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About Serine Protease
From Wikipedia, the free encyclopedia
Serine proteases (or serine endopeptidases)
are enzymes that cleave peptide
bonds in proteins, in whichserine serves as
the nucleophilic amino acid at the
(enzyme's) active site.[1] They are found
ubiquitously in both eukaryotes and prokaryotes.
Crystal structure of Trypsin, a typical serine protease.
Serine proteases fall into two broad categories based on their structure: chymotrypsin-like (trypsin-like)
or subtilisin-like.[2] In humans, they are responsible for co-ordinating various physiological functions,
including digestion, immune response, blood coagulation and reproduction.[1]
Contents
1 Classification
2 Substrate specificity
2.1 Trypsin-like
2.2Chymotrypsin-like
2.5Subtilisin-like
3 Catalytic mechanism
3.1Additional stabilizing effects
4 Regulation of Serine Protease activity
2.3Thrombin-like
4.1Zymogen activation
2.4Elastase-like
4.2Inhibition
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5 Role in disease
7References
6 Diagnostic use
Classification
The MEROPS protease classification system counts 16 superfamilies (as of 2013) each containing
manyfamilies. Each superfamily uses the catalytic triad or dyad in a different protein fold and so
representconvergent evolution of the catalytic mechanism. The majority belong to the S1 family of
the PA clan(superfamily) of proteases.
For superfamilies, P = superfamily containing a mixture of nucleophile class families, S = purely serine
proteases. superfamily. Within each superfamily, families are designated by their catalytic nucleophile (S
= serine proteases).
Families of Serine proteases
Superfamily Families
Examples
SB
S8, S53
Subtilisin (Bacillus licheniformis)
SC
S9, S10, S15, S28, S33, Prolyl oligopeptidase (Sus scrofa)
SE
D-Ala-D-Ala peptidase C (Escherichia coli)
SF
S11, S12, S13
S37
S24, S26
SH
S21, S73, S77, S78, S80
Cytomegalovirus assemblin (human herpesvirus 5)
SJ
S16, S50, S69
Lon-A peptidase (Escherichia coli)
SK
S14, S41, S49
Clp protease (Escherichia coli)
SO
S74
Phage K1F endosialidase CIMCD self-cleaving protein
SP
S59
SR
S60
Nucleoporin 145 (Homo sapiens)
(Enterobacteriaphage K1F)
Lactoferrin (Homo sapiens)
SS
S66
Murein tetrapeptidase LD-carboxypeptidase (Pseudomonas
ST
S54
PA
Signal peptidase I (Escherichia coli)
Rhomboid-1 (Drosophila melanogaster)
aeruginosa)
S1, S3, S6, S7, S29, S30, Chymotrypsin A (Bos taurus)
S31, S32, S39, S46, S55,
S64, S65, S75
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PB
S45, S63
Penicillin G acylase precursor (Escherichia coli)
PC
S51
Dipeptidase E (Escherichia coli)
PE
P1
DmpA aminopeptidase (Ochrobactrum anthropi)
unassigned
S48, S62, S68, S71, S72,
S81
SubstrateS79,
specificity
Serine proteases are characterised by a distinctive structure, consisting of two beta-barrel domains that
converge at the catalytic active site. These enzymes can be further categorised based on their substrate
specificity as either trypsin-like, chymotrypsin-like or elastase-like.[3]
Trypsin-like
Trypsin-like proteases cleave peptide bonds following a positively charged amino acid
(lysine or arginine).[4] This specificity is driven by the residue which lies at the base of the enzyme's S1
pocket (generally a negatively charged aspartic acid or glutamic acid).
Chymotrypsin-like
The S1 pocket of chymotrypsin-like enzymes is more hydrophobic than in trypsin-like proteases. This
results in a specificity for medium to large sized hydrophobic residues, such
as tyrosine, phenylalanine and tryptophan.
Thrombin-like
These include thrombin, tissue activating plasminogen and plasmin. They have been found to have roles
in coagulation and digestion as well as in the pathophysiology of neurodegenerative disorders such as
Alzheimer's and Parkinson's induced dementia.
Elastase-like
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Elastase-like proteases have a much smaller S1 cleft than either trypsin- or chymotrypsin-like proteases.
Consequently, residues such as alanine,glycine and valine tend to be preferred.
Subtilisin-like
Subtilisin is a serine protease in prokaryotes. Subtilisin is evolutionarily 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 serine proteases
is the catalytic triad. The triad is
located in the active site of the
enzyme, where catalysis occurs, and
is preserved in all superfamilies of
serine protease enzymes. The triad
is a coordinated structure consisting
of three amino
acids: His 57, Ser 195 (hence the name "serine protease") and Asp 102. These three key amino acids
each play an essential role in the cleaving ability of the proteases. While the amino acid members of the
triad are located far from one another on the sequence of the protein, due to folding, they will be very
close to one another in the heart of the enzyme. The particular geometry of the triad members are highly
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characteristic to their specific function: it was shown that the position of just four points of the triad
characterize the function of the containing enzyme.[5]
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:
1.
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.
2.
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.
3.
The carboxyl group on the aspartic acid in turn hydrogen bonds with the histidine, making the
nitrogen atom mentioned above much more electron egative.
The whole reaction can be summarized as follows:
1.
The polypeptide substrate binds to the surface of the serine protease enzyme such that the
scissile bond is inserted into the active site of the enzyme, with the carbonyl carbon of this bond
positioned near the nucleophilic serine.
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2.
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.
3.
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.
4.
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.
5.
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 histidinejust 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. BothGly 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,
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lowering the activation energy of the reaction. This "preferential binding" is responsible for much of the
catalytic efficiency of the enzyme.
Regulation of Serine Protease activity
Host organisms must ensure that the activity of serine proteases is adequately regulated. This is
achieved by a requirement for initial protease activation, and the secretion of inhibitors.
Zymogen activation
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.
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Zymogen
Enzyme
Notes
When
trypsinogen
enters
the small
intestine from
the
pancreas, enteropeptidase secretions from the duodenal mucosa
cleave the lysine 15 - isoleucine 16 peptide bond of the zymogen.
As a result, the zymogen trypsinogen breaks down into trypsin.
Trypsinogen
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.
After the Arg 15 - Ile 16 bond in the chymotrypsinogen zymogen is
cleaved
by
trypsin,
the
newly
generated
structure
called
Chymotrypsinogen chymotrypsin
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 chymotrypsinand elastase. Therefore, it is essential that this activation does
not occur prematurely. There are several protective measures taken by the organism to prevent
self-digestion:

The activation of trypsinogen by trypsin is relatively slow
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
The zymogens are stored in zymogen granules, capsules that have walls that are thought to be
resistant to proteolysis.
Inhibition
There are certain inhibitors that 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.
Serine proteases are paired with serine protease inhibitors, which turn off their activity when they are no
longer needed.[6]
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 acovalent 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.
A family of arthropod serine peptidase inhibitors, called pacifastin, has been identified
in locusts and crayfish, and may function in the arthropodimmune system.[7]
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 can lead to protein C deficiency and predisposing to thrombosis.
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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.
Serum prostate-specific antigen is used in prostate cancer screening, risk stratification, and

post-treatment monitoring.
Serine protease, as released by mast cells, is an important diagnostic marker for type 1

hypersensitivity reactions (e.g. anaphylaxis). More useful than e.g. histamine due to the
longer half-life, meaning it remains in the system for a clinically useful length of time.
See also

Serine hydrolase

Protease

cysteine-

threonine-

aspartic-

metallo-

PA clan

Convergent evolution

Proteolysis

Catalytic triad

The Proteolysis Map

Proteases in angiogenesis

Intramembrane proteases

Protease inhibitor (pharmacology)
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
Protease inhibitor (biology)

TopFIND - database of protease specificity, substrates, products and inhibitors

MEROPS - Database of protease evolutionary groups
References
1.
Hedstrom, L. (Dec 2002). "Serine protease mechanism and specificity.". Chem Rev. 102 (12):
4501–24. doi:10.1021/cr000033x. PMID 12475199.
2.
Madala PK, Tyndall JD, Nall T, Fairlie DP (Jun 2010). "Update 1 of: Proteases universally recognize beta
strands in their active sites". Chem Rev. 110 (6): PR1–31. doi:10.1021/cr900368a. PMID 20377171.
3.
Ovaere P, Lippens S, Vandenabeele P, Declercq W (Aug 2009). "The emerging roles of serine protease
cascades in the epidermis". Trends Biochem Sci. 34 (9):
453–63. doi:10.1016/j.tibs.2009.08.001. PMID 19726197.
4.
Evnin, Luke B.; Vásquez, John R.; Craik, Charles S. (1990). "Substrate specificity of trypsin investigated by
using a genetic selection". Proceedings of the National Academy of Sciences of the United States of
America. 87 (17): 6659–63. doi:10.1073/pnas.87.17.6659. JSTOR 2355359. PMC 54596 .PMID 2204062.
5.
Iván, Gábor.; Szabadka, Zoltán; Ordög, Rafael; Grolmusz, Vince; Náray-Szabó, Gábor (2009). "Four Spatial
Points That Define Enzyme Families". Biochemical and Biophysical Research Communications. 383 (4):
417–420. doi:10.1016/j.bbrc.2009.04.022. PMID 19364497.
6.
Kimball's Biology Pages, Serine Proteases [self-published source?]
7.
Breugelmans B, Simonet G, van Hoef V, van Soest S, Vanden BJ (2009). "Pacifastin-related peptides:
structural and functional characteristics of a family of serine peptidase inhibitors.". Peptides. 30 (3):
622–32. doi:10.1016/j.peptides.2008.07.026. PMID 18775459.
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