54 by: Caitlin Larrigan

Student – Caitlin Larrigan

Enzyme: Pepsin

Enzyme Commission Number: EC 3.4.23.1

Pepsin is one of three principle endopeptidases found in the body, the other two being trypsin and chymotrypsin [3]. It is found in the stomach where it is responsible for the
breakdown of proteins in ingested food. It is first made as pepsinogen in gastric chief cells occupying the stomach lining walls, deep within the mucosal layer [3]. Also within the stomach lining are parietal cells, which secrete hydrochloric acid, lowering the pH of the stomach to 1.5-2 [3]. It is this acidic pH that activates pepsinogen to become pepsin.
Once activated pepsin works to hydrolyze peptide bonds by acid-base catalysis [1]. Its abilities to do so can be attributed to two key aspartic acid residues, located within its active site
[1]. The active site of pepsin is a well-defined cleft, easy to identify on the enzyme’s globular structure [2]. The structure is divided into two domains that are visible as an N-lobe and C-lobe. Pepsin does not have a distinct “hydrophobic pocket,” however the N-lobe and C-lobe regions contain hydrophobic cores contributing to stability and functionality of the enzyme [2]. The aspartic acid residues at positions 32 and 215 are able to act simultaneously as a proton donor and acceptor, which is crucial in carrying out pepsin’s catalytic mechanism. This
process primarily takes place between the carboxyl ends of the two aspartic acids [1]. It should be noted that even though they have the same formula, they do not have the same pKa values because of the different residues surrounding each aspartic acid [4]. Water will nucleophilically attack the carbonyl carbon of the substrate while Asp32 accepts a proton from the water. Asp215 will donate a proton to the substrate’s carbonyl carbon [4]. The amide dehydrate intermediate formed is then broken down. To do so, Asp215 will accept a proton from the intermediate as Asp32 donates its proton to the amide which results in the cleaving of the peptide bond in the substrate [4]. The specifics of the catalytic mechanism of pepsin are outlined in more detail in the video linked below.

References:
1. Antonov, V. K., Ginodman, L. M., Kapitannikov, V. K., Barshevskaya, T.N., Gurova, A.G., & Rumsh, L.D. (1978). Mechanism of Pepsin Catalysis: General base Catalysis by the Active-site Carboxylate Ion. FEBS Letters.
2. Fujinaga, M., Chernaia, M. M., Tarasova, N. I., Mosimann, S. C., & James, M. N. (1995). Crystal structure of human pepsin and its complex with pepstatin. Protein science : a publication of the Protein Society, 4(5), 960–972. https://doi.org/10.1002/pro.5560040516
3. Heda, R., Toro, F., & Tombazzi, C. R. (2023). Physiology, pepsin. In StatPearls (Updated May 1, 2023). StatPearls Publishing. https://www.ncbi.nlm.nih.gov/books/NBK537005/
4. Hollands, T. R., & Fruton, J. S. (1969). On the mechanism of pepsin action. Proceedings of the National Academy of Sciences of the United States of America, 62(4), 1116–1120. https://doi.org/10.1073/pnas.62.4.1116