PTGS2 (COX-2), converts
arachidonic acid (AA) to prostaglandin endoperoxide H2. PTGSs are targets for
NSAIDs and PTGS2 (COX-2) specific inhibitors called coxibs. PTGS-2 is a sequence homodimer. Each
monomer of the enzyme has a
peroxidase and a PTGS (COX)
active site. The PTGS (COX) enzymes catalyze the conversion of
arachidonic acid to
prostaglandins in two steps. First, hydrogen is abstracted from carbon 13 of arachidonic acid, and then two molecules of oxygen are added by the PTGS2 (COX-2), giving PGG2. Second,
PGG2 is reduced to
PGH2 in the peroxidase active site. The synthesized PGH2 is converted to prostaglandins (
PGD2,
PGE2,
PGF2α),
prostacyclin (PGI2), or
thromboxane A2 by tissue-specific isomerases.(Figure 2)[6]
While metabolizing arachidonic acid primarily to PGG2, COX-2 also converts this fatty acid to small amounts of a racemic mixture of
15-Hydroxyicosatetraenoic acids (i.e., 15-HETEs) composed of ~22% 15(R)-HETE and ~78% 15(S)-HETE
stereoisomers as well as a small amount of 11(R)-HETE.[7] The two 15-HETE stereoisomers have intrinsic biological activities but, perhaps more importantly, can be further metabolized to a major class of agents, the
lipoxins. Furthermore,
aspirin-treated COX-2 metabolizes arachidonic acid almost exclusively to 15(R)-HETE which product can be further metabolized to epi-
lipoxins.[8] The lipoxins and epi-lipoxins are potent anti-inflammatory agents and may contribute to the overall activities of the two COX's as well as to aspirin.[citation needed]
COX-2 is naturally inhibited by
calcitriol (the active form of Vitamin D).[9][10]
Mechanism
Both the peroxidase and PTGS activities are inactivated during catalysis by mechanism-based, first-order processes, which means that PGHS-2 peroxidase or PTGS activities fall to zero within 1–2 minutes, even in the presence of sufficient substrates.[12][13][14]
The conversion of arachidonic acid to PGG2 can be shown as a series of
radical reactions analogous to polyunsaturated
fatty acidautoxidation.[15] The 13-pro(S) -hydrogen is abstracted and dioxygen traps the
pentadienyl radical at carbon 11. The 11-peroxyl radical cyclizes at carbon 9 and the carbon-centered radical generated at C-8 cyclizes at carbon 12, generating the
endoperoxide. The
allylic radical generated is trapped by dioxygen at carbon 15 to form the 15-(S) -peroxyl radical; this radical is then reduced to
PGG2 . This is supported by the following evidence: 1) a significant
kinetic isotope effect is observed for the abstraction of the 13-pro (S)-hydrogen; 2) carbon-centered radicals are trapped during
catalysis;[16] 3) small amounts of
oxidation products are formed due to the oxygen trapping of an allylic radical intermediate at positions 13 and 15.[17][18]
Another mechanism in which the 13-pro (S)-hydrogen is
deprotonated and the
carbanion is
oxidized to a
radical is theoretically possible. However, oxygenation of 10,10-difluoroarachidonic acid to 11-(S)-hydroxyeicosa-5,8,12,14-tetraenoic acid is not consistent with the generation of a carbanion intermediate because it would eliminate fluoride to form a conjugated diene.[19] The absence of endoperoxide-containing products derived from 10,10-difluoroarachidonic acid has been thought to indicate the importance of a C-10 carbocation in
PGG2 synthesis.[20] However, the cationic mechanism requires that endoperoxide formation comes before the removal of the 13-pro (S)-hydrogen. This is not consistent with the results of the isotope experiments of
arachidonic acid oxygenation.[21]
Structure
PTGS2 (COX-2) exists as a homodimer, each monomer with a molecular mass of about 70 kDa. The tertiary and quaternary structures of PTGS1 (COX-1) and PTGS2 (COX-2) enzymes are almost identical. Each subunit has three different structural domains: a short
N-terminal epidermal growth factor (
EGF) domain; an
α-helical membrane-binding moiety; and a
C-terminal catalytic domain. PTGS (COX, which can be confused with "
cytochrome oxidase") enzymes are
monotopic membrane proteins; the membrane-binding domain consists of a series of
amphipathic α helices with several
hydrophobicamino acids exposed to a membrane monolayer. PTGS1 (COX-1) and PTGS2 (COX-2) are bifunctional enzymes that carry out two consecutive chemical reactions in spatially distinct but mechanistically
coupled active sites. Both the
cyclooxygenase and the
peroxidase active sites are located in the catalytic domain, which accounts for approximately 80% of the protein. The catalytic domain is
homologous to mammalian peroxidases such as
myeloperoxidase.[23][24]
It has been found that human PTGS2 (COX-2) functions as a conformational heterodimer having a catalytic monomer (E-cat) and an allosteric monomer (E-allo).
Heme binds only to the
peroxidase site of E-cat while substrates, as well as certain
inhibitors (e.g.
celecoxib), bind the COX site of E-cat. E-cat is regulated by E-allo in a way dependent on what ligand is bound to E-allo.
Substrate and non-substrate
fatty acid (FAs) and some PTGS (COX) inhibitors (e.g.
naproxen) preferentially bind to the PTGS (COX) site of E-allo.
Arachidonic acid can bind to E-cat and E-allo, but the affinity of AA for E-allo is 25 times that for Ecat. Palmitic acid, an efficacious stimulator of
huPGHS-2, binds only E-allo in palmitic acid/murine PGHS-2 co-crystals. Non-substrate FAs can potentiate or
attenuate PTGS (COX) inhibitors depending on the
fatty acid and whether the inhibitor binds E-cat or E-allo. Studies suggest that the concentration and composition of the free fatty acid pool in the environment in which PGHS-2 functions in cells, also referred to as the FA tone, is a key factor regulating the activity of PGHS-2 and its response to PTGS (COX) inhibitors.[22]
Clinical significance
PTGS2 (COX-2) is unexpressed under normal conditions in most cells, but elevated levels are found during
inflammation. PTGS1 (COX-1) is constitutively expressed in many tissues and is the predominant form in gastric mucosa and in the kidneys. Inhibition of PTGS1 (COX-1) reduces the
basal production of cytoprotective
PGE2 and
PGI2 in the
stomach, which may contribute to
gastric ulceration. Since PTGS2 (COX-2) is generally expressed only in cells where
prostaglandins are upregulated (e.g., during inflammation), drug-candidates that selectively inhibit PTGS2 (COX-2) were suspected to show fewer
side-effects[24] but proved to substantially increase risk for cardiovascular events such as heart attack and stroke. Two different mechanisms may explain contradictory effects. Low-dose aspirin protects against heart attacks and strokes by blocking PTGS1 (COX-1) from forming a prostaglandin called thromboxane A2. It sticks platelets together and promotes clotting; inhibiting this helps prevent heart disease. On the other hand, PTGS2 (COX-2) is a more important source of prostaglandins, particularly prostacyclin which is found in blood vessel lining. Prostacyclin relaxes or unsticks platelets, so
selective COX-2 inhibitors (coxibs) increase risk of cardiovascular events due to clotting.[26]
The expression of PTGS2 (COX-2) is upregulated in many cancers. The overexpression of PTGS2 (COX-2) along with increased angiogenesis and SLC2A1 (GLUT-1) expression is significantly associated with gallbladder carcinomas.[28] Furthermore, the product of PTGS2 (COX-2),
PGH2 is converted by
prostaglandin E2 synthase into
PGE2, which in turn can stimulate cancer progression. Consequently, inhibiting PTGS2 (COX-2) may have benefit in the prevention and treatment of these types of cancer.[29][30]
COX-2 expression was found in human idiopathic epiretinal membranes.[31] Cyclooxygenases blocking by
lornoxicam in acute stage of inflammation reduced the frequency of membrane formation by 43% in the
dispase model of
PVR and by 31% in the
concanavalin one.
Lornoxicam not only normalized the expression of cyclooxygenases in both models of PVR, but also neutralized the changes of the
retina and the
choroid thickness caused by the injection of pro-inflammatory agents. These facts underline the importance of cyclooxygenases and prostaglandins in the development of PVR.[32]
PTGS2 gene upregulation has also been linked with multiple stages of human reproduction. Presence of gene is found in the
chorionic plate, in the
amnion epithelium,
syncytiotrophoblasts, villous fibroblasts, chorionic
trophoblasts,
amniotic trophoblasts, as well as the
basal plate of the placenta, in the
decidual cells and
extravillous cytotrophoblasts. During the process of
chorioamnionitis/deciduitis, the upregulation of PTGS2 in the
amnion and choriodecidua is one of three limited effects of inflammation in the
uterus. Increased expression of the PTGS2 gene in the
fetal membranes is connected to the presence of inflammation, causing uterine prostaglandin gene expression and immunolocalization of
prostaglandin pathway proteins in chorionic trophoblast cells and adjacent decidua, or choriodecidua. PTGS2 is linked with the inflammatory system and has been observed in inflammatory
leukocytes. It has been noted that there is a positive correlation with PTGS2 expression in the amnion during spontaneous labour and was discovered to have increased expression with gestational age following the presence of labour with no change observed in amnion and choriodecidua during either preterm or term labour. Additionally,
oxytocin stimulates the expression of PTGS2 in
myometrial cells.[33]
The mutant allele PTGS2 5939C carriers among the Han Chinese population have been shown to have a higher risk of
gastric cancer. In addition, a connection was found between Helicobacter pylori infection and the presence of the 5939C allele.[34]
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