptide from pepsinogen has been observed. This may be result of bimolecular activation mechanism” (Barrett, Alan J., et al, 2004).
After pepsinogen is converted to pepsin, pepsin is ready to react and breakdown proteins into smaller peptide chains. As discussed before, the active site is composed of two aspartic acid residues at locations 32 and 215 in the primary structure. Although, in the primary structure Asp 32 and Asp 215 are far apart when the structure folds, both of these amino acids come together and form the active site. With the active site being negatively charged because of the two aspartic acids present, pepsin prefers to catalyze large hydrophobic amino acid chains such as phenylalanine and leucine (Barrett, Alan J., et al, 2004). The reaction begins with a water molecule being hydrogen-bonded to both Asp 32 and Asp 215 (Barrett, Alan J., et al, 2004), as seen in Figure 6. Asp 32 accepts a proton from the water, making it a better nucleophile. The hydroxyl now is a better nucleophile than the water molecule and can attack the carbonyl carbon on the polypeptide forming a tetrahedral intermediate as seen in Figure 6. Asp 215 donates a proton to the substrate to stabilize the tetrahedral intermediate (“Carboxyl Proteinase [Molecular Biology]”). With the formed tetrahedral intermediate, Asp 32 is protonated, and Asp 215 is deprotonated. “Hydrogen donation from Asp 215 to substrate nitrogen resulted in the breaking of the peptide bond” (Barrett, Alan J., et al, 2004). After, the breaking of the peptide bond, pepsin is free to breakdown another polypeptide. In the reaction of pepsin breaking down proteins, it is seen that the proximity effect takes place. The carbonyl of the substrate is positioned in-between the two aspartic acids to be able to perform the correct reaction. Another enzymatic mechanism that is seen is the general-acid and general-base catalysis. As seen in Figure 6, Asp 32 accepts a proton from the water molecule making the water molecule a better nucleophile and Asp 32 a general base. On the other hand, Asp 215 acts as a general acid and donates its proton to the substrate. Nucleophilic and electrophilic catalyst is an enzyme mechanism that is also seen in this reaction. The water being deprotonated by Asp 32 makes it a better nucleophile and is able to attack the carbonyl of the substrate. Figure 6: Pepsin Breakdown Mechanism (“Carboxyl Proteinase [Molecular Biology]”).
All in all, pepsin has a very important role in the digestion of polypeptides to provide the body with the nutrients it needs. Pepsin’s structure is very important for the function it accomplishes. Pepsinogen, the zymogen of the pepsin, is formed in the chief cells of the gastric mucosa in the lining of the stomach. Pepsinogen has 47 extra amino acid residues that are not present in pepsin. When pepsinogen is converted to pepsin by the reaction discussed previously, pepsin is ready to breakdown polypeptides. When pepsin is active, the nucleophilic reaction takes place where Asp 32 and Asp 215 work together to break the hydrophobic side chain of amino acids such as phenylalanine and leucine.
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