Trypsin

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Trypsin
ExPASy
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KEGGKEGG entry
MetaCycmetabolic pathway
PRIAMprofile
PDB structuresRCSB PDB PDBe PDBsum
Gene OntologyAmiGO / QuickGO
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PMCarticles
PubMedarticles
NCBIproteins
Trypsin
Identifiers
SymbolTrypsin
SCOP2
1c2g / SCOPe / SUPFAM
CDDcd00190
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary

Trypsin is an

glycerin.[5]

Function

In the

peptide bonds, breaking down proteins into smaller peptides. The peptide products are then further hydrolyzed into amino acids via other proteases, rendering them available for absorption into the blood stream. Tryptic digestion is a necessary step in protein absorption, as proteins are generally too large to be absorbed through the lining of the small intestine.[6]

Trypsin is produced as the inactive

proteolytic cleavage. The trypsin then activates additional trypsin, chymotrypsin and carboxypeptidase
.

Mechanism

The enzymatic mechanism is similar to that of other serine proteases. These enzymes contain a

aspartate-102, and serine-195.[7] This catalytic triad was formerly called a charge relay system, implying the abstraction of protons from serine to histidine and from histidine to aspartate, but owing to evidence provided by NMR that the resultant alkoxide form of serine would have a much stronger pull on the proton than does the imidazole ring of histidine, current thinking holds instead that serine and histidine each have effectively equal share of the proton, forming short low-barrier hydrogen bonds therewith.[8][page needed] By these means, the nucleophilicity of the active site serine is increased, facilitating its attack on the amide carbon during proteolysis. The enzymatic reaction that trypsin catalyzes is thermodynamically favorable, but requires significant activation energy (it is "kinetically
unfavorable"). In addition, trypsin contains an "oxyanion hole" formed by the backbone amide hydrogen atoms of Gly-193 and Ser-195, which through hydrogen bonding stabilize the negative charge which accumulates on the amide oxygen after nucleophilic attack on the planar amide carbon by the serine oxygen causes that carbon to assume a tetrahedral geometry. Such stabilization of this tetrahedral intermediate helps to reduce the energy barrier of its formation and is concomitant with a lowering of the free energy of the transition state. Preferential binding of the transition state is a key feature of enzyme chemistry.

The negative aspartate residue (Asp 189) located in the catalytic pocket (S1) of trypsin is responsible for attracting and stabilizing positively charged lysine and/or arginine, and is, thus, responsible for the specificity of the enzyme. This means that trypsin predominantly cleaves

C-terminal side") of the amino acids lysine and arginine except when either is bound to a C-terminal proline,[9] although large-scale mass spectrometry data suggest cleavage occurs even with proline.[10] Trypsin is considered an endopeptidase, i.e., the cleavage occurs within the polypeptide chain rather than at the terminal amino acids located at the ends of polypeptides
.

Properties

Human trypsin has an optimal operating temperature of about 37 °C.[11] In contrast, the Atlantic cod has several types of trypsins for the poikilotherm fish to survive at different body temperatures. Cod trypsins include trypsin I with an activity range of 4 to 65 °C (40 to 150 °F) and maximal activity at 55 °C (130 °F), as well as trypsin Y with a range of 2 to 30 °C (36 to 86 °F) and a maximal activity at 21 °C (70 °F).[12]

As a protein, trypsin has various molecular weights depending on the source. For example, a molecular weight of 23.3 kDa is reported for trypsin from bovine and porcine sources.

The activity of trypsin is not affected by the enzyme inhibitor tosyl phenylalanyl chloromethyl ketone, TPCK, which deactivates chymotrypsin.

Trypsin should be stored at very cold temperatures (between −20 and −80 °C) to prevent autolysis, which may also be impeded by storage of trypsin at pH 3 or by using trypsin modified by reductive methylation. When the pH is adjusted back to pH 8, activity returns.

Isozymes

These human genes encode proteins with trypsin enzymatic activity:

Chr. 7 q32-qter
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StructuresSwiss-model
DomainsInterPro
Chr. 7 q35
Search for
StructuresSwiss-model
DomainsInterPro
Chr. 9 p13
Search for
StructuresSwiss-model
DomainsInterPro

Other

isoforms
of trypsin may also be found in other organisms.

Clinical significance

Activation of trypsin from proteolytic cleavage of trypsinogen in the pancreas can lead to a series of events that cause pancreatic self-digestion, resulting in

meconium ileus, which involves intestinal obstruction (ileus) due to overly thick meconium, which is normally broken down by trypsin and other proteases, then passed in feces.[13]

Applications

Trypsin is available in high quantity in pancreases, and can be purified rather easily. Hence, it has been used widely in various biotechnological processes.

In a tissue culture lab, trypsin is used to resuspend cells adherent to the cell culture dish wall during the process of harvesting cells.[14] Some cell types adhere to the sides and bottom of a dish when cultivated in vitro. Trypsin is used to cleave proteins holding the cultured cells to the dish, so that the cells can be removed from the plates.

Trypsin can also be used to dissociate dissected cells (for example, prior to cell fixing and sorting).

Trypsin can be used to break down

translucent
. The rate of reaction can be measured by using the amount of time needed for the milk to turn translucent.

Trypsin is commonly used in biological research during proteomics experiments to digest proteins into peptides for mass spectrometry analysis, e.g. in-gel digestion. Trypsin is particularly suited for this, since it has a very well defined specificity, as it hydrolyzes only the peptide bonds in which the carbonyl group is contributed either by an arginine or lysine residue.

Trypsin can also be used to dissolve blood clots in its microbial form and treat inflammation in its pancreatic form.

In veterinary medicine, trypsin is an ingredient in wound spray products, such as Debrisol, to dissolve dead tissue and pus in wounds in horses, cattle, dogs, and cats.[15]

In food

Commercial protease preparations usually consist of a mixture of various protease enzymes that often includes trypsin. These preparations are widely used in food processing:[16]

  • as a baking enzyme to improve the workability of dough
  • in the extraction of seasonings and flavorings from vegetable or animal proteins and in the manufacture of sauces
  • to control aroma formation in cheese and milk products
  • to improve the texture of fish products
  • to tenderize meat
  • during cold stabilization of beer
  • in the production of hypoallergenic food where proteases break down specific allergenic proteins into nonallergenic peptides, for example, proteases are used to produce hypoallergenic baby food from cow's milk, thereby diminishing the risk of babies developing milk allergies.

Trypsin inhibitor

Main article: Trypsin inhibitor

To prevent the action of active trypsin in the pancreas, which can be highly damaging, inhibitors such as

α1-antitrypsin in the serum are present as part of the defense against its inappropriate activation. Any trypsin prematurely formed from the inactive trypsinogen is then bound by the inhibitor. The protein-protein interaction between trypsin and its inhibitors is one of the tightest bound, and trypsin is bound by some of its pancreatic inhibitors nearly irreversibly.[17] In contrast with nearly all known protein assemblies, some complexes of trypsin bound by its inhibitors do not readily dissociate after treatment with 8M urea.[18]

Trypsin inhibitors can serve as tools when addressing metabolic and obesity disorders. Metabolic disorders, obesity, and being overweight are known to increase non-communicable chronic disease prevalence.[19] It is of public health policy interest to explore various ways to mitigate this occurrence including use of trypsin inhibitors. These inhibitors have capabilities of reducing colon, breast, skin, nad prostate cancer by way of radioprotective and anticarcinogenic activity. Trypsin inhibitors can act as regulatory mechanisms to control release of neutrophil proteases and avoid significant tissue damage.[19] In regards to cardiovascular conditions associated with unproductive serine protease activity, trypsin inhibitors can block their activity in platelet aggregation, fibrinolysis, coagulation, and blood coagulation.

The multifunctionality of trypsin inhibitors includes being potential protease inhibitors for AMP activity.[20] While the antibacterial action mechanisms of trypsin inhibitors are unclear, studies have aimed to study their mechanisms as potential applications in bacterial infection treatments.[20] Research and scanning microscopy showed antibacterial effects on bacterial membranes from Staphylococcus aureus.[20] Trypsin inhibitors from amphibian skin showed bacterial death promotion that affected Staphylococcus aureus' cell wall and membrane.[20] Studies also analyzed antibacterial actions in trypsin inhibitor peptides, proteins, and E. coli. The results showed sufficient bacterial growth prevention. However, trypsin inhibitors have to meet certain criteria to be utilized in foods and medical treatments.[20]

Trypsin alternatives

Main article: Trypsin inhibitor

Trypsin digestion of extra cellular matrix is a common practice in cell culture. However this enzymatic degradation of the cells can negatively effect cell viability and surface markers, especially in stem cells. There are gentler alternatives than trypsin such as Accutase which doesn't effect surface markers such as cd14, cd117, cd49f, cd292.[21][22] However Accutase decreases the surface levels of FasL and Fas receptor on macrophages, these receptors are associated with cell cytotoxicity in the immune system and can also facilitate apoptosis-related cell death.[23]

ProAlanase could also serve as an alternative to Trypsin in proteomic applications.[24] ProAlanase is an Aspergillus niger fungus protease that can achieve high proteolytic activity and specificity for digestion under the correct conditions.[24]ProAnalase, the acidic prolyl-endopeptidase protease, previously studied as An-PEP, has been observed in various experiments to define its specificity.[24] ProAnalase performed optimally in LC-MS applications with short digestion times and highly acidic pH.[24]

See also

References

  1. PMID 15044735
    .
  2. PMID 7845208
    .
  3. ^ The German physiologist Wilhelm Kühne (1837-1900) discovered trypsin in 1876. See: Kühne W (1877). "Über das Trypsin (Enzym des Pankreas)". Verhandlungen des Naturhistorisch-medicinischen Vereins zu Heidelberg. new series. 1 (3): 194–198 – via Google Books.Open access icon
  4. ISBN 978-0-12-391909-0
    .
  5. ^ Kühne W (March 6, 1876). "Ueber das Trypsin (Enzym des Pankreas)" [About trypsin (enzyme of the pancreas)]. In Naturhistorisch-medizinischen Verein (ed.). Verhandlungen des Naturhistorisch-medizinischen Vereins zu Heidelberg [Negotiations by the Natural History Medical Association in Heidelberg] (in German). Heidelberg, Germany: Carl Winter's Universitätsbuchhandlung (published 1877). pp. 194–8 – via Archive.org.
  6. ^ "Digestion of Proteins". Elective course (Clinical biochemistry). Ternopil National Medical University. July 14, 2015. Archived from the original on August 8, 2020. Retrieved April 11, 2020.
  7. S2CID 3343824
    .
  8. OCLC 690489261
    .
  9. ^ "Sequencing Grade Modified Trypsin" (PDF). promega.com. 2007-04-01. Archived from the original (PDF) on 2003-05-19. Retrieved 2009-02-08.
  10. PMID 18067249. Archived from the original
    (PDF) on 2020-08-13. Retrieved 2017-10-25.
  11. PMID 21319805
    .
  12. S2CID 42480996
    .
  13. PMID 11729110
    .
  14. ^ "Trypsin-EDTA (0.25%)". Stem Cell Technologies. Retrieved 2012-02-23.
  15. ^ "Debrisol". drugs.com.
  16. ^ "Protease - GMO Database". GMO Compass. European Union. 2010-07-10. Archived from the original on 2015-02-24. Retrieved 2012-01-01.
  17. ISBN 978-0-471-58651-7
    .
  18. PMID 5528741
    .
  19. ^
    PMID 30734596
    .
  20. ^
    PMID 35168466
    .
  21. PMID 23481571
    .
  22. PMID 30983404
    .
  23. PMID 35383242
    .
  24. ^
    PMID 33020190
    .

Further reading

External links
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