Enteropeptidase

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enteropeptidase
1EKB.png
Crystal structure of Enteropeptidase with an inhibitor
Identifiers
EC no. 3.4.21.9
CAS no. 9014-74-8
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 / QuickGO
Search
PMC articles
PubMed articles
NCBI proteins
protease, serine, 7 (enteropeptidase)
Identifiers
SymbolTMPRSS15
NCBI gene 5651
HGNC 9490
OMIM 606635
RefSeq NM_002772
UniProt P98073
Other data
Locus Chr. 21 q21
Search for
Structures Swiss-model
Domains InterPro

Enteropeptidase (also called enterokinase) is an enzyme produced by cells of the duodenum and is involved in digestion in humans and other animals. Enteropeptidase converts trypsinogen (a zymogen) into its active form trypsin, resulting in the subsequent activation of pancreatic digestive enzymes. [1] [2] Absence of enteropeptidase results in intestinal digestion impairment. [3]

Contents

Enteropeptidase is a serine protease (EC 3.4.21.9) consisting of a disulfide-linked heavy-chain of 82-140 kDa that anchors enterokinase in the intestinal brush border membrane and a light-chain of 35–62 kDa that contains the catalytic subunit. [4] Enteropeptidase is a part of the chymotrypsin-clan of serine proteases, and is structurally similar to these proteins. [5]

Historical significance

Enteropeptidase was discovered by Ivan Pavlov, who was awarded the 1904 Nobel Prize in Physiology or Medicine for his studies of gastrointestinal physiology. It is the first known enzyme to activate other enzymes, and it remains a remarkable example of how serine proteases have been crafted to regulate metabolic pathways. [6] The inert function of digestive enzymes within the pancreas was known, as compared to their potent activity within the intestine, but the basis of this difference was unknown. In 1899, Pavlov's student, N. P. Schepowalnikov, demonstrated that canine duodenal secretions dramatically stimulated the digestive activity of pancreatic enzymes, especially trypsinogen. The active principle was recognized as a special enzyme in the intestine that could activate other enzymes. Pavlov named it enterokinase. The debate of whether enterokinase was a cofactor or enzyme was resolved by Kunitz, who showed that the activation of trypsinogen by enterokinase was catalytic. In the 1950s, cattle trypsinogen was shown to be activated autocatalytically by cleavage of an N-terminal hexapeptide. [7] The more precise IUBMB name enteropeptidase has been in existence since 1970. However, the original name ‘enterokinase’ has a long history and remains in common use. [8]

Enzyme structure

Enteropeptidase is a type II transmembrane serine protease (TTSP) localized to the brush border of the duodenal and jejunal mucosa and synthesized as a zymogen, proenteropeptidase, which requires activation by duodenase or trypsin. [9] TTSPs are synthesized as single chain zymogens with N-terminal propeptide sequences of different lengths. These enzymes are activated by cleavage at the carboxyl side of lysine or arginine residues present in a highly conserved activation motif. Once activated, TTSPs are predicted to remain membrane-bound through a conserved disulfide bond linking the pro- and catalytic domains. [10]

In the case of cattle enteropeptidase the primary translation product comprises 1035 residues with an expected mass of 114.9 kDa. [11] The detected apparent mass of about 160 kDa is consistent with the specified carbohydrate content of 30 - 40%, with equal amounts of neutral and amino sugars. [12] [13] The activation cleavage site after Lys800 splits the heavy and light chains of mature cattle enteropeptidase. There are 17 potential N-linked glycosylation sites in the heavy chain and three in the light chain; most of these are conserved in other species. The heavy chain has a hydrophobic section near the N-terminus that supports the transmembrane anchor. [14] [15] The heavy chain influences the specificity of enteropeptidase. Native enteropeptidase is resistant to soybean trypsin inhibitor. However, the isolated light chain is subtle whether prepared by limited reduction of the natural protein [16] or by mutagenesis and expression in COS cells. [17] Native enteropeptidase and the isolated light chain have similar activity toward Gly-(Asp)4-Lys-NHNap, but the secluded light chain has distinctly decreased activity toward trypsinogen . An analogous selective defect in the recognition of trypsinogen can be produced in two-chain enteropeptidase by heating or by acetylation. [18] This behavior implies that the catalytic center and one or more secondary substrate-binding sites are essential for optimal recognition of trypsinogen.

Human enteropeptidase - light chain Human enteropeptidase - light chain.png
Human enteropeptidase - light chain

Activity

Despite its alternative name (enterokinase), enteropeptidase is a serine protease that catalyses the hydrolysis of peptide bonds in proteins and, unlike other kinases, does not catalyze transfer of phosphate groups. Enteropeptidase exhibits trypsin-like activity, cleaving proteins following a lysine at a specific cleavage site (Asp-Asp-Asp-Asp-Lys). [19] This cleavage results in trypsindependent activation of other pancreatic zymogens, such as chymotrypsinogen, proelastase, procarboxypeptidase and prolipase in the lumen of the gut. [20] As the pro-region of trypsinogen contains this sequence, enteropeptidase catalyses its activation in vivo:

trypsinogen → trypsin + pro-region (Val-Asp-Asp-Asp-Asp-Lys)

Genetics and disease relevance

In humans, enteropeptidase is encoded by the TMPRSS15 gene (also known as ENTK, and previously as PRSS7) on chromosome 21q21. Some nonsense and frameshift mutations in this gene lead to a rare recessive disorder characterised by severe failure to thrive in affected infants, due to enteropeptidase deficiency. [21] Enteropeptidase mRNA expression is limited to the proximal small intestine, and the protein is found in enterocytes of duodenum and proximal jejunum. Upon secretion from the pancreas into the duodenum, trypsinogen encounters enteropeptidase and is activated. Trypsin then cleaves and activates other pancreatic serine protease zymogens (chymotrypsinogen and proelastases), metalloprotease zymogens (procarboxypeptidases) and prolipases. By means of this simple two-step cascade, the destructive activity of these digestive hydrolases is confined to the lumen of the intestine. The physiological importance of this pathway is demonstrated by the severe intestinal malabsorption caused by congenital deficiency of enteropeptidase. [22] [23] This condition can be life-threatening, but responds to oral supplementation with pancreatic extract.

Applications

Enteropeptidase's specificity makes it an ideal tool in biochemical applications; a fusion protein containing a C-terminal affinity tag (such as poly-His) linked by this sequence can be cleaved by enteropeptidase to obtain the target protein following protein purification. [19] On the converse, the N-terminal pro-sequence of proteases that must be cleaved prior to activation can be mutated to enable activation with enteropeptidase. [24]

Related Research Articles

<span class="mw-page-title-main">Chymotrypsin</span> Digestive enzyme

Chymotrypsin (EC 3.4.21.1, 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 duodenum, where it performs proteolysis, the breakdown of proteins and polypeptides. Chymotrypsin preferentially cleaves peptide amide bonds where the side chain of the amino acid N-terminal to the scissile amide bond (the P1 position) is a large hydrophobic amino acid (tyrosine, tryptophan, and phenylalanine). These amino acids contain an aromatic ring in their side chain that fits into a hydrophobic pocket (the S1 position) of the enzyme. It is activated in the presence of trypsin. The hydrophobic and shape complementarity between the peptide substrate P1 side chain and the enzyme S1 binding cavity accounts for the substrate specificity of this enzyme. Chymotrypsin also hydrolyzes other amide bonds in peptides at slower rates, particularly those containing leucine at the P1 position.

<span class="mw-page-title-main">Proteolysis</span> Breakdown of proteins into smaller polypeptides or amino acids

Proteolysis is the breakdown of proteins into smaller polypeptides or amino acids. Uncatalysed, the hydrolysis of peptide bonds is extremely slow, taking hundreds of years. Proteolysis is typically catalysed by cellular enzymes called proteases, but may also occur by intra-molecular digestion.

<span class="mw-page-title-main">Trypsin</span> Family of digestive enzymes

Trypsin is an enzyme in the first section of the small intestine that starts the digestion of protein molecules by cutting long chains of amino acids into smaller pieces. It is a serine protease from the PA clan superfamily, found in the digestive system of many vertebrates, where it hydrolyzes proteins. Trypsin is formed in the small intestine when its proenzyme form, the trypsinogen produced by the pancreas, is activated. Trypsin cuts peptide chains mainly at the carboxyl side of the amino acids lysine or arginine. It is used for numerous biotechnological processes. The process is commonly referred to as trypsinogen proteolysis or trypsinization, and proteins that have been digested/treated with trypsin are said to have been trypsinized. Trypsin was discovered in 1876 by Wilhelm Kühne and was named from the Ancient Greek word for rubbing since it was first isolated by rubbing the pancreas with glycerin.

<span class="mw-page-title-main">Protease</span> Enzyme that cleaves other proteins into smaller peptides

A protease is an enzyme that catalyzes proteolysis, breaking down proteins into smaller polypeptides or single amino acids, and spurring the formation of new protein products. They do this by cleaving the peptide bonds within proteins by hydrolysis, a reaction where water breaks bonds. Proteases are involved in numerous biological pathways, including digestion of ingested proteins, protein catabolism, and cell signaling.

In biochemistry, a zymogen, also called a proenzyme, is an inactive precursor of an enzyme. A zymogen requires a biochemical change for it to become an active enzyme. The biochemical change usually occurs in Golgi bodies, where a specific part of the precursor enzyme is cleaved in order to activate it. The inactivating piece which is cleaved off can be a peptide unit, or can be independently-folding domains comprising more than 100 residues. Although they limit the enzyme's ability, these N-terminal extensions of the enzyme or a “prosegment” often aid in the stabilization and folding of the enzyme they inhibit.

<span class="mw-page-title-main">Serine protease</span> Class of enzymes

Serine proteases are enzymes that cleave peptide bonds in proteins. Serine serves as the nucleophilic amino acid at the (enzyme's) active site. They are found ubiquitously in both eukaryotes and prokaryotes. Serine proteases fall into two broad categories based on their structure: chymotrypsin-like (trypsin-like) or subtilisin-like.

<span class="mw-page-title-main">Digestive enzyme</span> Class of enzymes

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 animals and in the tracts of carnivorous plants, where they aid in the digestion of food, as well as inside cells, especially in their lysosomes, where they function to maintain cellular survival. Digestive enzymes of diverse specificities are found in the saliva secreted by the salivary glands, in the secretions of cells lining the stomach, in the pancreatic juice secreted by pancreatic exocrine cells, and in the secretions of cells lining the small and large intestines.

Trypsinogen is the precursor form of trypsin, a digestive enzyme. It is produced by the pancreas and found in pancreatic juice, along with amylase, lipase, and chymotrypsinogen. It is cleaved to its active form, trypsin, by enteropeptidase, which is found in the intestinal mucosa. Once activated, the trypsin can cleave more trypsinogen into trypsin, a process called autoactivation. Trypsin cleaves the peptide bond on the carboxyl side of basic amino acids such as arginine and lysine.

<span class="mw-page-title-main">Acrosin</span> Mammalian protein found in Homo sapiens

Acrosin is a digestive enzyme that acts as a protease. In humans, acrosin is encoded by the ACR gene. Acrosin is released from the acrosome of spermatozoa as a consequence of the acrosome reaction. It aids in the penetration of the Zona Pellucida.

<span class="mw-page-title-main">Catalytic triad</span> Set of three coordinated amino acids

A catalytic triad is a set of three coordinated amino acids that can be found in the active site of some enzymes. Catalytic triads are most commonly found in hydrolase and transferase enzymes. An acid-base-nucleophile triad is a common motif for generating a nucleophilic residue for covalent catalysis. The residues form a charge-relay network to polarise and activate the nucleophile, which attacks the substrate, forming a covalent intermediate which is then hydrolysed to release the product and regenerate free enzyme. The nucleophile is most commonly a serine or cysteine amino acid, but occasionally threonine or even selenocysteine. The 3D structure of the enzyme brings together the triad residues in a precise orientation, even though they may be far apart in the sequence.

The prothrombinase enzyme complex consists of factor Xa (a serine protease) and factor Va (a protein cofactor). The complex assembles on negatively charged phospholipid membranes in the presence of calcium ions. The prothrombinase complex catalyzes the conversion of prothrombin (factor II), an inactive zymogen, to thrombin (factor IIa), an active serine protease. The activation of thrombin is a critical reaction in the coagulation cascade, which functions to regulate hemostasis in the body. To produce thrombin, the prothrombinase complex cleaves two peptide bonds in prothrombin, one after Arg271 and the other after Arg320. Although it has been shown that factor Xa can activate prothrombin when unassociated with the prothrombinase complex, the rate of thrombin formation is severely decreased under such circumstances. The prothrombinase complex can catalyze the activation of prothrombin at a rate 3 x 105-fold faster than can factor Xa alone. Thus, the prothrombinase complex is required for the efficient production of activated thrombin and also for adequate hemostasis.

<span class="mw-page-title-main">Cysteine protease</span> Class of enzymes

Cysteine proteases, also known as thiol proteases, are hydrolase enzymes that degrade proteins. These proteases share a common catalytic mechanism that involves a nucleophilic cysteine thiol in a catalytic triad or dyad.

A trypsin inhibitor (TI) is a protein and a type of serine protease inhibitor (serpin) that reduces the biological activity of trypsin by controlling the activation and catalytic reactions of proteins. Trypsin is an enzyme involved in the breakdown of many different proteins, primarily as part of digestion in humans and other animals such as monogastrics and young ruminants. Serpins – including trypsin inhibitors – are irreversible and suicide substrate-like inhibitors.

<span class="mw-page-title-main">Aprotinin</span> Antifibrinolytic molecule

The drug aprotinin, is a small protein bovine pancreatic trypsin inhibitor (BPTI), or basic trypsin inhibitor of bovine pancreas, which is an antifibrinolytic molecule that inhibits trypsin and related proteolytic enzymes. Under the trade name Trasylol, aprotinin was used as a medication administered by injection to reduce bleeding during complex surgery, such as heart and liver surgery. Its main effect is the slowing down of fibrinolysis, the process that leads to the breakdown of blood clots. The aim in its use was to decrease the need for blood transfusions during surgery, as well as end-organ damage due to hypotension as a result of marked blood loss. The drug was temporarily withdrawn worldwide in 2007 after studies suggested that its use increased the risk of complications or death; this was confirmed by follow-up studies. Trasylol sales were suspended in May 2008, except for very restricted research use. In February 2012 the European Medicines Agency (EMA) scientific committee reverted its previous standpoint regarding aprotinin, and has recommended that the suspension be lifted. Nordic became distributor of aprotinin in 2012.

<span class="mw-page-title-main">Plasma kallikrein</span>

Plasma kallikrein is an enzyme that catalyses the following chemical reaction:

Pancreatic elastase is a form of elastase that is produced in the acinar cells of the pancreas, initially produced as an inactive zymogen and later activated in the duodenum by trypsin. Elastases form a subfamily of serine proteases, characterized by a distinctive structure consisting of two beta barrel domains converging at the active site that hydrolyze amides and esters amongst many proteins in addition to elastin, a type of connective tissue that holds organs together. Pancreatic elastase 1 is a serine endopeptidase, a specific type of protease that has the amino acid serine at its active site. Although the recommended name is pancreatic elastase, it can also be referred to as elastase-1, pancreatopeptidase, PE, or serine elastase.

<span class="mw-page-title-main">TEV protease</span> Highly specific protease

TEV protease is a highly sequence-specific cysteine protease from Tobacco Etch Virus (TEV). It is a member of the PA clan of chymotrypsin-like proteases. Due to its high sequence specificity, TEV protease is frequently used for the controlled cleavage of fusion proteins in vitro and in vivo.

<span class="mw-page-title-main">Trypsin 1</span> Protein-coding gene in the species Homo sapiens

Trypsin-1, also known as cationic trypsinogen, is a protein that in humans is encoded by the PRSS1 gene. Trypsin-1 is the main isoform of trypsinogen secreted by pancreas, the others are trypsin-2, and trypsin-3 (meso-trypsinogen).

<span class="mw-page-title-main">TMPRSS11D</span> Protein-coding gene in the species Homo sapiens

Transmembrane protease, serine 11D is an enzyme that in humans is encoded by the TMPRSS11D gene.

<span class="mw-page-title-main">Kazal domain</span>

The Kazal domain is an evolutionary conserved protein domain usually indicative of serine protease inhibitors. However, kazal-like domains are also seen in the extracellular part of agrins, which are not known to be protease inhibitors.

References

  1. ↑ Kunitz M (March 1939). "Formation of trypsin from crystalline trypsinogen by means of enterokinase". J. Gen. Physiol. 22 (4): 429–46. doi:10.1085/jgp.22.4.429. PMC   2141988 . PMID   19873112.
  2. ↑ Kiel B (1971). "Trypsin". In Boyer PS (ed.). The Enzymes, 3: Hydrolysis - Peptide Bonds. Amsterdam: Elsevier. pp. 249–75. ISBN   978-0-12-122703-6.
  3. ↑ Light A, Janska H (March 14, 1989). "Enterokinase (enteropeptidase) : comparative aspects". Trends Biochem. Sci. 14 (3): 110–2. doi:10.1016/0968-0004(89)90133-3. PMID   2658218.
  4. ↑ Huang L, Ruan H, Gu W, Xu Z, Cen P, Fan L (2007). "Functional expression and purification of bovine enterokinase light chain in recombinant Escherichia coli". Prep. Biochem. Biotechnol. 37 (3): 205–17. doi:10.1080/10826060701386695. PMID   17516250. S2CID   25387669.
  5. ↑ Rawlings ND, Barrett AJ (February 1993). "Evolutionary families of peptidases". Biochem. J. 290 (1): 205–18. doi:10.1042/bj2900205. PMC   1132403 . PMID   8439290.
  6. ↑ Lu D, Fütterer K, Korolev S, Zheng X, Tan K, Waksman G, Sadler JE (Sep 17, 1999). "Crystal structure of enteropeptidase light chain complex with an analog of the trypsinogen activation peptide". J Mol Biol. 292 (2): 361–73. doi:10.1006/jmbi.1999.3089. PMID   10493881.
  7. ↑ Yamashina I. (May 1956). "The action of enterokinase on trypsinogen" (PDF). Biochim Biophys Acta. 20 (2): 433–4. doi:10.1016/0006-3002(56)90329-8. PMID   13328891.
  8. ↑ Rawlings ND, Salvesen G (2013). Handbook of Proteolytic Enzymes (3rd ed.). Academic Press. ISBN   978-0-12-382219-2 . Retrieved February 20, 2014.
  9. ↑ Zamolodchikova TS, Sokolova EA, Lu D, Sadler JE (Jan 28, 2000). "Activation of recombinant proenteropeptidase by duodenase". FEBS Lett. 466 (2–3): 295–9. doi: 10.1016/s0014-5793(00)01092-9 . PMID   10682847. S2CID   254189.
  10. ↑ Hooper JD, Clements JA, Quiqley JP, Antalis TM (Jan 12, 2001). "Type II transmembrane serine proteases. Insights into an emerging class of cell surface proteolytic enzymes". J Biol Chem. 276 (2): 857–60. doi: 10.1074/jbc.r000020200 . PMID   11060317.
  11. ↑ Kitamoto Y, Yuan X, Wu Q, McCourt DW, Sadler JE (August 2, 1994). "Enterokinase, the initiator of intestinal digestion, is a mosaic protease composed of a distinctive assortment of domains". Proc Natl Acad Sci USA. 91 (16): 7588–92. Bibcode:1994PNAS...91.7588K. doi: 10.1073/pnas.91.16.7588 . PMC   44447 . PMID   8052624.
  12. ↑ Anderson LE, Walsh KA, Neurath H (July 26, 1977). "Bovine enterokinase. Purification, specificity and some molecular properties". Biochemistry. 16 (15): 3354–60. doi:10.1021/bi00634a011. PMID   889800.
  13. ↑ Liepnieks JJ, Light A (March 10, 1979). "The preparation and properties of bovine enterokinase". J Biol Chem. 254 (5): 1677–83. doi: 10.1016/S0021-9258(17)37826-2 . PMID   762166.
  14. ↑ Fonseca P, Light A (March 10, 1983). "Incorporation of bovine enterokinase in reconstituted soybean phospholipid vesicles". J Biol Chem. 258 (5): 3069–74. doi: 10.1016/S0021-9258(18)32831-X . PMID   6338012.
  15. ↑ Lu D, Yuan X, Zheng X, Sadler JE (December 12, 1997). "Bovine proenteroptidase is activated by trypsin, and the specificity of enteropeptidase depends on the heavy chain". J Biol Chem. 272 (50): 31293–300. doi: 10.1074/jbc.272.50.31293 . PMID   9395456.
  16. ↑ Light A, Fonseca P (November 10, 1984). "The preparation and properties of the catalytic subunit of bovine enterokinase". J Biol Chem. 259 (21): 13195–8. doi: 10.1016/S0021-9258(18)90676-9 . PMID   6386810.
  17. ↑ LaVallie ER, Rehemtulla A, Racie LA, DiBlasio EA, Ferenz C, Grant KL, Light A, McCoy JM (November 5, 1993). "Cloning and functional expression of a cDNA encoding the catalytic subunit of bovine enterokinase". J Biol Chem. 268 (31): 23311–7. doi: 10.1016/S0021-9258(19)49464-7 . PMID   8226855.
  18. ↑ Baratti J, Maroux S (December 8, 1976). "On the catalytic and binding sites of porcine enteropeptidase". Biochim Biophys Acta. 452 (2): 488–96. doi:10.1016/0005-2744(76)90199-6. PMID   12810.
  19. 1 2 Terpe K (2003). "Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems" (PDF). Appl Microbiol Biotechnol. 60 (5): 523–33. doi:10.1007/s00253-002-1158-6. PMID   12536251. S2CID   206934268.
  20. ↑ Kunitz M, Northrop JH (Jul 20, 1936). "Isolation from beef pancreas of crystalline trypsinogen, trypsin, a trypsin inhibitor, and an inhibitor-trypsin compound". J Gen Physiol. 19 (6): 991–1007. doi:10.1085/jgp.19.6.991. PMC   2141477 . PMID   19872978.
  21. ↑ Holzinger A, Maier EM, Bück C, Mayerhofer PU, Kappler M, Haworth JC, Moroz SP, Hadorn HB, Sadler JE, Roscher AA (January 2002). "Mutations in the proenteropeptidase gene are the molecular cause of congenital enteropeptidase deficiency". Am. J. Hum. Genet. 70 (1): 20–5. doi:10.1086/338456. PMC   384888 . PMID   11719902.
  22. ↑ Hadorn B, Tarlow MJ, Lloyd JK, Wolff OH (April 19, 1969). "Intestinal enterokinase deficiency". Lancet. 1 (7599): 812–3. doi:10.1016/s0140-6736(69)92071-6. PMID   4180366.
  23. ↑ Haworth JC, Gourley B, Hadorn B, Sumida C (March 1971). "Malabsorption and growth failure due to intestinal enterokinase deficiency". J. Pediatr. 78 (3): 481–90. doi:10.1016/s0022-3476(71)80231-7. PMID   4322674.
  24. ↑ Wang ZM, Rubin H, Schechter NM (Nov 1995). "Production of active recombinant human chymase from a construct containing the enterokinase cleavage site of trypsinogen in place of the native propeptide sequence". Biol Chem Hoppe-Seyler. 376 (11): 681–84. doi:10.1515/bchm3.1995.376.11.681. PMID   8962677.
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