Summary: Trypsin
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Trypsin Edit Wikipedia article
| trypsin | |||||||||
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| Identifiers | |||||||||
| EC number | 3.4.21.4 | ||||||||
| CAS number | 9002-07-7 | ||||||||
| 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 | ||||||||
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| Trypsin | |||||||||
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| Identifiers | |||||||||
| Symbol | Trypsin | ||||||||
| Pfam | PF00089 | ||||||||
| InterPro | IPR001254 | ||||||||
| SMART | SM00020 | ||||||||
| PROSITE | PDOC00124 | ||||||||
| MEROPS | S1 | ||||||||
| SCOP | 1c2g | ||||||||
| SUPERFAMILY | 1c2g | ||||||||
| CDD | cd00190 | ||||||||
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Trypsin (EC 3.4.21.4) is a serine protease from the PA clan superfamily, found in the digestive system of many vertebrates, where it hydrolyzes proteins.[2][3] Trypsin is formed in the small intestine when its proenzyme form, the trypsinogen produced by the pancreas, is activated. Trypsin cleaves peptide chains mainly at the carboxyl side of the amino acids lysine or arginine, except when either is followed by proline. It is used for numerous biotechnological processes. The process is commonly referred to as trypsin proteolysis or trypsinisation, and proteins that have been digested/treated with trypsin are said to have been trypsinized.[4] Trypsin was discovered in 1876 by Wilhelm Kühne.[5]
Contents
Function
In the duodenum, trypsin catalyzes the hydrolysis of 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 zymogen trypsinogen in the pancreas. When the pancreas is stimulated by cholecystokinin, it is then secreted into the first part of the small intestine (the duodenum) via the pancreatic duct. Once in the small intestine, the enzyme enteropeptidase activates trypsinogen into trypsin by proteolytic cleavage. Autocatalysis does not happen with trypsin, as trypsinogen is a poor substrate, therefore enzymatic damage to the pancreas is avoided.[6]
Mechanism
The enzymatic mechanism is similar to that of other serine proteases. These enzymes contain a catalytic triad consisting of histidine-57, 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] 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 stabilisation 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 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 proteins at the carboxyl side (or "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. This is important because, in some applications, like mass spectrometry, the specificity of cleavage is important.
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:
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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 pancreatitis. One consequence of the autosomal recessive disease cystic fibrosis is a deficiency in transport of trypsin and other digestive enzymes from the pancreas. This leads to the disorder termed 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 casein in breast milk. If trypsin is added to a solution of milk powder, the breakdown of casein causes the milk to become 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 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:[15]
- 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
To prevent the action of active trypsin in the pancreas, which can be highly damaging, inhibitors such as BPTI and SPINK1 in the pancreas and 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.[16] 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.[17]
References
- ^ PDB: 1UTN; Leiros HK, Brandsdal BO, Andersen OA, Os V, Leiros I, Helland R, Otlewski J, Willassen NP, Smalås AO (April 2004). "Trypsin specificity as elucidated by LIE calculations, X-ray structures, and association constant measurements". Protein Science. 13 (4): 1056–70. doi:10.1110/ps.03498604. PMC 2280040
. PMID 15044735. - ^ Rawlings ND, Barrett AJ (1994). "Families of serine peptidases". Methods in Enzymology. Methods in Enzymology. 244: 19–61. doi:10.1016/0076-6879(94)44004-2. ISBN 978-0-12-182145-6. PMID 7845208.
- ^ The German physiologist Wilhelm Kühne (1837-1900) discovered trypsin in 1876. See: W. Kühne (1877) "Über das Trypsin (Enzym des Pankreas)", Verhandlungen des naturhistorisch-medicinischen Vereins zu Heidelberg, new series, vol. 1, no. 3, pages 194-198.
- ^ Engelking, Larry R. (2015-01-01). Textbook of Veterinary Physiological Chemistry (Third Edition). Boston: Academic Press. pp. 39–44. ISBN 9780123919090.
- ^ "Verhandlungen des Naturhistorisch-medizinischen Vereins zu Heidelberg". archive.org. Retrieved 2017-04-24.
- ^ a b "Digestion of Proteins". intranet.tdmu.edu.ua. Retrieved 2017-04-24.
- ^ Polgár L (October 2005). "The catalytic triad of serine peptidases". Cellular and Molecular Life Sciences. 62 (19-20): 2161–72. doi:10.1007/s00018-005-5160-x. PMID 16003488.
- ^ Voet D, Voet JG (2011). Biochemistry (4th ed.). Hoboken, NJ: John Wiley & Sons. ISBN 9780470570951. OCLC 690489261.
- ^ "Sequencing Grade Modified Trypsin" (PDF). www.promega.com. 2007-04-01. Retrieved 2009-02-08.
- ^ Rodriguez J, Gupta N, Smith RD, Pevzner PA (January 2008). "Does trypsin cut before proline?" (PDF). Journal of Proteome Research. 7 (1): 300–5. doi:10.1021/pr0705035. PMID 18067249.
- ^ Hustoft HK, Malerod H, Wilson SR, Reubsaet L, Lundanes E, Greibrokk T. "A Critical Review of Trypsin Digestion for LC-MS Based Proteomics" (PDF). Norway: University of Oslo. p. 80.
- ^ Gudmundsdóttir A, Pálsdóttir HM (2005). "Atlantic cod trypsins: from basic research to practical applications". Marine Biotechnology. 7 (2): 77–88. doi:10.1007/s10126-004-0061-9. PMID 15759084.
- ^ Noone PG, Zhou Z, Silverman LM, Jowell PS, Knowles MR, Cohn JA (December 2001). "Cystic fibrosis gene mutations and pancreatitis risk: relation to epithelial ion transport and trypsin inhibitor gene mutations". Gastroenterology. 121 (6): 1310–9. doi:10.1053/gast.2001.29673. PMID 11729110.
- ^ "Trypsin-EDTA (0.25%)". Stem Cell Technologies. Retrieved 2012-02-23.
- ^ "Protease - GMO Database". GMO Compass. European Union. 2010-07-10. Retrieved 2012-01-01.
- ^ Voet D, Voet JG (1995). Biochemistry (2nd ed.). John Wiley & Sons. pp. 396–400. ISBN 978-0-471-58651-7.
- ^ Levilliers N, Péron M, Arrio B, Pudles J (October 1970). "On the mechanism of action of proteolytic inhibitors. IV. Effect of 8 M urea on the stability of trypsin in trypsin-inhibitor complexes". Archives of Biochemistry and Biophysics. 140 (2): 474–83. doi:10.1016/0003-9861(70)90091-3. PMID 5528741.
External links
- The MEROPS online database for peptidases and their inhibitors: Trypsin 1 S01.151, Trypsin 2 S01.258, Trypsin 3 S01.174
- Trypsin Inhibitors and Trypsin Assay Method at Sigma-Aldrich
- Trypsin at the US National Library of Medicine Medical Subject Headings (MeSH)
This page is based on a Wikipedia article. The text is available under the Creative Commons Attribution/Share-Alike License.
This tab holds the annotation information that is stored in the Pfam database. As we move to using Wikipedia as our main source of annotation, the contents of this tab will be gradually replaced by the Wikipedia tab.
Trypsin Provide feedback
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Literature references
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Rawlings ND, Barrett AJ; , Meth Enzymol 1994;244:19-61.: Families of Serine Peptidases PUBMED:7845208 EPMC:7845208
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Sprang S, Standing T, Fletterick RJ, Stroud RM, Finer-Moore J, Xuong NH, Hamlin R, Rutter WJ, Craik CS; , Science 1987;237:905-909.: The Three Dimensional Structure of Asnl02 Trypsin: Role of Aspl02 in Serine Protease Catalysis PUBMED:3112942 EPMC:3112942
Internal database links
| SCOOP: | DUF1986 DUF316 Peptidase_S29 Peptidase_S32 Peptidase_S46 Peptidase_S7 Trypsin_2 |
| Similarity to PfamA using HHSearch: | DUF316 DUF1986 Trypsin_2 |
External database links
| HOMSTRAD: | serbact sermam |
| PROSITE: | PDOC00124 |
| SCOP: | 1mct |
This tab holds annotation information from the InterPro database.
InterPro entry IPR001254
This entry represents the active-site-containing domain found in the trypsin family members. The catalytic activity of the serine proteases from the trypsin family is provided by a charge relay system involving an aspartic acid residue hydrogen-bonded to a histidine, which itself is hydrogen-bonded to a serine. The sequences in the vicinity of the active site serine and histidine residues are well conserved in this family of proteases [PUBMED:3136396]. A partial list of proteases known to belong to the trypsin family is shown below.
- Acrosin.
- Blood coagulation factors VII, IX, X, XI and XII, thrombin, plasminogen, and protein C.
- Cathepsin G.
- Chymotrypsins.
- Complement components C1r, C1s, C2, and complement factors B, D and I.
- Complement-activating component of RA-reactive factor.
- Cytotoxic cell proteases (granzymes A to H).
- Duodenase I.
- Elastases 1, 2, 3A, 3B (protease E), leukocyte (medullasin).
- Enterokinase (EC 3.4.21.9) (enteropeptidase).
- Hepatocyte growth factor activator.
- Hepsin.
- Glandular (tissue) kallikreins (including EGF-binding protein types A, B, and C, NGF-gamma chain, gamma-renin, prostate specific antigen (PSA) and tonin).
- Plasma kallikrein.
- Mast cell proteases (MCP) 1 (chymase) to 8.
- Myeloblastin (proteinase 3) (Wegener's autoantigen).
- Plasminogen activators (urokinase-type, and tissue-type).
- Trypsins I, II, III, and IV.
- Tryptases.
All the above proteins belong to family S1 in the classification of peptidases [PUBMED:7845208] and originate from eukaryotic species. It should be noted that bacterial proteases that belong to family S2A are similar enough in the regions of the active site residues that they can be picked up by the same patterns. These proteases are listed below.
- Achromobacter lyticus protease I.
- Lysobacter alpha-lytic protease.
- Streptogrisin A and B (Streptomyces proteases A and B).
- Streptomyces griseus glutamyl endopeptidase II.
- Streptomyces fradiae proteases 1 and 2.
Gene Ontology
The mapping between Pfam and Gene Ontology is provided by InterPro. If you use this data please cite InterPro.
| Molecular function | serine-type endopeptidase activity (GO:0004252) |
| Biological process | proteolysis (GO:0006508) |
Domain organisation
Below is a listing of the unique domain organisations or architectures in which this domain is found. More...
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Pfam Clan
This family is a member of clan Peptidase_PA (CL0124), which has the following description:
This clan contains a diverse set of peptidases with the trypsin fold.
The clan contains the following 26 members:
DUF1986 DUF31 DUF316 Peptidase_C107 Peptidase_C24 Peptidase_C3 Peptidase_C30 Peptidase_C37 Peptidase_C3G Peptidase_C4 Peptidase_C62 Peptidase_S29 Peptidase_S3 Peptidase_S30 Peptidase_S31 Peptidase_S32 Peptidase_S39 Peptidase_S46 Peptidase_S55 Peptidase_S6 Peptidase_S64 Peptidase_S7 Peptidase_S76 Pico_P2A Trypsin Trypsin_2Alignments
We store a range of different sequence alignments for families. As well as the seed alignment from which the family is built, we provide the full alignment, generated by searching the sequence database (reference proteomes) using the family HMM. We also generate alignments using four representative proteomes (RP) sets, the UniProtKB sequence database, the NCBI sequence database, and our metagenomics sequence database. More...
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We make a range of alignments for each Pfam-A family. You can see a description of each above. You can view these alignments in various ways but please note that some types of alignment are never generated while others may not be available for all families, most commonly because the alignments are too large to handle.
| Seed (70) |
Full (43638) |
Representative proteomes | UniProt (81070) |
NCBI (203617) |
Meta (4136) |
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| RP15 (10346) |
RP35 (20339) |
RP55 (34710) |
RP75 (43902) |
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| PP/heatmap | 1 | ||||||||
1Cannot generate PP/Heatmap alignments for seeds; no PP data available
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| Seed (70) |
Full (43638) |
Representative proteomes | UniProt (81070) |
NCBI (203617) |
Meta (4136) |
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| RP15 (10346) |
RP35 (20339) |
RP55 (34710) |
RP75 (43902) |
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| Raw Stockholm | |||||||||
| Gzipped | |||||||||
You can also download a FASTA format file containing the full-length sequences for all sequences in the full alignment.
HMM logo
HMM logos is one way of visualising profile HMMs. Logos provide a quick overview of the properties of an HMM in a graphical form. You can see a more detailed description of HMM logos and find out how you can interpret them here. More...
Trees
This page displays the phylogenetic tree for this family's seed alignment. We use FastTree to calculate neighbour join trees with a local bootstrap based on 100 resamples (shown next to the tree nodes). FastTree calculates approximately-maximum-likelihood phylogenetic trees from our seed alignment.
Note: You can also download the data file for the tree.
Curation and family details
This section shows the detailed information about the Pfam family. You can see the definitions of many of the terms in this section in the glossary and a fuller explanation of the scoring system that we use in the scores section of the help pages.
Curation
| Seed source: | SCOP and Prosite |
| Previous IDs: | trypsin; |
| Type: | Domain |
| Sequence Ontology: | SO:0000417 |
| Author: |
Lutfiyya LL, Sonnhammer ELL 
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| Number in seed: | 70 |
| Number in full: | 43638 |
| Average length of the domain: | 205.60 aa |
| Average identity of full alignment: | 23 % |
| Average coverage of the sequence by the domain: | 55.65 % |
HMM information
| HMM build commands: |
build method: hmmbuild -o /dev/null HMM SEED
search method: hmmsearch -Z 45638612 -E 1000 --cpu 4 HMM pfamseq
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| Model details: |
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| Model length: | 221 | ||||||||||||
| Family (HMM) version: | 26 | ||||||||||||
| Download: | download the raw HMM for this family |
Species distribution
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Colour assignments
Archea
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Eukaryota
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Viruses
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Unclassified
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Selections
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Interactions
There are 67 interactions for this family. More...
Omptin Trypsin EGF FXa_inhibition WAP Kunitz_legume Serpin CLIP Sema A2M_recep Ecotin A2M_recep PDZ EGF LRR_8 NGF Fib_alpha Ecotin MG2 Pacifastin_I Serpin Kazal_2 Kringle TIL Bowman-Birk_leg Pro_Al_protease Kringle Triabin Kunitz_BPTI Sushi Staphylokinase Tissue_fac Antistasin SH3_1 VWA Tissue_fac Thrombin_light Kazal_1 Pacifastin_I PAN_1 Anophelin Coagulase FXa_inhibition PDZ PDZ_2 Thrombin_light Bowman-Birk_leg NGF potato_inhibit Sushi Hepsin-SRCR Hirudin Globin Peptidase_M14 Propep_M14 Pro_Al_protease Kunitz_BPTI LRR_8 Ldl_recept_a SSI Prot_inhib_II CompInhib_SCIN Haemadin Squash A2M Kunitz_legume Hepsin-SRCRStructures
For those sequences which have a structure in the Protein DataBank, we use the mapping between UniProt, PDB and Pfam coordinate systems from the PDBe group, to allow us to map Pfam domains onto UniProt sequences and three-dimensional protein structures. The table below shows the structures on which the Trypsin domain has been found. There are 2733 instances of this domain found in the PDB. Note that there may be multiple copies of the domain in a single PDB structure, since many structures contain multiple copies of the same protein sequence.
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