Protease

Protease
A protease is any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that link amino acids together in the polypeptide chain.
Classification
Proteases are currently classified into six groups:
• Serine proteases
• Threonine proteases
• Cysteine proteases
• Aspartic acid proteases
• Metalloproteases
• Glutamic acid 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 (peptidases) or a water molecule (aspartic acid, metallo- and glutamic acid peptidases) nucleophilic so that it can attack the peptide carbonyl 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.
Occurrence
Proteases occur naturally 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). Peptidases 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 the peptide bonds in proteins and therefore break the proteins down into their constituent monomers.
Proteases are also a type of exotoxin, which is a virulence factor in bacteria pathogenesis. Bacteria exotoxic proteases destroy extracellular structures. Protease enzymes are also found used extensively in the bread industry in Bread improver.
Proteases, also known as proteinases or proteolytic enzymes, are a large group of enzymes. Proteases belong to the class of enzymes known as hydrolases, which catalyse the reaction of hydrolysis of various bonds with the participation of a water molecule.
Proteases are involved in digesting long protein chains into short fragments, splitting the peptide bonds that link amino acid residues. Some of them can detach the terminal amino acids from the protein chain (exopeptidases, such as aminopeptidases, carboxypeptidase www A); the others attack internal peptide bonds of a protein (endopeptidases, such as trypsin, chymotrypsin, pepsin, papain, elastase).
Proteases are divided into four major groups according to the character of their catalytic active site and conditions of action: serine proteinases, cysteine (thiol) proteinases, aspartic proteinases, and metalloproteinases. Attachment of a protease to a certain group depends on the structure of catalytic site and the amino acid (as one of the constituents) essential for its activity.
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.
Inhibitors
The function of peptidases is inhibited by protease inhibitor enzymes. Examples of protease inhibitors are the class of serpins (serine protease or peptidase inhibitors), incorporating alpha 1-antitrypsin. Other serpins are complement 1-inhibitor, antithrombin, alpha 1-antichymotrypsin, plasminogen activator inhibitor 1 (coagulation, fibrinolysis) and the recently discovered 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 among them, depend on proteases in their reproductive cycle. Thus, protease inhibitors are developed as antiviral means.


Degradation
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 peptidase activity.
Serine protease
Crystal structure of Trypsin, a typical serine protease.
Serine proteases or serine endopeptidases (newer name) are proteases (enzymes that cut peptide bonds in proteins) in which one of the amino acids at the active site is serine.
They are found in both single-cell and complex organisms, in both cells with nuclei (eukaryotes) and without nuclei (prokaryotes).
Serine proteases are grouped into clans that share structural similarities (homology) and are then further subgrouped into families with simiar sequences.
The major clans found in humans include the chymotrypsin-like, the subtilisin-like, the alpha/beta hydrolase, and signal peptidase clans.
In evolutionary history, serine proteases were originally digestive enzymes. In mammals, they evolved by gene duplication to serve functions in blood clotting, the immune system, and inflammation.
Serine proteases are paired with serine protease inhibitors, which turn off their activity when they are no longer needed.[1]
Digestive serine proteases
Members
Chymotrypsin-clan
The three serine proteases of the chymotrypsin-like clan that have been studied in greatest detail are chymotrypsin, trypsin, and elastase. All three enzymes are synthesized by the pancreatic acinar cells, secreted in the small intestine, and are responsible for catalyzing the hydrolysis of peptide bonds. All three of these enzymes are similar in structure, as shown through their X-ray structures. The differing aspect lies in the peptide bond that is being cleaved; this is called the scissile bond. The different enzymes, like most enzymes, are highly specific in the reactions they catalyze. Each of these digestive serine proteases targets different regions of a polypeptide chain, based upon the side chains of the amino acid residues surrounding the site of cleavage:
• Chymotrypsin is responsible for cleaving peptide bonds following a bulky hydrophobic amino acid residue. Preferred residues include phenylalanine, tryptophan, and tyrosine, which fit into a snug hydrophobic pocket.
• Trypsin is responsible for cleaving peptide bonds following a positively-charged amino acid residue. Instead of having the hydrophobic pocket of the chymotrypsin, there exists an aspartic acid residue at the base of the pocket. This can then interact with positively-charged residues such as arginine and lysine on the substrate peptide to be cleaved.
• Elastase is responsible for cleaving peptide bonds following a small neutral amino acid residue, such as Alanine, glycine, and valine. (These amino acid residues form much of the connective tissues in meat). The pocket that is in "trypsin" and "chymotrypsin" is now partially filled with valine and threonine, rendering it a mere depression, which can accommodate these smaller amino acid residues.
The combination of these three enzymes make an incredibly effective digestive team and are primarily responsible for the digestion of proteins.
Subtilisin
Subtilisin is a serine protease in prokaryotes. Subtilisin is evolutionary unrelated to the chymotrypsin-clan, but shares the same catalytic mechanism utilising a catalytic triad, to create a nucleophilic serine. This is the classic example used to illustrate convergent evolution, since the same mechanism evolved twice independently during evolution.
Catalytic mechanism
The main player in the catalytic mechanism in the chymotrypsin and subtillisin clan enzymes mentioned above is the catalytic triad. The triad is located in the active site of the enzyme, where catalysis occurs, and is preserved in all serine protease enzymes. The triad is a coordinated structure consisting of three essential amino acids: histidine (His 57), serine (Ser 195) (hence the name "serine protease") and aspartic acid (Asp 102). Located very near one another near the heart of the enzyme, these three key amino acids each play an essential role in the cleaving ability of the proteases.
In the event of catalysis, an ordered mechanism occurs in which several intermediates are generated. The catalysis of the peptide cleavage can be seen as a ping-pong catalysis, in which a substrate binds (in this case, the polypeptide being cleaved), a product is released (the N-terminus "half" of the peptide), another substrate binds (in this case, water), and another product is released (the C-terminus "half" of the peptide).
Each amino acid in the triad performs a specific task in this process:

*The serine has an -OH group that is able to act as a nucleophile, attacking the carbonyl carbon of the scissile peptide bond of the substrate.
• A pair of electrons on the histidine nitrogen has the ability to accept the hydrogen from the serine -OH group, thus coordinating the attack of the peptide bond.
• The carboxyl group on the aspartic acid in turn hydrogen bonds with the histidine, making the pair of electrons mentioned above much more electronegative.
The whole reaction can be summarized as follows:
• The polypeptide substrate binds to the surface of the serine protease enzyme such that scissile bond is inserted into the active site of the enzyme, with the carbonyl carbon of this bond positioned near the nucleophilic serine.
• The serine -OH attacks the carbonyl carbon, and the nitrogen of the histidine accepts the hydrogen from the -OH of the [serine] and a pair of electrons from the double bond of the carbonyl oxygen moves to the oxygen. As a result, a tetrahedral intermediate is generated.
• The bond joining the nitrogen and the carbon in the peptide bond is now broken. The covalent electrons creating this bond move to attack the hydrogen of the histidine, breaking the connection. The electrons that previously moved from the carbonyl oxygen double bond move back from the negative oxygen to recreate the bond, generating an acyl-enzyme intermediate.
• Now, water comes in to the reaction. Water replaces the N-terminus of the cleaved peptide, and attacks the carbonyl carbon. Once again, the electrons from the double bond move to the oxygen making it negative, as the bond between the oxygen of the water and the carbon is formed. This is coordinated by the nitrogen of the histidine. which accepts a proton from the water. Overall, this generates another tetrahedral intermediate.
• In a final reaction, the bond formed in the first step between the serine and the carbonyl carbon moves to attack the hydrogen that the histidine just acquired. The now electron-deficient carbonyl carbon re-forms the double bond with the oxygen. As a result, the C-terminus of the peptide is now ejected.
Additional stabilizing effects
It was discovered that additional amino acids of the protease, Gly 193 and Ser 195, are involved in creating what is called an oxyanion hole. Both Gly 193 and Ser 195 can donate backbone hydrogens for hydrogen bonding. When the tetrahedral intermediate of step 1 and step 3 are generated, the negative oxygen ion, having accepted the electrons from the carbonyl double bond fits perfectly into the oxyanion hole. In effect, serine proteases preferentially bind the transition state and the overall structure is favored, lowering the activation energy of the reaction. This "preferential binding" is responsible for much of the catalytic efficiency of the enzyme.
Zymogens
There are certain inhibitors which resemble the tetrahedral intermediate, and thus fill up the active site, preventing the enzyme from working properly. Trypsin, a powerful digestive enzyme, is generated in the pancreas. Inhibitors prevent self-digestion of the pancreas itself.
Zymogens are the usually inactive precursors of an enzyme. If the digestive enzymes were active when synthesized, they would immediately start chewing up the synthesizing organs and tissues. Acute pancreatitis is such a condition, in which there is premature activation of the digestive enzymes in the pancreas, resulting in self-digestion (autolysis). It also complicates postmortem investigations, as the pancreas often digests itself before it can be assessed visually.
Zymogens are large, inactive structures, which have the ability to break apart or change into the smaller activated enzymes. The difference between zymogens and the activated enzymes lies in the fact that the active site for catalysis of the zymogens is distorted. As a result, the substrate polypeptide cannot bind effectively, and proteolysis does not occur. Only after activation, during which the conformation and structure of the zymogen change and the active site is opened, can proteolysis occur.
Zymogen Enzyme Notes
Trypsinogen trypsin
When trypsinogen enters the small intestine from the pancreas, secretions from the duodenal mucosa cleaves the lysine 15 - isoleucine 16 peptide bond of the zymogen. As a result, the zymogen trypsinogen breaks down into trypsin. Recall that trypsin is also responsible for cleaving lysine peptide bonds, and thus, once a small amount of trypsin is generated, it participates in cleavage of its own zymogen, generating even more trypsin. The process of trypsin activation can thus be called autocatalytic.

Chymotrypsinogen chymotrypsin
After the Arg 15 - Ile 16 bond in the chymotrypsinogen zymogen is cleaved by trypsin, the newly generated structure called a pi-chymotrypsin undergoes autolysis (self digestion), yielding active chymotrypsin.
Proelastase elastase
It is activated by cleavage through trypsin.
As can be seen, trypsinogen activation to trypsin is essential, because it activates its own reaction, as well as the reaction of both chymotrypsin and elastase. It is therefore essential that this activation doesn't occur prematurely. There are several protective measures taken by the organism to prevent self-digestion:
• The activation of trypsinogen by trypsin is relatively slow
• The zymogens are stored in zymogen granules, capsules that have walls that are thought to be resistant to proteolysis.
Inhibition
Serine proteases are inhibited by a diverse group of inhibitors, including synthetic chemical inhibitors for research or therapeutic purposes, and also natural proteinaceous inhibitors. One family of natural inhibitors called "serpins" (abbreviated from serine protease inhibitors) can form a covalent bond with the serine protease, inhibiting its function. The best-studied serpins are antithrombin and alpha 1-antitrypsin, studied for their role in coagulation/thrombosis and emphysema/A1AT respectively. Artificial irreversible small molecule inhibitors include AEBSF and PMSF.
Role in disease
Mutations may lead to decreased or increased activity of enzymes. This may have different consequences, depending on the normal function of the serine protease. For example, mutations in protein C, when leading to insufficient protein levels or activity, predispose to thrombosis.
Diagnostic use
Determination of serine protease levels may be useful in the context of particular diseases.
• Coagulation factor levels may be required in the diagnosis of hemorrhagic or thrombotic conditions.
• Fecal elastase is employed to determine the exocrine activity of the pancreas, e.g. in cystic fibrosis or chronic pancreatitis.
• Prostate specific antigen is used to determine prostate cancer risk


Aspartate protease
Aspartic proteases are a family of eukaryotic protease enzymes that utilize an aspartate residue for catalysis of their peptide substrates. In general, they have two highly-conserved aspartates in the active site and are optimally active at acidic pH. Nearly all known aspartyl proteases are inhibited by pepstatin.
Eukaryotic aspartic proteases include pepsins, cathepsins, and renins. They have a two-domain structure, probably arising from ancestral duplication. Retroviral and retrotransposon proteases (Pfam PF00077) are much smaller and appear to be homologous to a single domain of the eukaryotic aspartyl proteases.
Examples
• HIV-1 protease - a major drug-target for treatment of HIV
• Chymosin (or "rennin", with two "n"s)
• Renin (with one "n")
• Cathepsin D
• Pepsin
• Plasmepsin
Mechanism


Proposed mechanism of peptide cleavage by aspartyl proteases[1]
While a number of different mechanisms for aspartyl proteases have been proposed, the most widely accepted is a general acid-base mechanism involving coordination of a water molecule between the two highly-conserved aspartate residues.[1][2] One aspartate activates the water by abstracting a proton, enabling the water to attack the carbonyl carbon of the substrate scissile bond, generating a tetrahedral oxyanion intermediate. Rearrangement of this intermediate leads to protonation of the scissile amide.