Protease


A protease is an enzyme that catalyzes proteolysis, the breakdown of proteins into smaller polypeptides or single amino acids. They do this by cleaving the peptide bonds within proteins by hydrolysis, a reaction where water breaks bonds. Proteases are involved in many biological functions, including digestion of ingested proteins, protein catabolism, and cell signalling.
Without additional helping mechanisms, proteolysis would be very slow, taking hundreds of years. Proteases can be found in all forms of life and viruses. They have independently evolved multiple times, and different classes of protease can perform the same reaction by completely different catalytic mechanisms.

Hierarchy of proteases

Based on catalytic residue

Proteases can be classified into seven broad groups:
Proteases were first grouped into 84 families according to their evolutionary relationship in 1993, and classified under four catalytic types: serine, cysteine, aspartic, and metallo proteases. 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 or a water molecule 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 superfamilies, and some superfamilies show divergent evolution to multiple different nucleophiles.

Peptide lyases

A seventh catalytic type of proteolytic enzymes, asparagine peptide lyase, was described in 2011. Its proteolytic mechanism is unusual since, rather than hydrolysis, it performs an elimination reaction. During this reaction, the catalytic asparagine forms a cyclic chemical structure that cleaves itself at asparagine residues in proteins under the right conditions. Given its fundamentally different mechanism, its inclusion as a peptidase may be debatable.

Evolutionary phylogeny

An up-to-date classification of protease evolutionary superfamilies is found in the MEROPS database. In this database, proteases are classified firstly by 'clan' based on structure, mechanism and catalytic residue order. Within each 'clan', proteases are classified into families based on sequence similarity. Each family may contain many hundreds of related proteases.
Currently more than 50 clans are known, each indicating an independent evolutionary origin of proteolysis.

Classification based on optimal pH

Alternatively, proteases may be classified by the optimal pH in which they are active:
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 ; others attack internal peptide bonds of a protein.

Catalysis

is achieved by one of two mechanisms:
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\....
Conversely some proteases are highly specific and only cleave substrates with a certain sequence. Blood clotting and viral polyprotein processing 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....

Degradation and autolysis

Proteases, being themselves proteins, are cleaved by other protease molecules, sometimes of the same variety. This acts as a method of regulation of protease activity. Some proteases are less active after autolysis whilst others are more active.

Biodiversity of proteases

Proteases occur in all organisms, from prokaryotes to eukaryotes to viruses. These enzymes are involved in a multitude of physiological reactions from simple digestion of food proteins to highly regulated cascades. Proteases can either break specific peptide bonds, depending on the amino acid sequence of a protein, or completely break down a peptide to amino acids. The activity can be a destructive change, it can be an activation of a function, or it can be a signal in a signalling pathway.

Plants

Protease containing plant-solutions called vegetarian rennet has been in use for hundreds of years in Europe and middle-east for making kosher and halal Cheeses. Vegetarian rennet from Withania coagulans has been in use for thousands of years as Ayurvedic remedy for digestion and diabetes in the Indian subcontinent. It is also used to make Paneer.
Plant genomes encode hundreds of proteases, largely of unknown function. Those with known function are largely involved in developmental regulation. Plant proteases also play a role in regulation of photosynthesis.

Animals

Proteases are used throughout an organism for various metabolic processes. Acid proteases secreted into the stomach and serine proteases present in duodenum enable us to digest the protein in food. Proteases present in blood serum 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 and play several different roles in metabolic control. Some snake venoms are also proteases, such as pit viper haemotoxin and interfere with the victim's blood clotting cascade. 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.

Bacteria

Bacteria secrete proteases to hydrolyse the peptide bonds in proteins and therefore break the proteins down into their constituent 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.
Bacteria contain proteases responsible for general protein quality control by degrading unfolded or misfolded proteins.
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.

Viruses

Some viruses express their entire genome as one massive polyprotein and use a protease to cleave this into functional units. These proteases have high specificity and only cleave a very restricted set of substrate sequences. They are therefore a common target for protease inhibitors.

Uses

The field of protease research is enormous. Since 2004, approximately 8000 papers related to this field were published each year. Proteases are used in industry, medicine and as a basic biological research tool.
Digestive proteases are part of many laundry detergents and are also used extensively in the bread industry in bread improver. A variety of proteases are used medically both for their native function or for completely artificial functions. Highly specific proteases such as TEV protease and thrombin are commonly used to cleave fusion proteins and affinity tags in a controlled fashion.

Inhibitors

The activity of proteases is inhibited by protease inhibitors. One example of protease inhibitors is the serpin superfamily. It includes alpha 1-antitrypsin, alpha 1-antichymotrypsin, C1-inhibitor, antithrombin, 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.
Other natural protease inhibitors are used as defense mechanisms. Common examples are the trypsin inhibitors found in the seeds of some plants, most notable for humans being soybeans, a major food crop, where they act to discourage predators. Raw soybeans are toxic to many animals, including humans, until the protease inhibitors they contain have been denatured.