This document discusses an enzyme called cyclooxygenase. Prostaglandin Endoperoxide H Synthase (Commonly known as "Cyclooxygenase") is an enzyme essential for immune response and mainly, in prostaglandin biosynthesis (a metabolic pathway in Arachidonic acid metabolism). Read the document for further information.

1. CYCLOOXYGENASE: IMMUNO-PHYSIOLOGIC REGULATOR
Sarines, Johnathan C.
HUB31
De La Salle University-Dasmarinas
Dasmarinas, Cavite Philippines
I. INTRODUCTION
Cyclooxygenase (COX), also known as Prostaglandin Endoperoxide H Synthase, is an enzyme
that catalyzes the first two processes in prostaglandin biosynthesis, oxygenation of arachidonic
acid and prostaglandin G (1), (2). Endothelial cells in the blood vessels, platelets, leukocytes and
other sites of inflammation have this enzyme, while arachidonic acid is present in phospholipids
of the plasma membrane. Hydrolysis of phospholipids can lead to dissociation of the hydrophilic
phosphate head and the hydrophobic tail which may include arachidonic acid. Cyclooxygenase is
basically produced by two types of genes: COX-1 and COX-2 genes (3). COX-1 gene forms
COX-1, while COX-2 gene forms the isoform of COX-1, COX-2 (3). Both isozymes may have
similarities in terms of the reactions they catalyze, yet they differ in inhibitors, function, active
sites, gene expression, and sequence of amino acids. COX-1 and COX-2 can promote
degenerative disease such as cancer, however this remains unclear. Alzheimer’s disease can be
the overall effect of inflammatory action of COX product.
II. CYCLOOXYGENASE
A. Plasma membrane, Arachidonic acid and Prostaglandins
The Plasma membrane is made up of phospholipids which consist of hydrophilic/polar head and
2 hydrophobic/non-polar tails. The hydrophobic head can either be phosphatidic acid,
phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, or phosphatidylinositides.
On the other hand, the hydrophobic tails are saturated and unsaturated fatty acids Phospholipids
can be broken down through hydrolytic reaction catalysed by phospholipase, an esterase (4).
Through such process, fatty acids like arachidonic acid are synthesized.
Arachidonic acid (AA) is a polyunsaturated form of arachidic acid (C20) which has cis
configurations in C-5, 8, 11 and 14. Thus, it is also known as cis-5, 8, 11, 14-eicosatetraenoic
acid. Arachidonic acid is mainly involved in the biosynthesis of oxygenated arachidonic acid
derivatives via three pathways: cyclooxygenase, lipooxygenase and epoxygenase pathways (5),
(6). Cyclooxygenase (COX) catalyzes the conversion of arachidonic acid to prostaglandin G
(PGG2), an intermediate of other prostanoids, and PGG2 to PGH2 (1), (2), (7), (8). Prostaglandins
are prostanoids which is made up of cyclopentane and its two substituents: heptanoic acid and
octane (8). Prostaglandins control several physiological and immune responses like inflammation,
hyperalgesia, pyrexia, vasoconstriction and vasodilation, blood coagulation, renal functions,
neurotransmission, circadian rhythm, and gastrointestinal cytoprotection (1), (8).
B. Structure, coenzymes and co-factors
Cyclooxygenase is made up of four domains, namely: Catalytic domain, Dimerization Domain
with epidermal growth factor (EGF), and Membrane Binding Domain (MBD). Some domains are
glycosylated (with sugar unitsFrom the name itself, MBD connects COX and internal membrane
of endoplasmic reticulum through a series of four ampiphilic α-helices that relatively consist of 50
amino acids (9). This domain is linked with Dimerization Domain through the carboxylic end of
dimerization domain. Dimerization Domain, on the other hand, consists of two monomers that
bind together through non-covalent bonds and three disulfide bridges in EGF. Amino acids
(estimately 50 amino acids) proline 65 to isoleucine 104 and leucine 156 to phenylalanine 176
comprise this domain, including the epidermal growth factors (9). In Figure 1, EGF is composed
of an anti-parallel β-pleated sheet. Four disulfide bridges, which are formed under oxidative
conditions, interconnect Dimerization Domain with the Catalytic Domain (9).

2. Figure 1 Half Structure of Cyclooxygenase (10)
Lastly, Catalytic Domain is made up of 480 amino acids, relatively, and a prosthetic group called
heme which is also present in Hemoglobin and Myoglobin. This is subdivided into two sites,
namely: Peroxidase and Cyclooxygenase Active Sites. Peroxidase active site is located on the
superior surface of the enzyme in which the heme is embedded. Heme is arranged on its location
through the bond formed by Ferric ion, proximal (His 423) and distal (His 241) histidines. Since
COX contains heme, Fe
3+
is the co-factor of COX. On the other hand, Cyclooxygenase active site
is a 25 x 10
-10
m long, 8 x 10
-10
m in diameter hydrophobic dead-end groove which differentiates
COX-1 and COX-2. This groove constricts when Arg 120, Glu 524, and Tyr 355 bonds non-
covalently, causing slimming of this active site. One of the differences of COX-1 and COX-2 is the
role of Arg 120. Arg 120, together with Tyr 355, is necessary for the attachment of Arachidonic
acid and competitive inhibitors, while Arg 120 is unnecessary for the substrate and inhibitor
attachment in COX-2 (9)
Figure 2 Cyclooxygenase Active Sites of COX-1 and COX-2 (9)
Figure 2 shows the cyclooxygenase active sites of COX-1 and COX-2 in which two-point
differences of isozymes are amino acid 523 (isoleucine for COX-1, valine for COX-2) and 513
(histidine for COX-1 and arginine for COX-2). The yellow “bulges” in COX-1 are formed due to Ile
523 which restricts the entry of selective COX-2 inhibitors that contain aromatic moieties. In the
case of COX-2, Val 523 does not form those “bulges”, thus entry of Coxibs is highly probable (9).
C. Mechanism of Catalysis
The metabolic activity of COX plays an important role in physiological responses of most organs
and inflammatory action of polymorphonuclear (PMN) leucocytes or immune responses. COX
inactivity might result to organ dysfunction and absence of inflammatory responses.

3. Figure 3 Mechanism of COX Catalysis (11)
Prior to the conversion of AA, Tyr 385 radical formation first occurs in the peroxidase active site,
illustrated in Figure 3, via conversion of Ferryl (IV)-oxo-porphyrin radical to Ferryl (IV)-oxo heme.
The first step of AA conversion involves hydrogen abstraction at C-13 due to tyrosyl radical and
the attachment of AA carboxyl moiety to Arg 120 and Tyr 355 (9). This would result to arachidonyl
radical and non-radical Tyr 385. After which, bisoxygenation takes place wherein oxygen gas (O2)
forms bonds with arachidonyl radical at C-9 and C-11. And, the last step is hydroperoxidation at
C-15 assisted by Ser 530 and Val 349 (9). From these series of steps, PGG2 is synthesized.
Conversion of PGG2 to PGH2 includes only one step which is reduction of hydroperoxide to
hydroxide through oxidation of heme. Conversion of AA and PGG2 may occur simultaneously as
AA conversion requires tyrosyl radical.
D. Kinetics of Reaction and Mode of Regulation
Arachidonic acid is usually at low concentration, approximately 1 – 10 μM. However, its
concentration can elevate up to 100 – 300 μM when AA derivative production is necessitated in
response to physiological and immune stimuli (12). COX inactivity can cause dysfunction of
organs and lack of immune response.
Activators of COX-1 and COX-2 include nitric oxide (NO) (9), Hydrogen peroxide (H2O2), PGH2
and some hydroperoxide – containing compounds. In the mechanism of COX catalysis,
hydroperoxide or peroxynitrite can act as endogenous oxidant which oxidizes heme. Those
activators can initiate this step which is a pre-requisite of arachidonic acid conversion. However,
in the case of NO, it may also act as an inhibitor through nitration of Tyr 385 (9).
Figure 4 Some Non-selective NSAIDs (right) and Coxibs (left) (9), (13)

4. Meanwhile, most of the COX inhibitors are synthetic in a form of Non-Steroidal Anti-inflammatory
Drugs (NSAIDs). NSAIDs are divided into major groups, namely: Classic NSAIDs and Selective
COX-2 inhibitors or sometimes called Coxibs (9). Classic NSAIDs, also known as non-selective
NSAIDs, can inhibit both COX isozymes, whereas Coxibs inhibit COX-2 only. Classic NSAIDs
include aspirin, ibruprofen, paracetamol, Indomethacin, Diclofenac, Naproxen, Piroxicam,
Mefenamic acid, Salsalate, Phenylbutazone, Ketoprofen and other non-selective NSAIDs. On the
other hand, Coxibs include Celecoxib, Valdecoxib, Rofecoxib, Deracoxib, Parecoxib, Etoricoxib,
Lumiracoxib and Meloxicam (7), (8), (9), (13). Aspirin is a popular NSAID which is well known as
a pain killer. It competitively inhibits (half-minimal) COX-1 at 0.3 μM and COX-2 at 50 μM by
acetylating Ser 530 in the cyclooxygenase active site (14). The orientation of Aspirin could
probably affect the IC50 of aspirin in both isozymes. Aspirin has a better orientation in COX-1
compared to COX-2 due to coordination with Arg 120. This permits acetylation of Ser 530 in
COX-1. Whereas, COX-2 have relatively larger active site compared to COX-1, however, the
orientation of aspirin is improper which makes COX-2 less sensitive to aspirin (9). Indomethacin,
another non-selective NSAIDs, competitively inhibits COX-1 at IC50 = 0.01 μM and COX-2 at IC50
= 0.6 μM (13). Coxibs can exclusively inhibit COX-2 due to the absence of yellow “bulges”
illustrated in Figure 2. In COX-2, Val 523 replaced Ile 523 which does not form those bulges that
restrict aromatic moiety-containing compounds. Thus, Coxibs can enter the cyclooxygenase
active site. Coxibs may not have a carboxyl group to attach itself to Ser 530 through an ester
bond, yet series of non-covalent bonds stabilizes the the binding of Coxibs (9).
NSAIDs, nitric oxide, H2O2, PGG2 and some hydroperoxide – containing compounds serve as
allosteric regulators of COX wherein NSAIDs are deactivators and the rest are activators. Thus,
one of the modes of regulation of COX is allosteric modification. Aside from allosteric regulation,
COX-1 and COX-2 can be transcriptionally controlled by Protein-Tyrosine-Kinase (PTK),
vanadate (VO4
3+
), cytokines and interleukins (9), (15).
Figure 5 Pathways of Arachidonic acid metabolism (5)
Figure 5 shows the pathways of AA metabolism. When cyclooxygenase pathway is restricted due
to inhibitors, AA can either be converted to hydroperoxyeicosatetraenoic acid (HPETE) via
Lipoxygenase pathway or Epoxyeicosatrienoic acid (EET) via Cytochrome P450 and epoxygenase
pathway.
E. Significance to Human Health and Associated Diseases
1. Significance to Human Health
Cyclooxygenase catalyzes the cyclization and bisoxygenation of AA, forming PGG2, and
reduction of PGG2 to PGH2. PGH2 derives prostaglandins, prostacyclins and thromboxanes that
have specific functions. PGE2 and PGI2 are responsible for inflammatory action, hyperalgesia,
pyrexia, vasoconstriction and vasodilation of visceral muscles (except in respiratory tract),
gastrointestinal mucus secretion, angiogenesis induction, speeding up of renal blood flow,
regulation of gastric acid secretion, and GI cytoprotection. On the other hand, thromboxanes
regulate thrombosis, vasocontraction of blood vessels and inflammation. Brochocontraction and
brochorelaxation in the respiratory tract are best facilitated by PGD2 and PGF2 (9).
2. Associated Diseases
Cyclooxygenase had been speculated for being involved in turmorigenesis via induced
angiogenesis and neurodegenerative diseases known as Alzheimer’s disease (AD) through
inflammation of neurons. However, those speculations remain obscure and unexplained. Studies
show that intake of NSAIDs could actually decrease the risk of having cancer and AD (9).