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 similar 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.
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:
- 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:
serine protease reaction mechanism
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.
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.
