Pepsin

pepsin A

Pepsin in complex with pepstatin.[1]
Identifiers
EC number 3.4.23.1
CAS number 9001-75-6
Databases
IntEnz IntEnz view
BRENDA BRENDA entry
ExPASy NiceZyme view
KEGG KEGG entry
MetaCyc metabolic pathway
PRIAM profile
PDB structures RCSB PDB PDBe PDBsum
Gene Ontology AmiGO / EGO
pepsin B
Identifiers
EC number 3.4.23.2
CAS number 9025-48-3
Databases
IntEnz IntEnz view
BRENDA BRENDA entry
ExPASy NiceZyme view
KEGG KEGG entry
MetaCyc metabolic pathway
PRIAM profile
PDB structures RCSB PDB PDBe PDBsum
pepsin C (gastricsin)
Identifiers
EC number 3.4.23.3
CAS number 9012-71-9
Databases
IntEnz IntEnz view
BRENDA BRENDA entry
ExPASy NiceZyme view
KEGG KEGG entry
MetaCyc metabolic pathway
PRIAM profile
PDB structures RCSB PDB PDBe PDBsum

Pepsin is an enzyme that breaks down proteins into smaller peptides (that is, a protease). It is produced in the stomach and is one of the main digestive enzymes in the digestive systems of humans and many other animals, where it helps digest the proteins in food.

It is one of three principal proteases in the human digestive system, the other two being chymotrypsin and trypsin. During the process of digestion, these enzymes, each of which is specialized in severing links between particular types of amino acids, collaborate to break down dietary proteins into their components, i.e., peptides and amino acids, which can be readily absorbed by the small intestine. Pepsin is most efficient in cleaving peptide bonds between hydrophobic and preferably aromatic amino acids such as phenylalanine, tryptophan, and tyrosine.[2]

Pepsin's proenzyme, pepsinogen, is released by the chief cells in the stomach wall, and upon mixing with the hydrochloric acid of the gastric juice, pepsinogen activates to become pepsin. Pepsin is an aspartic protease, using a catalytic aspartate in its active site.[3]

History

Pepsin was one of the first enzymes to be discovered. It was discovered in 1836 by Theodor Schwann. Schwann coined its name from the Greek word πέψις pepsis, meaning "digestion" (from πέπτειν peptein "to digest").[4][5][6][7] Scientists around this time began discovering many biochemical compounds that play a significant role in biological processes, and pepsin was one of them. An acidic substance that was able to convert nitrogen-based foods into water-soluble material was determined to be pepsin.[8]

In 1928, it became one of the first enzymes to be crystallized when John H. Northrop crystallized it using dialysis, filtration, and cooling.[9]

Precursor

Pepsin is expressed as a zymogen called pepsinogen, whose primary structure has an additional 44 amino acids.

In the stomach, chief cells release pepsinogen. This zymogen is activated by hydrochloric acid (HCl), which is released from parietal cells in the stomach lining. The hormone gastrin and the vagus nerve trigger the release of both pepsinogen and HCl from the stomach lining when food is ingested. Hydrochloric acid creates an acidic environment, which allows pepsinogen to unfold and cleave itself in an autocatalytic fashion, thereby generating pepsin (the active form). Pepsin cleaves the 44 amino acids from pepsinogen to create more pepsin.

Activity and stability

Pepsin is most active in acidic environments between 37 °C and 42 °C.[10][11] Accordingly, its primary site of synthesis and activity is in the stomach (pH 1.5 to 2). Pepsin will digest up to 20% of ingested amide bonds by cleaving preferentially after the N-terminal[12]:96 of aromatic amino acids such as phenylalanine, tryptophan, and tyrosine.[12]:675 Pepsin exhibits preferential cleavage for hydrophobic, preferably aromatic, residues in P1 and P1' positions. Increased susceptibility to hydrolysis occurs if there is a sulfur-containing amino acid close to the peptide bond, which has an aromatic amino acid. Pepsin cleaves Phe1Val, Gln4His, Glu13Ala, Ala14Leu, Leu15Tyr, Tyr16Leu, Gly23Phe, Phe24 in the insulin B chain. Pepsin exhibits maximal activity at pH 2.0 and is inactive at pH 6.5 and above, however pepsin is not fully denatured or irreversibly inactivated until pH 8.0.[13] Therefore, pepsin in solution of up to pH 8.0 can be reactivated upon re-acidification. The stability of pepsin at high pH has significant implications on disease attributed to laryngopharyngeal reflux. Pepsin remains in the larynx following a gastric reflux event.[14][15] At the mean pH of the laryngopharynx (pH = 6.8) pepsin would be inactive but could be reactivated upon subsequent acid reflux events resulting in damage to local tissues.

In laryngopharyngeal reflux

Pepsin is one of the primary causes of mucosal damage during laryngopharyngeal reflux.[16][17] Pepsin remains in the larynx (pH 6.8) following a gastric reflux event.[14][15] While enzymatically inactive in this environment, pepsin would remain stable and could be reactivated upon subsequent acid reflux events.[13] Exposure of laryngeal mucosa to enzymatically active pepsin, but not irreversibly inactivated pepsin or acid, results in reduced expression of protective proteins and thereby increases laryngeal susceptibility to damage.[13][14][15]

Pepsin may also cause mucosal damage during weakly acidic or non-acid gastric reflux. Weak or non-acid reflux is correlated with reflux symptoms and mucosal injury.[18][19][20][21] Under non-acid conditions (neutral pH), pepsin is internalized by cells of the upper airways such as the larynx and hypopharynx by a process known as receptor-mediated endocytosis.[22] The receptor by which pepsin is endocytosed is currently unknown. Upon cellular uptake, pepsin is stored in intracellular vesicles of low pH at which its enzymatic activity would be restored. Pepsin is retained within the cell for up to 24 hours.[23] Such exposure to pepsin at neutral pH and endoyctosis of pepsin causes changes in gene expression associated with inflammation, which underlies signs and symptoms of reflux,[24] and tumor progression.[25] This and other research[26] implicates pepsin in carcinogenesis attributed to gastric reflux.

Pepsin in airway specimens is considered to be a sensitive and specific marker for laryngopharyngeal reflux.[27][28] Research to develop new pepsin-targeted therapeutic and diagnostic tools for gastric reflux is ongoing. A rapid non-invasive pepsin diagnostic called Peptest is now available which determines the presence of pepsin in saliva samples.[29]

Storage

Pepsins should be stored at very low temperatures (between −80 °C and −20 °C) to prevent autolysis (self-digestion).

Inhibitors

Pepsin may be inhibited by high pH (see "Activity" and "Stability", above) or by inhibitor compounds. Pepstatin is a low molecular weight compound and potent inhibitor specific for acid proteases with a Ki of about 10−10 M for pepsin. The statyl residue of pepstatin is thought to be responsible for pepstatin inhibition of pepsin; statine is a potential analog of the transition state for catalysis by pepsin and other acid proteases. Pepstatin does not covalently bind pepsin and inhibition of pepsin by pepstatin is therefore reversible.[30] 1-bis(diazoacetyl)-2-penylethane reversibly inactivates pepsin at pH 5, a reaction which is accelerated by the presence of Cu(II).[31]

Pepsin also undergoes feedback inhibition; a product of protein digestion slows down the reaction by inhibiting pepsin.[32][33]

Sucralfate also inhibits pepsin activity.

Applications

Commercial pepsin is extracted from the glandular layer of hog stomachs. It is a component of rennet used to curdle milk during the manufacture of cheese. Pepsin is used for a variety of applications in food manufacturing: to modify and provide whipping qualities to soy protein and gelatin,[34] to modify vegetable proteins for use in nondairy snack items, to make precooked cereals into instant hot cereals,[35] and to prepare animal and vegetable protein hydrolysates for use in flavoring foods and beverages. It is used in the leather industry to remove hair and residual tissue from hides and in the recovery of silver from discarded photographic films by digesting the gelatin layer that holds the silver.[36] Pepsin was historically an additive of Beemans gum brand chewing gum by Dr. Edward E. Beeman.

Pepsin is commonly used in the preparation of F(ab')2 fragments from antibodies. In some assays, it is preferable to use only the antigen-binding (Fab) portion of the antibody. For these applications, antibodies may be enzymatically digested to produce either an Fab or an F(ab')2 fragment of the antibody. To produce an F(ab')2 fragment, IgG is digested with pepsin, which cleaves the heavy chains near the hinge region. One or more of the disulfide bonds that join the heavy chains in the hinge region are preserved, so the two Fab regions of the antibody remain joined together, yielding a divalent molecule (containing two antibody binding sites), hence the designation F(ab')2. The light chains remain intact and attached to the heavy chain. The Fc fragment is digested into small peptides. Fab fragments are generated by cleavage of IgG with papain instead of pepsin. Papain cleaves IgG above the hinge region containing the disulfide bonds that join the heavy chains, but below the site of the disulfide bond between the light chain and heavy chain. This generates two separate monovalent (containing a single antibody binding site) Fab fragments and an intact Fc fragment. The fragments can be purified by gel filtration, ion exchange, or affinity chromatography.[37]

Fab and F(ab')2 antibody fragments are used in assay systems where the presence of the Fc region may cause problems. In tissues such as lymph nodes or spleen, or in peripheral blood preparations, cells with Fc receptors (macrophages, monocytes, B lymphocytes, and natural killer cells) are present which can bind the Fc region of intact antibodies, causing background staining in areas that do not contain the target antigen. Use of F(ab')2 or Fab fragments ensures that the antibodies are binding to the antigen and not Fc receptors. These fragments may also be desirable for staining cell preparations in the presence of plasma, because they are not able to bind complement, which could lyse the cells. F(ab')2, and to a greater extent Fab, fragments allow more exact localization of the target antigen, i.e., in staining tissue for electron microscopy. The divalency of the F(ab')2 fragment enables it to cross-link antigens, allowing use for precipitation assays, cellular aggregation via surface antigens, or rosetting assays.[38]

Genes

The following three genes encode identical human pepsinogen A enzymes:

A fourth human gene encodes gastricsin also known as pepsinogen C:

References

  1. ↑ PDB: 1PSO​; Fujinaga M, Chernaia MM, Tarasova NI, Mosimann SC, James MN (May 1995). "Crystal structure of human pepsin and its complex with pepstatin". Protein Sci. 4 (5): 960–72. doi:10.1002/pro.5560040516. PMC 2143119Freely accessible. PMID 7663352.
  2. ↑ Dunn BM (Nov 2001). "Overview of pepsin-like aspartic peptidases". Current Protocols in Protein Science. Chapter 21: Unit 21.3. doi:10.1002/0471140864.ps2103s25. ISBN 0471140864. PMID 18429164.
  3. ↑ "Enzyme entry 3.4.23.1". Retrieved 2008-12-14.
  4. ↑ Florkin M (Mar 1957). "[Discovery of pepsin by Theodor Schwann]". Revue Médicale De Liège (in French). 12 (5): 139–44. PMID 13432398.
  5. ↑ Asimov I (1980). "page 95". A short history of biology. Westport, Conn: Greenwood Press. ISBN 0-313-22583-4.
  6. ↑ Harper, Douglas. "pepsin". Online Etymology Dictionary.
  7. ↑ πέψις, πέπτειν. Liddell, Henry George; Scott, Robert; A Greek–English Lexicon at the Perseus Project.
  8. ↑ Fruton JS (Jun 2002). "A history of pepsin and related enzymes". The Quarterly Review of Biology. 77 (2): 127–47. doi:10.1086/340729. JSTOR 3071644. PMID 12089768.
  9. ↑ Northrop JH (May 1929). "Crystalline pepsin". Science. 69 (1796): 580. Bibcode:1929Sci....69..580N. doi:10.1126/science.69.1796.580. PMID 17758437.
  10. ↑ "Effects of pH". Retrieved 2010-04-29.
  11. ↑ "Information on EC 3.4.23.1 - pepsin A". Brenda-enzymes. Retrieved 2008-12-14.
  12. 1 2 Cox M, Nelson DR, Lehninger AL (2008). Lehninger principles of biochemistry. San Francisco: W.H. Freeman. ISBN 0-7167-7108-X.
  13. 1 2 3 Johnston N, Dettmar PW, Bishwokarma B, Lively MO, Koufman JA (Jun 2007). "Activity/stability of human pepsin: implications for reflux attributed laryngeal disease". The Laryngoscope. 117 (6): 1036–9. doi:10.1097/MLG.0b013e31804154c3. PMID 17417109.
  14. 1 2 3 Johnston N, Knight J, Dettmar PW, Lively MO, Koufman J (Dec 2004). "Pepsin and carbonic anhydrase isoenzyme III as diagnostic markers for laryngopharyngeal reflux disease". The Laryngoscope. 114 (12): 2129–34. doi:10.1097/01.mlg.0000149445.07146.03. PMID 15564833.
  15. 1 2 3 Johnston N, Dettmar PW, Lively MO, Postma GN, Belafsky PC, Birchall M, Koufman JA (Jan 2006). "Effect of pepsin on laryngeal stress protein (Sep70, Sep53, and Hsp70) response: role in laryngopharyngeal reflux disease". The Annals of Otology, Rhinology, and Laryngology. 115 (1): 47–58. doi:10.1177/000348940611500108. PMID 16466100.
  16. ↑ Goldberg HI, Dodds WJ, Gee S, Montgomery C, Zboralske FF (Feb 1969). "Role of acid and pepsin in acute experimental esophagitis". Gastroenterology. 56 (2): 223–30. PMID 4884956.
  17. ↑ Lillemoe KD, Johnson LF, Harmon JW (Aug 1982). "Role of the components of the gastroduodenal contents in experimental acid esophagitis". Surgery. 92 (2): 276–84. PMID 6808683.
  18. ↑ Tamhankar AP, Peters JH, Portale G, Hsieh CC, Hagen JA, Bremner CG, DeMeester TR (Nov 2004). "Omeprazole does not reduce gastroesophageal reflux: new insights using multichannel intraluminal impedance technology". Journal of Gastrointestinal Surgery. 8 (7): 890–7; discussion 897–8. doi:10.1016/j.gassur.2004.08.001. PMID 15531244.
  19. ↑ Kawamura O, Aslam M, Rittmann T, Hofmann C, Shaker R (Jun 2004). "Physical and pH properties of gastroesophagopharyngeal refluxate: a 24-hour simultaneous ambulatory impedance and pH monitoring study". The American Journal of Gastroenterology. 99 (6): 1000–10. doi:10.1111/j.1572-0241.2004.30349.x. PMID 15180717.
  20. ↑ Oelschlager BK, Quiroga E, Isch JA, et al. Gastroesophageal and pharyngeal reflux detection using impedance and 24-hour pH monitoring in asymptomatic subjects: defining the normal environment. J Gastrointest Surg 2006;10:54–62.
  21. ↑ Mainie I, Tutuian R, Shay S, Vela M, Zhang X, Sifrim D, Castell DO (Oct 2006). "Acid and non-acid reflux in patients with persistent symptoms despite acid suppressive therapy: a multicentre study using combined ambulatory impedance-pH monitoring". Gut. 55 (10): 1398–402. doi:10.1136/gut.2005.087668. PMC 1856433Freely accessible. PMID 16556669.
  22. ↑ Johnston N, Wells CW, Blumin JH, Toohill RJ, Merati AL (Dec 2007). "Receptor-mediated uptake of pepsin by laryngeal epithelial cells". The Annals of Otology, Rhinology, and Laryngology. 116 (12): 934–8. doi:10.1177/000348940711601211. PMID 18217514.
  23. ↑ Johnston N, Wells CW, Samuels TL, Blumin JH (Aug 2010). "Rationale for targeting pepsin in the treatment of reflux disease". The Annals of Otology, Rhinology, and Laryngology. 119 (8): 547–58. doi:10.1177/000348941011900808. PMID 20860281.
  24. ↑ Samuels TL, Johnston N (Nov 2009). "Pepsin as a causal agent of inflammation during nonacidic reflux". Otolaryngology--Head and Neck Surgery. 141 (5): 559–63. doi:10.1016/j.otohns.2009.08.022. PMID 19861190.
  25. ↑ Balkwill F, Mantovani A (Feb 2001). "Inflammation and cancer: back to Virchow?". Lancet. 357 (9255): 539–45. doi:10.1016/S0140-6736(00)04046-0. PMID 11229684.
  26. ↑ Adams J, Heintz P, Gross N, Andersen P, Everts E, Wax M, Cohen J (Mar 2000). "Acid/pepsin promotion of carcinogenesis in the hamster cheek pouch". Archives of Otolaryngology--Head & Neck Surgery. 126 (3): 405–9. doi:10.1001/archotol.126.3.405. PMID 10722017.
  27. ↑ Knight J, Lively MO, Johnston N, Dettmar PW, Koufman JA (Aug 2005). "Sensitive pepsin immunoassay for detection of laryngopharyngeal reflux". The Laryngoscope. 115 (8): 1473–8. doi:10.1097/01.mlg.0000172043.51871.d9. PMID 16094128.
  28. ↑ Samuels TL, Johnston N (Mar 2010). "Pepsin as a marker of extraesophageal reflux". The Annals of Otology, Rhinology, and Laryngology. 119 (3): 203–8. PMID 20392035.
  29. ↑ Bardhan KD, Strugala V, Dettmar PW (2012). "Reflux revisited: advancing the role of pepsin". International Journal of Otolaryngology. 2012: 1. doi:10.1155/2012/646901. PMC 3216344Freely accessible. PMID 22242022.
  30. ↑ Marciniszyn J, Hartsuck JA, Tang J (1977). "Pepstatin inhibition mechanism". Advances in Experimental Medicine and Biology. 95: 199–210. doi:10.1007/978-1-4757-0719-9_12. PMID 339690.
  31. ↑ Husain SS, Ferguson JB, Fruton JS (Nov 1971). "Bifunctional inhibitors of pepsin". Proceedings of the National Academy of Sciences of the United States of America. 68 (11): 2765–8. Bibcode:1971PNAS...68.2765H. doi:10.1073/pnas.68.11.2765. PMC 389520Freely accessible. PMID 4941985.
  32. ↑ Northrop HJ (1932). "The story of the isolation of crystalline pepsin and trypsin". The Scientific Monthly. 35 (4): 333–340. Bibcode:1932SciMo..35..333N.
  33. ↑ Greenwell P, Knowles JR, Sharp H (Jun 1969). "The inhibition of pepsin-catalysed reactions by products and product analogues. Kinetic evidence for ordered release of products". The Biochemical Journal. 113 (2): 363–8. PMC 1184643Freely accessible. PMID 4897199.
  34. ↑ Kun LY (2006). Microbial Biotechnology: Principles And Applications. World Scientific Publishing Company. ISBN 981-256-677-5.
  35. ↑ US patent 2259543, Billings HJ, "Fortified Cereal", published 1938, assigned to Cream of Wheat Corporation
  36. ↑ Smith ER (Sep 1933). "Gelatinase and the gates-gilman-cowgill method of pepsin estimation". The Journal of General Physiology. 17 (1): 35–40. doi:10.1085/jgp.17.1.35. PMC 2141270Freely accessible. PMID 19872760.
  37. ↑ Lane DS, Harlow E (1988). Antibodies: a laboratory manual. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory. pp. A2926. ISBN 0-87969-314-2.
  38. ↑ "Enzyme Explorer- Pepsin". Sigma-Aldrich.
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