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A protease (also termed peptidase or proteinase) is any enzyme that performs proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that link amino acids together in the polypeptide chain forming the protein. Proteases have evolved multiple times, and different classes of protease can perform the same reaction by completely different catalytic mechanisms. Proteases can be found in animals, plants, bacteria, archea and viruses.
Proteases are currently[update] classified into six broad groups:
- Serine proteases - using a serine alcohol
- Threonine proteases - using a threonine secondary alcohol
- Cysteine proteases - using a cysteine thiol
- Aspartate proteases - using an aspartate carboxylic acid
- Glutamic acid proteases - using a glutamate carboxylic acid
- Metalloproteases - using a metal, usually zinc
The threonine and glutamic-acid proteases were not described until 1995 and 2004, respectively. The mechanism used to cleave a peptide bond involves making an amino acid residue that has the cysteine and threonine (proteases) or a water molecule (aspartic acid, metallo- and glutamic acid proteases) nucleophilic so that it can attack the peptide carboxyl group. One way to make a nucleophile is by a catalytic triad, where a histidine residue is used to activate serine, cysteine, or threonine as a nucleophile. This is not an evolutionary grouping, however, as the nucleophile types have evolved convergently in different superfamies, and some superfamilies show divergent evolution to multiple different nucleophiles.
An up to date classification of protease evolutionary superfamilies is found in the MEROPS database. In this database, proteases are classified firstly by 'clan' (superfamily) based on structure, mechanism and catalytic residue order (e.g. the PA clan where P indicates a mixture of nucleophile families). Within each 'clan', proteases are classified into families based on sequence similarity (e.g. the S1 and C3 families within the PA clan). Each family may contain many hundreds of related proteases (e.g. trypsin, elastase, thrombin and streptogrisin within the S1 family).
Currently more than 50 clans are known, each indicating an independent evolutionary origin of proteolysis.
Alternatively, proteases may be classified by the optimal pH in which they are active:
- Acid proteases
- Neutral proteases involved in type 1 hypersensitivity. Here, it is released by mast cells and causes activation of complement and kinins. This group includes the calpains.
- Basic proteases (or alkaline proteases)
Function and mechanism
Proteases are involved in digesting long protein chains into shorter fragments by splitting the peptide bonds that link amino acid residues. Some detach the terminal amino acids from the protein chain (exopeptidases, such as aminopeptidases, carboxypeptidase A); others attack internal peptide bonds of a protein (endopeptidases, such as trypsin, chymotrypsin, pepsin, papain, elastase).
Catalysis is achieved by one of two mechanisms:
- Aspartic, glutamic and metallo proteases activate a water molecule which performs a nucleophilic attack on the peptide bond to hydrolyse it.
- Serine, threonine and cysteine proteases use a nucleophilic residue in a (usually in a catalytic triad). That residue performs a nucleophilc attack to covalently link the protease to the substrate protein, releasing the first half of the product. This covalent acyl-enzyme intermediate is then hydrolysed by activated water to complete catalysis by releasing the second half of the product and regenerating the free enzyme.
Proteolysis can be highly promiscuous such that a wide range of protein substrates are hydrolysed. This is the case for digestive enzymes such as trypsin which have to be able to cleave the array of proteins ingested into smaller peptide fragments. Promiscuous proteases typically bind to a single amino acid on the substrate and so only have specificity for that residue. For example trypsin is specific for the sequences ...K\... or ...R\... ('\'=cleavage site)
Conversely some proteases are highly specific and only cleave substrates with a certain sequence. Blood clotting (such as thrombin) and viral polyprotein processing (such as TEV protease) requires this level of specificity in order to achieve precise cleavage events. This is achieved by proteases having a long binding cleft or tunnel with several pockets along it which bind the specified residues. For example TEV protease is specific for the sequence ...ENLYFQ\S... ('\'=cleavage site).
Proteases occur in all organisms. These enzymes are involved in a multitude of physiological reactions from simple digestion of food proteins to highly regulated cascades (e.g., the blood-clotting cascade, the complement system, apoptosis pathways, and the invertebrate prophenoloxidase-activating cascade). Proteases can either break specific peptide bonds (limited proteolysis), depending on the amino acid sequence of a protein, or break down a complete peptide to amino acids (unlimited proteolysis). The activity can be a destructive change, abolishing a protein's function or digesting it to its principal components; it can be an activation of a function, or it can be a signal in a signaling pathway.
Bacteria also secrete proteases to hydrolyse (digest) the peptide bonds in proteins and therefore break the proteins down into their constituent monomers(amino acids). Bacterial and fungal proteases are particularly important to the global carbon and nitrogen cycles in the recycling of proteins, and such activity tends to be regulated by nutritional signals in these organisms. The net impact of nutritional regulation of protease activity among the thousands of species present in soil can be observed at the overall microbial community level as proteins are broken down in response to carbon, nitrogen, or sulfur limitation.
A secreted bacterial protease may also act as an exotoxin, and be an example of a virulence factor in bacterial pathogenesis. Bacterial exotoxic proteases destroy extracellular structures. Protease enzymes are also used extensively in the bread industry in bread improver.
Proteases are used throughout an organism for various metabolic processes. Acid proteases secreted into the stomach (such as pepsin) and serine proteases present in duodenum (trypsin and chymotrypsin) enable us to digest the protein in food; proteases present in blood serum (thrombin, plasmin, Hageman factor, etc.) play important role in blood-clotting, as well as lysis of the clots, and the correct action of the immune system. Other proteases are present in leukocytes (elastase, cathepsin G) and play several different roles in metabolic control. Proteases determine the lifetime of other proteins playing important physiological role like hormones, antibodies, or other enzymes—this is one of the fastest "switching on" and "switching off" regulatory mechanisms in the physiology of an organism. By complex cooperative action the proteases may proceed as cascade reactions, which result in rapid and efficient amplification of an organism's response to a physiological signal.
Proteases are part of many laundry detergents.
The activity of proteases is inhibited by protease inhibitors. One example of protease inhibitors is the serpin superfamily, which includes alpha 1-antitrypsin, C1-inhibitor, antithrombin, alpha 1-antichymotrypsin, plasminogen activator inhibitor-1, and neuroserpin.
Natural protease inhibitors include the family of lipocalin proteins, which play a role in cell regulation and differentiation. Lipophilic ligands, attached to lipocalin proteins, have been found to possess tumor protease inhibiting properties. The natural protease inhibitors are not to be confused with the protease inhibitors used in antiretroviral therapy. Some viruses, with HIV/AIDS among them, depend on proteases in their reproductive cycle. Thus, protease inhibitors are developed as antiviral means.
Proteases, being themselves proteins, are known to be cleaved by other protease molecules, sometimes of the same variety. This may be an important method of regulation of protease activity.
The field of protease research is enormous. Barrett and Rawlings estimated that approximately 8001 papers related to this field are published each year.
- Catalytic triad
- The Proteolysis Map
- TopFIND, a scientific database covering proteases, their cleavage site specificity, substrates, inhibitors and protein termini originating from their activity
- David Ho, an AIDS researcher famous for pioneering the use of protease inhibitors in treating HIV-infected patients
- Proteases in angiogenesis
- Heat stabilization, a technology for heat inactivation of protease activity
- Rawlings ND, Barrett AJ, Bateman A (January 2010). "MEROPS: the peptidase database". Nucleic Acids Res. 38 (Database issue): D227–33. doi:10.1093/nar/gkp971. PMC 2808883. PMID 19892822.
- Mitchell, Richard Sheppard; Kumar, Vinay; Abbas, Abul K.; Fausto, Nelson (2007). Robbins Basic Pathology. Philadelphia: Saunders. p. 122. ISBN 1-4160-2973-7. 8th edition.
- Sims, G.K. 2006. Nitrogen Starvation Promotes Biodegradation of N-Heterocyclic Compounds in Soil. Soil Biology & Biochemistry 38:2478-2480.
- Sims, G. K., and M. M. Wander. 2002. Proteolytic activity under nitrogen or sulfur limitation. Appl. Soil Ecol. 568:1-5.
- Barrett A.J., Rawlings ND, Woessner JF. The Handbook of Proteolytic Enzymes, 2nd ed. Academic Press, 2003. ISBN 0-12-079610-4.
- Hedstrom L. Serine Protease Mechanism and Specificity. Chem Rev 2002;102:4501-4523.
- Southan C. A genomic perspective on human proteases as drug targets. Drug Discov Today 2001;6:681-688.
- Hooper NM. Proteases in Biology and Medicine. London: Portland Press, 2002. ISBN 1-85578-147-6.
- Puente XS, Sanchez LM, Overall CM, Lopez-Otin C. Human and Mouse Proteases: a Comparative Genomic Approach. Nat Rev Genet 2003;4:544-558.
- Ross J, Jiang H, Kanost MR, Wang Y. Serine proteases and their homologs in the Drosophila melanogaster genome: an initial analysis of sequence conservation and phylogenetic relationships. Gene 2003;304:117-31.
- Puente XS, Lopez-Otin C. A Genomic Analysis of Rat Proteases and Protease Inhibitors. Genome Biol 2004;14:609-622.
- Lucía Feijoo-Siota, Tomás G. Villa Native and Biotechnologically Engineered Plant Proteases with Industrial Applications. Food and Bioprocess Technology 2010.
- International Proteolysis Society
- Merops - the peptidase database
- List of protease inhibitors
- Protease cutting predictor
- List of proteases and their specificities (see also )
- Proteolysis MAP from Center for Proteolytic Pathways
- Proteolysis Cut Site database - curated expert annotation from users
- Protease cut sites graphical interface
- TopFIND protease database covering cut sites, substrates and protein termini
- Proteases at the US National Library of Medicine Medical Subject Headings (MeSH)