protease

A protease (also called a peptidase or proteinase) is an

enzyme

that

catalyzes

(increases reaction rate or "speeds up")

proteolysis

, breaking down

proteins

into smaller polypeptides or single

amino acid

s, 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 many biological functions, including digestion of ingested proteins, protein catabolism (breakdown of old proteins), and cell signaling.

In the absence of functional accelerants, proteolysis would be very slow, taking hundreds of years. Proteases can be found in all forms of life and

virus

es. 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:
*

Serine protease

s - using a

serine

alcohol

* Cysteine proteases - using a

cysteine

thiol

* Threonine proteases - using a

threonine

secondary alcohol

*

Aspartic protease

s - using an aspartate carboxylic acid
*

Glutamic protease

s - using a glutamate carboxylic acid
* Metalloproteases - using a

metal

, usually

zinc

* Asparagine peptide lyases - using an

asparagine

to perform an

elimination reaction

(not requiring water)

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 (proteases) or a water molecule ( aspartic acid, metallo- and 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 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' (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.

Classification based on optimal pH

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 cell

s and causes activation of

complement

and kinins. This group includes the calpains.
*''Basic proteases'' (or ''alkaline proteases'')

Enzymatic 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

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 hydrolyze it.
*Serine, threonine, and cysteine proteases use a nucleophilic residue (usually in a

catalytic triad

). That residue performs a nucleophilic attack to

covalent

ly link the protease to the substrate protein, releasing the first half of the product. This covalent acyl-enzyme intermediate is then hydrolyzed by activated water to complete catalysis by releasing the second half of the product and regenerating the free enzyme

Specificity

Proteolysis can be highly promiscuous such that a wide range of protein substrates are hydrolyzed. 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 that bind to specified residues. For example, TEV protease is specific for the sequence ...ENLYFQ\S... ('\'=cleavage site).

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 (e.g. TEV protease) whilst others are more active (e.g. trypsinogen).

Biodiversity of proteases

Proteases occur in all organisms, from prokaryotes to

eukaryote

s to

virus

. 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 completely break down a 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 signalling pathway.

Plants

Protease-containing plant-solutions called

vegetarian rennet

have been in use for hundreds of years in

Europe

and the

Middle East

for making kosher and halal Cheeses. Vegetarian rennet from ''Withania coagulans'' has been in use for thousands of years as a

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 (such as

pepsin

) and serine proteases present in the

duodenum

(trypsin and

chymotrypsin

) enable us to digest the protein in food. Proteases present in blood serum (thrombin,

plasmin

, Hageman factor, etc.) play an 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. 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 roles 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 a complex cooperative action, proteases can catalyze 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 acid

s. 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 (e.g. the AAA+

proteasome

) 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

(for example, exfoliative toxin). Bacterial exotoxic proteases destroy extracellular structures.

Viruses

The genomes of some viruses encode one massive polyprotein, which needs a protease to cleave this into functional units (e.g. the hepatitis C virus virus and the

picornavirus

es). These proteases (e.g. TEV protease) 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 (e.g. controlling blood clotting) or for completely artificial functions (''e.g.'' for the targeted degradation of pathogenic proteins). 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 inhibitor (biology), protease inhibitors. One example of protease inhibitors is the serpin superfamily. It includes alpha 1-antitrypsin (which protects the body from excessive effects of its own inflammation, inflammatory proteases), alpha 1-antichymotrypsin (which does likewise), C1-inhibitor (which protects the body from excessive protease-triggered activation of its own

complement system

), antithrombin (which protects the body from excessive coagulation), plasminogen activator inhibitor 1, plasminogen activator inhibitor-1 (which protects the body from inadequate coagulation by blocking protease-triggered fibrinolysis), 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 inhibitor (biology), protease inhibitors are not to be confused with the protease inhibitor (pharmacology), protease inhibitors used in antiretroviral therapy. Some virus (biology), viruses, with HIV/AIDS among them, depend on proteases in their reproductive cycle. Thus, protease inhibitor (pharmacology), protease inhibitors are developed as Antiviral drug, antiviral therapeutic agents.

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 Soybean#Nutrition, toxic to many animals, including humans, until the protease inhibitors they contain have been denatured.

See also

* Ligase
*Protease
**cysteine protease, cysteine-
**serine protease, serine-
**threonine protease, threonine-
**aspartic protease, aspartic-
**glutamic protease, glutamic-
**metalloprotease, metallo-
* PA clan
* Convergent evolution
* Proteolysis
* Catalytic triad
* The Proteolysis Map
* Proteases in angiogenesis
* Intramembrane proteases
* Protease inhibitor (pharmacology)
* Protease inhibitor (biology)
* TopFIND - database of protease specificity, substrates, products and inhibitors
* MEROPS - Database of protease evolutionary groups

References

External links

International Proteolysis Society

MEROPS - the peptidase database



Protease cutting predictor


(see als



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
*

{{Authority control

Proteases,
EC 3.4, *
Post-translational modification