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Pepsin is an endopeptidase that breaks down
protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, res ...
s into smaller
peptide Peptides (, ) are short chains of amino acids linked by peptide bonds. Long chains of amino acids are called proteins. Chains of fewer than twenty amino acids are called oligopeptides, and include dipeptides, tripeptides, and tetrapeptides. ...
s. It is produced in the gastric chief cells of the stomach lining and is one of the main
digestive enzyme Digestive enzymes are a group of enzymes that break down polymeric macromolecules into their smaller building blocks, in order to facilitate their absorption into the cells of the body. Digestive enzymes are found in the digestive tracts of anima ...
s in the
digestive system The human digestive system consists of the gastrointestinal tract plus the accessory organs of digestion (the tongue, salivary glands, pancreas, liver, and gallbladder). Digestion involves the breakdown of food into smaller and smaller compone ...
s of humans and many other animals, where it helps
digest Digest may refer to: Biology *Digestion of food *Restriction digest Literature and publications *'' The Digest'', formerly the English and Empire Digest *Digest size magazine format * ''Digest'' (Roman law), also known as ''Pandects'', a digest ...
the proteins in
food Food is any substance consumed by an organism for nutritional support. Food is usually of plant, animal, or fungal origin, and contains essential nutrients, such as carbohydrates, fats, proteins, vitamins, or minerals. The substance is in ...
. Pepsin is an
aspartic protease Aspartic proteases are a catalytic type of protease enzymes that use an activated water molecule bound to one or more aspartate residues for catalysis of their peptide substrates. In general, they have two highly conserved aspartates in the activ ...
, using a catalytic aspartate in its
active site In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate ( binding site) ...
. It is one of three principal endopeptidases (enzymes cutting proteins in the middle) in the human digestive system, the other two being
chymotrypsin Chymotrypsin (, chymotrypsins A and B, alpha-chymar ophth, avazyme, chymar, chymotest, enzeon, quimar, quimotrase, alpha-chymar, alpha-chymotrypsin A, alpha-chymotrypsin) is a digestive enzyme component of pancreatic juice acting in the duod ...
and
trypsin Trypsin is an enzyme in the first section of the small intestine that starts the digestion of protein molecules by cutting these long chains of amino acids into smaller pieces. It is a serine protease from the PA clan superfamily, found in the d ...
. There are also exopeptidases which remove individual amino acids at both ends of proteins (
carboxypeptidase A carboxypeptidase ( EC number 3.4.16 - 3.4.18) is a protease enzyme that hydrolyzes (cleaves) a peptide bond at the carboxy-terminal (C-terminal) end of a protein or peptide. This is in contrast to an aminopeptidases, which cleave peptide b ...
s produced by the pancreas and
aminopeptidase Aminopeptidases are enzymes that catalyze the cleavage of amino acids from the amino terminus ( N-terminus) of proteins or peptides (exopeptidases). They are widely distributed throughout the animal and plant kingdoms and are found in many sub ...
s secreted by the small intestine). During the process of digestion, these enzymes, each of which is specialized in severing links between particular types of
amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha ...
s, collaborate to break down dietary proteins into their components, i.e., peptides and amino acids, which can be readily absorbed by the small intestine. The cleavage specificity of pepsin is broad, but some
amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha ...
s like
tyrosine -Tyrosine or tyrosine (symbol Tyr or Y) or 4-hydroxyphenylalanine is one of the 20 standard amino acids that are used by cells to synthesize proteins. It is a non-essential amino acid with a polar side group. The word "tyrosine" is from the G ...
,
phenylalanine Phenylalanine (symbol Phe or F) is an essential α-amino acid with the formula . It can be viewed as a benzyl group substituted for the methyl group of alanine, or a phenyl group in place of a terminal hydrogen of alanine. This essential amin ...
and
tryptophan Tryptophan (symbol Trp or W) is an α-amino acid that is used in the biosynthesis of proteins. Tryptophan contains an α-amino group, an α-carboxylic acid group, and a side chain indole, making it a polar molecule with a non-polar aromatic ...
increase the probability of cleavage. Pepsin's
proenzyme In biochemistry, a zymogen (), also called a proenzyme (), is an inactive precursor of an enzyme. A zymogen requires a biochemical change (such as a hydrolysis reaction revealing the active site, or changing the configuration to reveal the activ ...
, pepsinogen, is released by the gastric chief cells in the stomach wall, and upon mixing with the
hydrochloric acid Hydrochloric acid, also known as muriatic acid, is an aqueous solution of hydrogen chloride. It is a colorless solution with a distinctive pungent smell. It is classified as a strong acid. It is a component of the gastric acid in the dige ...
of the gastric juice, pepsinogen activates to become pepsin.


History

Pepsin was one of the first enzymes to be discovered, by
Theodor Schwann Theodor Schwann (; 7 December 181011 January 1882) was a German physician and physiologist. His most significant contribution to biology is considered to be the extension of cell theory to animals. Other contributions include the discovery of ...
in 1836. Schwann coined its name from the
Greek Greek may refer to: Greece Anything of, from, or related to Greece, a country in Southern Europe: *Greeks, an ethnic group. *Greek language, a branch of the Indo-European language family. **Proto-Greek language, the assumed last common ancestor ...
word ''pepsis'', meaning "
digestion Digestion is the breakdown of large insoluble food molecules into small water-soluble food molecules so that they can be absorbed into the watery blood plasma. In certain organisms, these smaller substances are absorbed through the small intest ...
" (from ''peptein'' "to digest"). An acidic substance that was able to convert nitrogen-based foods into water-soluble material was determined to be pepsin. In 1928, it became one of the first enzymes to be crystallized when
John H. Northrop John Howard Northrop (July 5, 1891 – May 27, 1987) was an American biochemist who, with James Batcheller Sumner and Wendell Meredith Stanley, won the 1946 Nobel Prize in Chemistry. The award was given for these scientists' isolation, crystal ...
crystallized it using dialysis, filtration, and cooling.


Precursor

Pepsin is expressed as a
zymogen In biochemistry, a zymogen (), also called a proenzyme (), is an inactive precursor of an enzyme. A zymogen requires a biochemical change (such as a hydrolysis reaction revealing the active site, or changing the configuration to reveal the activ ...
called pepsinogen, whose
primary structure Protein primary structure is the linear sequence of amino acids in a peptide or protein. By convention, the primary structure of a protein is reported starting from the amino-terminal (N) end to the carboxyl-terminal (C) end. Protein biosynth ...
has an additional 44
amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha ...
s compared to the active enzyme. In the stomach, gastric chief cells release pepsinogen. This zymogen is activated by
hydrochloric acid Hydrochloric acid, also known as muriatic acid, is an aqueous solution of hydrogen chloride. It is a colorless solution with a distinctive pungent smell. It is classified as a strong acid. It is a component of the gastric acid in the dige ...
(HCl), which is released from parietal cells in the stomach lining. The hormone
gastrin Gastrin is a peptide hormone that stimulates secretion of gastric acid (HCl) by the parietal cells of the stomach and aids in gastric motility. It is released by G cells in the pyloric antrum of the stomach, duodenum, and the pancreas. Gast ...
and the
vagus nerve The vagus nerve, also known as the tenth cranial nerve, cranial nerve X, or simply CN X, is a cranial nerve that interfaces with the parasympathetic control of the heart, lungs, and digestive tract. It comprises two nerves—the left and righ ...
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. Pepsinogens are mainly grouped in 5 different groups based on their primary structure: pepsinogen A (also called pepsinogen I), pepsinogen B, progastricsin (also called pepsinogen II and pepsinogen C), prochymosin (also called prorennin) and pepsinogen F (also called pregnancy-associated glycoprotein).


Activity and stability

Pepsin is most active in acidic environments between pH 1.5 to 2.5. Accordingly, its primary site of synthesis and activity is in the stomach ( pH 1.5 to 2). In humans the concentration of pepsin in the stomach reaches 0.5 – 1 mg/mL. Pepsin is inactive at pH 6.5 and above, however pepsin is not fully denatured or irreversibly inactivated until pH 8.0. Therefore, pepsin in solutions 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. 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. Pepsin exhibits a broad cleavage specificity. Pepsin will digest up to 20% of ingested amide bonds. Residues in the P1 and P1' positions are most important in determining cleavage probability. Generally, hydrophobic amino acids at P1 and P1' positions increase cleavage probability.
Phenylalanine Phenylalanine (symbol Phe or F) is an essential α-amino acid with the formula . It can be viewed as a benzyl group substituted for the methyl group of alanine, or a phenyl group in place of a terminal hydrogen of alanine. This essential amin ...
,
leucine Leucine (symbol Leu or L) is an essential amino acid that is used in the biosynthesis of proteins. Leucine is an α-amino acid, meaning it contains an α- amino group (which is in the protonated −NH3+ form under biological conditions), an α- ...
and
methionine Methionine (symbol Met or M) () is an essential amino acid in humans. As the precursor of other amino acids such as cysteine and taurine, versatile compounds such as SAM-e, and the important antioxidant glutathione, methionine plays a critical ...
at the P1 position, and
phenylalanine Phenylalanine (symbol Phe or F) is an essential α-amino acid with the formula . It can be viewed as a benzyl group substituted for the methyl group of alanine, or a phenyl group in place of a terminal hydrogen of alanine. This essential amin ...
,
tryptophan Tryptophan (symbol Trp or W) is an α-amino acid that is used in the biosynthesis of proteins. Tryptophan contains an α-amino group, an α-carboxylic acid group, and a side chain indole, making it a polar molecule with a non-polar aromatic ...
and
tyrosine -Tyrosine or tyrosine (symbol Tyr or Y) or 4-hydroxyphenylalanine is one of the 20 standard amino acids that are used by cells to synthesize proteins. It is a non-essential amino acid with a polar side group. The word "tyrosine" is from the G ...
at the P1' position result in the highest cleavage probability. Cleavage is disfavoured by positively charged
amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha ...
s
histidine Histidine (symbol His or H) is an essential amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated –NH3+ form under biological conditions), a carboxylic acid group (which is in the ...
,
lysine Lysine (symbol Lys or K) is an α-amino acid that is a precursor to many proteins. It contains an α-amino group (which is in the protonated form under biological conditions), an α-carboxylic acid group (which is in the deprotonated − ...
and
arginine Arginine is the amino acid with the formula (H2N)(HN)CN(H)(CH2)3CH(NH2)CO2H. The molecule features a guanidino group appended to a standard amino acid framework. At physiological pH, the carboxylic acid is deprotonated (−CO2−) and both the am ...
at the P1 position.


In laryngopharyngeal reflux

Pepsin is one of the primary causes of mucosal damage during laryngopharyngeal reflux. Pepsin remains in the larynx (pH 6.8) following a gastric reflux event. While enzymatically inactive in this environment, pepsin would remain stable and could be reactivated upon subsequent acid reflux events. 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. 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. 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 Receptor-mediated endocytosis (RME), also called clathrin-mediated endocytosis, is a process by which cells absorb metabolites, hormones, proteins – and in some cases viruses – by the inward budding of the plasma membrane ( invagination). Th ...
. 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. Such exposure to pepsin at neutral pH and endocyctosis of pepsin causes changes in gene expression associated with inflammation, which underlies signs and symptoms of reflux, and tumor progression. This and other research implicates pepsin in carcinogenesis attributed to gastric reflux. Pepsin in airway specimens is considered to be a sensitive and specific marker for laryngopharyngeal reflux. 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.


Inhibitors

Pepsin may be inhibited by high pH (see Activity and stability) or by inhibitor compounds. Pepstatin is a low molecular weight compound and potently inhibitor specific for acid proteases with an inhibitory dissociation constant (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 In chemistry, the transition state of a chemical reaction is a particular configuration along the reaction coordinate. It is defined as the state corresponding to the highest potential energy along this reaction coordinate. It is often marked ...
for catalysis by pepsin and other acid proteases. Pepstatin does not covalently bind pepsin and inhibition of pepsin by pepstatin is therefore reversible. 1-bis(diazoacetyl)-2-phenylethane reversibly inactivates pepsin at pH 5, a reaction which is accelerated by the presence of Cu(II). Porcine pepsin is inhibited by pepsin inhibitor-3 (PI-3) produced by the large roundworm of pig ('' Ascaris suum''). PI-3 occupies the active site of pepsin using its N-terminal residues and thereby blocks substrate binding. Amino acid residues 1 - 3 (Gln-Phe-Leu) of mature PI-3 bind to P1' - P3' positions of pepsin. The N-terminus of PI-3 in the PI-3:pepsin complex is positioned by
hydrogen bond In chemistry, a hydrogen bond (or H-bond) is a primarily electrostatic force of attraction between a hydrogen (H) atom which is covalently bound to a more electronegative "donor" atom or group (Dn), and another electronegative atom bearing a l ...
s which form an eight-stranded
β-sheet The beta sheet, (β-sheet) (also β-pleated sheet) is a common motif of the regular protein secondary structure. Beta sheets consist of beta strands (β-strands) connected laterally by at least two or three backbone hydrogen bonds, forming a gen ...
, where three strands are contributed by pepsin and five by PI-3. A product of protein digestion by pepsin inhibits the reaction. Sucralfate, a drug used to treat stomach ulcers and other pepsin-related conditions, also inhibits pepsin activity.


Applications

Commercial pepsin is extracted from the glandular layer of hog stomachs. It is a component of
rennet Rennet () is a complex set of enzymes produced in the stomachs of ruminant mammals. Chymosin, its key component, is a protease enzyme that curdles the casein in milk. In addition to chymosin, rennet contains other enzymes, such as pepsin an ...
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, to modify vegetable proteins for use in nondairy snack items, to make precooked cereals into instant hot cereals, 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. Pepsin was historically an additive of Beeman's gum brand
chewing gum Chewing gum is a soft, cohesive substance designed to be chewed without being swallowed. Modern chewing gum is composed of gum base, sweeteners, softeners/ plasticizers, flavors, colors, and, typically, a hard or powdered polyol coating. Its t ...
by Dr. Edwin E. Beeman. Pepsin is commonly used in the preparation of
F(ab')2 fragment The fragment antigen-binding region (Fab region) is a region on an antibody that binds to antigens. It is composed of one constant and one variable domain of each of the heavy and the light chain. The variable domain contains the paratope (the ant ...
s from antibodies. In some assays, it is preferable to use only the antigen-binding (Fab) portion of the
antibody An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of t ...
. 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 Papain, also known as papaya proteinase I, is a cysteine protease () enzyme present in papaya (''Carica papaya'') and mountain papaya (''Vasconcellea cundinamarcensis''). It is the namesake member of the papain-like protease family. It has wide ...
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. 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.


Genes

The following three genes encode identical human pepsinogen A enzymes: A fourth human gene encodes gastricsin also known as pepsinogen C:


References


External links

* The
MEROPS MEROPS is an online database for peptidases (also known as proteases, proteinases and proteolytic enzymes) and their inhibitors. The classification scheme for peptidases was published by Rawlings & Barrett in 1993, and that for protein inhibitors ...
online database for peptidases and their inhibitors: Pepsin
A01.001
Pepsin
A01.002
Pepsin C (Gastricsin
A01.003
* * * *
Beemans Gum

Pepsin: Molecule of the Month
, by David Goodsell, RCSB Protein Data Bank * * {{Authority control EC 3.4.23 Proteases Stomach