Current Pharmaceutical Design, 2004, 10, 577-588 577

Cyclooxygenase Enzymes: Regulation and Function

F.A. Fitzpatrick*

Department of Oncological Sciences & Department of Medicinal Chemistry, Huntsman Cancer Institute, University of
Utah School of Medicine, Salt Lake City, Utah 84112-0555, USA

Abstract: The cyclooxygenase isoenzymes, COX-1 and COX-2, catalyze the formation of prostaglandins, thromboxane,
and levuloglandins. The prostaglandins are autocoid mediators that affect virtually all known physiological and
pathological processes via their reversible interaction with G-protein coupled membrane receptors. The levuloglandins are
a newer class of products that appear to act via irreversible, covalent attachment to numerous proteins. COX enzymes are
clinically important because they are inhibited by aspirin and numerous other non-steroidal anti-inflammatory drugs. This
inhibition of COX confers relief from inflammatory, pyretic, thrombotic, neurodegenerative and oncological maladies.
About one hundred years have elapsed since Hoffman designed and synthesized acetylsalicylic (aspirin) as an agent
intended to lessen the gastrointestinal irritation of salicylates while maintaining their efficacy. During the past forty years
systematic advances in our understanding of the structure, regulation and function of COX isoenzymes have enabled the
design and synthesis of COX-2 selective inhibitors as agents intended to lessen the gastrointestinal irritation of aspirin and
non-selective NSAIDs. This review discusses: 1) how two separate catalytic processes in COX - peroxidase and
prostaglandin synthase - act in an integrated fashion manner to generate prostaglandins; 2) why irreversible inactivation of
COX is important constitutively and pharmacologically; 3) how cells have managed to use two closely related, almost
identical enzymes in ways that discriminate their physiological versus pathological roles; 4) how investigators have used
these advances to formulate and test medically important uses for old drugs (i.e. aspirin) and create new ones that still
seek to achieve Hoffman's original goal.

INTRODUCTION
The cyclooxygenase (COX) enzymes have elaborate
roles in human physiology and pathology [1], spanning the
cardiovascular [2], neuronal [3], renal [4], immune [5],
gastrointestinal [6] and reproductive systems [7, 8]. Since the
1960's, progressive improvements in our understanding of
their structure, function and regulation have enabled the
introduction of many chemical classes of non-steroidal anti-
inflammatory drugs (NSAIDS) into the pharmacopoeia [9-
11]. The recent discovery and design of the 'coxib' class of
NSAIDS exemplifies the doctrine of contemporary pharma-
ceutical design - the use of rational, objective, molecular
criteria in an urgent and highly integrated quest for a new
drug [12, 13]. Few dispute the advantages of this approach,
especially its superiority over empirical, biological appro-
aches that can distort our comprehension of structure-activity
relationships.
Oddly, the COX story begins in a mystical doctrine
popularized by a shoemaker in 17th century Germany. The
"Doctrine of Signatures" asserts that God annotated his
herbal creations with a sign (signature) indicating their uses.
Jakob Böhme (1575-1624) based his book "Signatura
Rerum: The Signature of all Things" on mystical visions that
illuminated this ancient idea. Enthusiasts adopted this
doctrine for medical purposes, convinced that one could
determine a plant's therapeutic utility from the physical traits
*Address correspondence to this author at the Department of Oncological
Sciences & Department of Medicinal Chemistry, Huntsman Cancer
Institute, University of Utah School of Medicine, Salt Lake City, Utah
84112-0555, USA; Tel: 801-581-6204; Fax: 801-585-0101;
E-mail: frank.fitzpatrick@hci.utah.edu
of its flowers, roots, or leaves; or the place that supported its
growth. Considering the immature state of chemistry at that
time, physicians had few better alternatives than the Doctrine
of Signatures as a guide for the discovery of medicines.
Thus, in 1763 Reverend Edward Stone reported to the Royal
Society in London his use of a powder prepared from the
dried bark of the white willow tree for the effective treatment
of fever in over 50 patients. The Doctrine of Signatures
recommended the willow tree to Dr. Stone because it
flourishes in damp locales where fevers, aches and pains are
common in the population. Whether divinely ordained or
accidental, the Doctrine of Signatures did prompt the identi-
fication of salicylic acid as the active ingredient of willow;
the "design" and synthesis of acetylsalicylic acid (aspirin) to
alleviate gastrointestinal toxicity while maintaining salicyl-
ate's analgesic efficacy, and ultimately the inauguration of
the modern pharmaceutical industry with formation of Bayer
& Co in 1899. After one hundred years aspirin is still widely
used as an anti-pyretic, anti-inflammatory and analgesic
agent [14].
The discovery of the COX enzymes and the prosta-
glandins (PG) occurred over 60 years after the introduction
of aspirin and salicylates into medical practice. When a
therapeutic agent has a sixty years head start and an
established record of efficacy for inflammation, pain and
fever how does understanding its mechanism of action
matter? This chapter will review our current concepts on the
regulation and function of COX enzymes. First, we will
present the basic catalytic mechanisms of the COX enzymes
and their regulation. Second, we will discuss how two
separate catalytic processes - peroxidase and PGH synthase -
act in an integrated manner. Third, we will discuss the

1381-6128/04 $45.00+.00 © 2004 Bentham Science Publishers Ltd.

578 Current Pharmaceutical Design, 2004, Vol. 10, No. 6
irreversible inactivation of COX catalysis by constitutive,
mechanism-based, "suicide" inactivation and compare this to
the irreversible inactivation by acetylation of COX. Fourth,
we will present some of the lessons learned from the
determination of the crystal structure of COX. Fifth, we will
present a glimpse at the transcriptional and post-translational
regulation of COX expression. This topic is covered in
greater detail elsewhere in this issue of the Journal. Sixth, we
will summarize some cellular regulatory mechanisms and
functions of the COX enzymes and consider a few
paradoxes. For example, do COX enzymes have functions
that are unrelated to PG formation? Are all NSAIDS
equivalent? Seventh, we will summarize how all of this
detailed information has advanced the design and use of
NSAIDS in modern medicine. For instance, our detailed
understanding of how COX enzymes catalyze PG formation
has enabled the use of low dose aspirin as a unique anti-
thrombotic therapeutic entity. Likewise, our detailed
understanding of the different roles and structures of the
COX-1 and COX-2 isoenzymes has enabled the design of
coxib class NSAIDS to avoid gastrointestinal toxicity while
maintaining analgesic and anti-inflammatory efficacy - the
identical medical rationale that inspired Hoffman's synthesis
of aspirin in 1897.
CATALYTIC MECHANISMS OF CYCLOOXY-
GENASE
In the sections below the term COX refers to both
members of the enzyme family, or to the enzyme used in
investigations that pre-dated the discovery of the COX-2
isoenzyme. The terms COX-1 and COX-2 refer to the
respective isoenzymes. The reader seeking more detailed
reviews on the catalytic mechanisms of COX enzymes
should refer to recent publications [15, 16].
COX enzymes interest scientists and physicians because
they generate biologically important autocoid lipid
mediators, the PG and thromboxanes (Tx). How did
investigators unravel the catalytic mechanism of these
enzymes? Between 1965-1968 investigators at the
Karolinska Institute and at Unilever Laboratories established
the absolute configuration of PGs [17] and identified
molecular oxygen and arachidonic acid as co-substrates in
PG biosynthesis [18, 19]. This information, plus peculiar
kinetics of oxygen consumption during PG biosynthesis,
suggested the existence of transient intermediates in the
biosynthetic pathway, which eventually led to the isolation
and structural elucidation of the prostaglandin
endoperoxides, PGG2 and PGH2 in 1973-74 [20, 21]. A
contemporaneous discovery that aspirin abolished PG
formation [22] prompted the idea that the PG biosynthesis
enzyme, COX, was a “molecular target” of great medical
significance. Within 2 years, the discovery of the vasoactive,
pro-thrombotic substance thromboxane A2 (TxA2) and its
physiological antagonist, PGI2, as products derived from the
COX catalysis accentuated its importance [23, 24].
In the 1970's investigators at the University of Michigan
sought experimental conditions for optimizing PG formation
by isolated purified COX enzyme [25-28]. The COX enzyme
purified to homogeneity from sheep vesicular glands had a
molecular weight of 70,000 daltons; contained appreciable
F.A. Fitzpatrick
levels of non-heme iron and trace levels of heme iron;
catalyzed the insertion of the two oxygen molecules
necessary for the formation of PGG2 / PGH2 from
arachidonic acid; and reacted covalently with [3H]
acetylsalicylic acid to form a radioactive adduct [28]. The
Unilever group, using a different purification scheme added
that COX was a glycoprotein with both peroxidase and PG
synthase activity [29]. They also established that loosely
associated haemin protected the enzyme from inactivation by
peroxide. These two reports laid the foundation for our
understanding of COX catalysis and its inhibition by
NSAIDs.
The availability of methods to purify COX permitted the
investigation of its reaction kinetics and its interaction with
NSAIDs. Investigators had noted that certain reducing agents
stimulated purified COX in a reversible, time-dependent
manner with modification of its Vmax and Km [30].
Scientists at Merck studied the stimulation of COX, and its
inhibition, in an integrated way using structurally distinct
classes of NSAIDS [31]. Their findings were among the first
to establish that 'stimulators', e.g. phenols, affected both
stimulation and inhibition of COX and that NSAIDS could
be divided into at least two distinct groups - introducing the
notion of inhibitor specificity. Fenamates inhibited COX
weakly in the absence of phenol but potently in its presence.
In contrast, aryl propionates (ibuprofen, flurbiprofen) and
indomethacin inhibited COX more potently in the absence of
phenol. The relative potency of COX stimulation by phenol
> norepinephrine > tryptophan > benzoquinone > anisole
corresponded with their synergistic effect on the fenamates
and their antagonist effect on the arylpropionates and
indomethacin. These data also suggested that constitutive
phenolic anti-oxidants in cells could modulate both COX
activity and its sensitivity to NSAIDS. Intrinsically, this
investigation also established that a better understanding of
COX kinetics could provide meaningful and unanticipated
information about NSAIDS.
One of the most important insights into the catalytic
mechanisms of COX came from studies using twenty carbon
polyunsaturated fatty acids (PUFA) with olefin
configurations different from arachidonic acid (5,8,11,14-
cis-eicosatetraenoic acid). Investigators showed that purified
COX, which catalyzed the formation of the PG
endoperoxides from arachidonic acid (20:4) in a bis-
dioxygenase reaction, also catalyzed the insertion of
molecular O 2 into the C-11 carbon of 8,11-cis-eicosadienoic
acid (20:2) in a mono-dioxygenase reaction [32]. Both
oxygenation reactions had similar pH optima, substrate Km
values, a need for peroxide, and an absolute requirement for
heme. Importantly, NSAIDS inhibited C20:2 oxygenase
activity. This investigation implied that abstraction of the 13-
proS hydrogen from arachidonic acid, followed by C-11
oxygenation is one of the rate-limiting steps of COX action.
It also drew attention to useful precedents with plant
lipoxygenase (LOX) enzymes [33] and provided a basis to
understand how COX catalysed the formation of the
peroxide bridge between the C-11 and C-9 atoms of
arachidonic acid.
These separate oxygenase activities (11-lipoxygenase and
15-lipoxygenase) of COX have interested investigators
Cyclooxygenase Enzymes
sporadically and for biological, enzymological, and
pharmacological reasons. For instance, COX isolated from
human endothelial arteries converts arachidonic acid into its
bis-dioxygenase products PGG2 / PGH2, along with its
mono-dioxygenase products 11-hydroxeicosatetraenoic acid
(11-HETE) and 15-HETE [34]. It is uncertain whether these
HETEs are biologically relevant. From an enzymological
perspective, the formation of multiple products indicates that
catalytic competency depends on the spatial arrangement of
arachidonic acid within the COX active site [35]. Briefly,
arachidonate can occupy the active site of COX-1 in at least
three different, catalytically competent arrangements that
yield PGG2, 11R-HETE, and 15-HETE, respectively.
Arachidonic acid assumes its spatial arrangement after its
entry into the active site. If its arrangement distorts the
optimal spacing between C-9 and C-11 the 11-peroxyl
radical cannot attack C-9 to generate an endoperoxide
efficiently. Instead, 11-HpETE is the default product. The
observation that preparations of COX enzyme could still
generate 15(R)-HETE after treatment with aspirin was one of
the important, early clues that there existed a second, unique
COX isoform, now termed COX-2 [36].
Structural studies indicate that the COX-1 active site is a
hydrophobic channel that protrudes from its membrane-
binding domain into its globular domain [37, 38]. A detailed
crystal structure of Co (3+)-heme COX-1 complexed with
arachidonic acid predicts that 19 active site residues interact
Fig. (1). Amino acid residues in COX 1 that govern substrate positioning and catalysis.
Current Pharmaceutical Design, 2004, Vol. 10, No. 6 579
with the substrate. About 95% of the interactions involve
hydrophobic residues. Site-directed mutational analyses have
positioned the residue that abstracts the proS hydrogen from
C-13 of arachidonate (Tyr-385); the residues that orients C-
13 of arachidonate for hydrogen abstraction (Gly-533 and
Tyr-348); the residue responsible for the electrostatic
interaction with the carboxylate anion of arachidonate (Arg-
120); and lastly the residues that govern the orientation of
arachidonate so that it is optimally arranged to yield PGG2
(Val-349, Trp-387, and Leu-534). When these latter residues
do not orient arachidonate optimally COX converts it into
11R-HETE, and 15-HETE as mentioned above. Fig. (1)
depicts the interactions between arachidonic acid and the
amino acid residues lining the COX-1 active site and the
catalytic reaction. Both isoforms of COX have similar
amino-acid sequences and catalytic mechanisms. A 3A°
crystal structure of murine apo-COX-2 also affirms that
arachidonic acid can assume orientations within the active
site that are catalytically incompetent. For instance, the
anionic carboxylate end of the substrate can form hydrogen
bonds with Tyr 385 and Ser 530, rather than the predicted
ion pair with Arg120. Instead, the Arg 120 residue contacts
the hydrophobic -end of the substrate [39]. In its
catalytically competent state COX-2 resembles COX-1, with
the carboxylate anion of arachidonic acid coordinated by
Arg 120 and Tyr 355 and the C13 pro (S)-hydrogen atom of
the substrate positioned near Tyr 385.
580 Current Pharmaceutical Design, 2004, Vol. 10, No. 6
BI-FUNCTIONAL CATALYSIS IN A SINGLE
ENZYME PROTEIN: INTEGRATED ACTIONS OF
PEROXIDASE AND CYCLOOXYGENASE
Once investigators established that tyrosine and tyrosyl
radicals initiated, sustained and terminated COX catalysis
[40, 41] it became possible to clarify how two separate
catalytic processes - peroxidase and PG synthase - work
coordinately to manage a radical reaction within a single
COX enzyme [42].
COX has an intrinsic peroxidase activity [25, 28, 29].
Early experiments, using limited proteolysis of COX,
investigated whether the peroxidase and COX catalytic
processes occurred at separate or overlapping sites in the
enzyme. Digestion of purified COX enzyme with trypsin
generated an amino terminal 33 kDa fragment and a 38 kDa
fragment. When purified neither the 33- nor 38-kDa
fragment had PGH synthase activity; however, treatment
with [3H]acetylsalicylic acid introduced [3H]acetyl substi-
tuents into the 38-kDa fragment, but not the 33-kDa
fragment suggesting a prominent part of PG synthase active
site occurred within this fragment [43]. Subsequent studies
by the same group showed that the peroxidase activity
declined rapidly, corresponding to the rate of proteolysis
[44]. Spectrometric analyses suggested that proteolysis
decreased heme binding at a site that controls peroxidase
activity. Since trypsin cleaved COX between Arg253 and
Gly254 the investigators proposed that the heme responsible
for the peroxidase activity of COX bound to the His residue
of the sequence His250-Tyr251-Pro252-Arg253. A compar-
able study affirmed that the two separate enzymatic pro-
cesses could be differentiated. Only the PG synthase activity
was lost during catalysis; the peroxidase activity was
retained. By contrast, only the peroxidase activity was lost
by limited proteolysis with trypsin, suggesting the presence
of separate PG synthase and peroxidase structural domains in
the COX enzyme [45]. It is important to note that one
investigator has reported the opposite result [46]. Namely,
proteolytic attack on the Arg 253 region of COX diminished
both PG synthase and peroxidase activities together. The
reason for the discrepancy is uncertain.
Improved methods for quantifying peroxidase activity
fortified the notion of a regulatory relationship between the
peroxidase and PG synthase activities of the COX enzyme
[47]. Kinetic and chemical studies established that the
peroxidase in COX is a classical heme peroxidase.
Comparison of a series of phenols, aromatic amines, beta-
dicarbonyls, naturally occurring compounds, and NSAIDS
indicated differences in their ability to act as reducing
substrates. Importantly, there was no structure-activity
correlation between agents that sustain peroxidase activity
and agents that inhibited COX. Thus, pharmacological data
implied that the efficient hydroperoxide-reducing activity
(peroxidase) might be responsible for the hydroperoxide
dependent stimulation of COX during the initial phase of
catalysis, as well as protection against hydroperoxide-
dependent inactivation of COX during the sustained phase of
catalysis.
Thus, the current mechanistic model places peroxidase
and COX catalysis at separate but electronically interrelated
active sites. The stimulating effect of peroxides is best
F.A. Fitzpatrick
attributed to oxidation of the heme group of the peroxidase,
which leads to controlled oxidation of the active site tyrosine
of COX. An iron-containing heme prosthetic group is the
oxidant that ultimately produces a tyrosine radical. Although
COX contains Fe+3 in its resting state, the oxidation of Tyr-
385 via the corresponding reduction of Fe3+ to Fe2+ is not
considered favorable. Instead, it is more likely that peroxides
convert the heme of COX into a ferryl-oxo complex, which
is more likely to oxidize Tyr-385.
There have been several insights into the physical
chemical and enzymatic role of radicals in COX catalysis
since the initial report on their occurrence [40]. For instance,
purified COX-1 and COX-2 enzymes that have been
reconstituted with manganese protoporphyrin IX and probed
with peroxides yield electron paramagentic resonance (EPR)
spectra that reflect radical-dependent oxidation [48]. This
study indicated that the peroxide-generated radicals in COX-
2 and MnCOX-1 each form the same carbon-centered
arachidonic acid radical that subsequently reacts with oxygen
to the lipid hydroperoxide. The EPR data also indicate that
the metastable arachidonate radical contains a planar
pentadienyl radical, as widely speculated.
Given the similarities between their primary sequences,
their crystal structures and their catalytic mechanisms the
constitutive regulation of COX-1 and COX-2 catalysis is
similar with only a few nuances. For instance, catalysis by
the COX-2 isoenzyme can proceed at hydroperoxide levels
10-fold lower than those needed to promote catalysis by
COX-1. Investigators have speculated that this may be a
mechanism to generate PG preferentially by COX-2 when
both isoforms occur in the same intracellular compartment
[49, 50]. It is unclear why cells would find this advanta-
geous. Likewise, the structural stability of COX-2, both in
the active site regions and in the subunits overall, is less than
that of COX-1 [51]. This may relate to their different
preferences for fatty acid substrates and inhibitors and the
larger size of the pocket in the active site of COX-2. A valine
residue at position 509 in COX-2 corresponds to the isoleu-
cine residue at position 523 in COX-1. However, mutage-
nesis studies in both COX-1 and COX-2 suggest that
exchange of valine and isoleucine at position 509/523 does
not account fully for all the differences in inhibitor
specificity between the isoforms [52]. Thus, differences in
structural stability may contribute to the ligand selectivity in
the respective enzyme active sites.
IRREVERSIBLE INACTIVATION OF COX: CONSTI-
TUTIVE 'SUICIDE' INACTIVATION
As discussed above COX enzymes use peroxides and
peroxidase in a radical dependent mechanism to stimulate
their initial rate of catalysis. Once these processes reach
maximal velocity how can they be blunted? “Suicide”
inactivation, or mechanism-based inactivation, appears to be
one of the rate-limiting processes governing COX.
Investigators described this aspect of COX catalysis in some
of the earliest studies of the enzyme [26]. It is now known
that many other eicosanoid biosynthetic enzymes undergo
"suicide" inactivation, e.g. Tx synthase [53], PGI synthase
[54], LTA4 hydrolase [55, 56], 5-LOX [57- 59], 12-LOX
[60] and 15- lipoxygenases [61-63]. Studies on 15-LOX may
Cyclooxygenase Enzymes
be particularly relevant because COX carries out this same
reaction. Suicide inactivation is a cumbersome regulatory
process; yet COX manages to use metastable radical inter-
mediates for catalysis that generates reactive hydroperoxide
or endoperoxide products (PGG2 and PGH 2). Thus, it is not
surprising that COX enzymes have consigned the chemical
intermediates in their Michaelis complex to two separate
fates. One fate is a normal catalytic cycle yielding product
and regenerating enzyme. A second fate is a catalytic cycle
leading to irreversible inactivation of the enzyme.
The first detailed studies of COX auto-inactivation, by
the Merck group, showed that the generation of PGH2 from
arachidonic acid released oxidizing equivalents. Phenol and
methional, which quench oxygen-centered radicals,
increased the initial rate and the extent of catalysis prior to
COX inactivation. Hence, it appeared that the COX was
irreversibly self-deactivated due to its oxidation by oxygen-
centered radicals that formed as a result of the peroxidase-
dependent reduction of the hydroperoxide on PGG2 [64].
This information aligns well with our current understanding
of how peroxide → peroxidase → tyrosine → arachidonic
acid propagate radical-dependent oxidation reactions.
However, it is doubtful that this is the only chemical process
that participates in the modification and inactivation of
COX. For example, PGG2 and PGH2 may react covalently
with proteins [65 -67]. Likewise, hydroperoxy-eicosate-
traenoic acids, like the 15-HpETE formed by COX-1 or
COX-2 under certain conditions may be able to oxidize
methionine residues [68]. It is not known if this actually
occurs in COX but a methionine residue at position 522
could be an attractive candidate for such a reaction.
Recently, investigators from Vanderbilt University have
provided a novel hypothesis relevant to the catalysis-
dependent, irreversible inactivation of COX [69]. The
endoperoxide PGH 2, can rearrange spontaneously into the γ-
keto aldehydes, levuglandin (LG) E2 and LGD2. Fig. (2)
depicts their structures. LG can react with the ε-amino group
of lysine to form both the lysyl-levuglandin Shiff base and
the pyrrole-derived lysyl-levuglandin lactam adducts. These
lysyl-LG adducts form on COX following its oxygenation of
arachidonic acid. Approximately 25-20% of the arachidonic
acid provided to COX becomes covalently associated with
O
O
OH
PGH2
O O
COOH
O OH
LGD2
Fig. (2). Structures of levuloglandins derived from PGH2.
Current Pharmaceutical Design, 2004, Vol. 10, No. 6 581
the enzyme and LG generated from PGH2 form covalent
adducts with a nearly 100% reaction yield, consistent with
the reactivity of these electrophiles. Importantly, the lysyl-
LG adducts retain reactivity so the Schiff base adduct with
lysine can undergo nucleophilic attack, and the oxidation of
the pyrrole adducts generates sites of electrophilic reactivity.
Thus, lysyl-LG adducts can form covalent bonds with other
nucleophiles, leading to intra- or even intermolecular cross-
linking. It is likely that some of the unidentified PGH2
adducts reported in earlier studies are actually LG adducts.
The site of LG attack on COX is uncertain, as is any
relationship between adduct stoichiometry and COX
inactivation. This type of reaction does occur in cells [70]
and tissue [71]. Thus, separate from its relevance to auto-
inactivation of COX this process must now be considered as
a significant aspect of many COX mediated maladies,
especially neurodegenerative disorders and cancer.
“Suicide” inactivation of COX has some implications
that have not yet been fully appreciated, or fully integrated,
into models of eicosanoid biosynthesis. For example, efforts
to correlate the amount of cellular COX enzyme with the
amount of product, e.g. PGE2, need to take into account the
catalytic history of the enzyme. For instance, if cells
accumulate COX-2 protein via de novo synthesis and if this
COX-2 propagates catalysis continuously then there should
co-exist two “pools” of enzyme - a pool that retains catalytic
activity and a pool with depleted catalytic activity. Right
now there is very little known about the intracellular
degradation of COX-2 protein. How is auto-inactivated COX
recognized and targeted for degradation? Is this different
from degradation of active COX? Does cellular
accumulation of “ suicide” inactivated enzyme account, in
part, for any poor correlation between protein expression and
product formation (e.g. COX-2 protein versus PGE2)? If
cells fail to degrade suicide-inactivated enzymes are there
any pathological consequences? The identification of LG
adducts to COX should allow investigators to address these
questions experimentally.
Analogous to its PG synthase activity, the peroxidase
activity of COX also has a self-limited number of catalytic
turnovers. Data suggest that the auto-inactivation process
originates with an enzyme (Intermediate II), which contains
COOH
COOH
O OH
LGE 2
582 Current Pharmaceutical Design, 2004, Vol. 10, No. 6
an oxyferryl heme and a tyrosyl radical that is converted
quickly (k = 0.5-2 s-1) to an Intermediate III, which retains an
intact heme group that is not covalently linked with COX-1
This implies that the heme group in Intermediate III is not
properly coordinated to the histidine ligand(s) but still
remains non-covalently associated with the protein [72].
Irreversible heme destruction occurs during COX catalysis,
but at a much slower rate than the conversion of Intermediate
II to III. Others have substantiated this model of COX
inactivation but provided some important insights. Under
conditions where COX inactivation is complete the enzyme
retains ∼ 60% of its peroxidase activity. The rates of
peroxidase inactivation were indistinguishable for native
COX-1 and an Y385F mutant which cannot form the Tyr385
radical of COX Intermediate II suggesting that another
species, corresponding to Intermediate III with radical
centered on a tyrosine other than Tyr385, is the precursor for
peroxidase inactivation [73].
Overall, COX-1 and COX-2 have adapted complex
chemical processes for the practical purpose of regulating
their own activation and inactivation in an orderly way. In
this regard they are among nature's most chemically
sophisticated creations.
IRREVERSIBLE INACTIVATION OF COX: PHAR-
MACOLOGICAL ACETYLATION
Irreversible inactivation of COX via acetylation is a less
complex phenomenon chemically but a more significant one
pharmacologically. This reaction accounts for the unique
value of aspirin in cardiovascular medicine and it has
allowed investigators to unmask some notable differences
between the active sites of COX-1 and COX-2.
Shortly after investigators proposed that aspirin inhibited
COX [22] investigators at Washington University showed
that aspirin irreversibly acetylated COX in microsomes of
seminal vesicles and human platelets [74]. The rate and
stoichiometry of COX acetylation correlated precisely with
inhibition of COX activity. At concentrations correlated with
its Michaelis constant the substrate, arachidonic acid,
prevented COX acetylation by aspirin. A reversible COX
inhibitor indomethacin also inhibited the acetylation
reaction. These data established that aspirin exerts its anti-
inflammatory and anti-platelet effects by acetylating the
COX active site. Subsequent investigations established the
serine 530 residue in the active site as the one modified
[75,76] and, incidentally, reinforced the fact that the PG
synthase activity and peroxidase activity of COX were
distinct and separable, pharmacologically. Treatment of
COX with an appreciable excess of other acetylating agents,
such as N-acetylimidazole, can inactivate both COX and
peroxidase activity. However, COX acetylated by N-
acetylimidazole contains acetyl adducts throughout the
protein. Thus, binding of the salicylate portion of aspirin in
the active site of COX governs its tendency to react solely at
Ser530 [77]. The acetylated serine 530 residue is not readily
reversed by hydrolysis - this is consistent with our current
understanding that the COX active site is a hydrophobic
pocket that evolved to accommodate a very hydrophobic
substrate - see Fig. (1). Hydrolysis is unlikely in such
conditions and the aspirin effect is difficult to reverse.
F.A. Fitzpatrick
Functionally, inhibition of COX by aspirin is "reversed"
only when de novo protein synthesis replaces inactive
enzyme with newly translated enzyme. This has had
profound implications for the use of aspirin in thrombosis
because platelets, which lack a nucleus, cannot regenerate
COX [78-81]. It is now incontestable that aspirin reduces the
incidence of vascular occlusive events in patients at risk of
thrombotic complications and that its survival benefit persist
through ten years of follow-up [82, 83]. These beneficial
effects are due to suppression of platelet aggregation from
the permanent depletion of COX activity for the full lifespan
of a platelet (∼ 6 days). The rational use of low-dose aspirin,
which spares the gastrointestinal tract while maintaining
anti-thrombotic efficacy, results directly from understanding
the molecular mechanism of aspirin in detail and the
availability of quantitative methods to relate biochemical
effects with clinical effects. Whether measured in economic
or human terms the detailed knowledge of COX catalysis
and its relationship to vascular biology has been a
remarkable medical achievement.
Can one design an irreversible inhibitor based on selec-
tive acetylation of the COX-2 active site? Investigators from
Vanderbilt University have made progress toward this goal
with their design and synthesis of o-(acetoxyphenyl)hept-2-
ynyl sulfide (APHS) which inhibits COX-2 selectively (100
× vs. COX-1) [84]. Structure-activity relationships (SAR)
indicate that the S-alkyl chain length is a determinant of
potency, with a heptyl chain optimal. Insertion of a triple
bond into the heptyl chain enhanced potency and selectivity.
Sulfides were also more potent and selective COX-2
inhibitors than the corresponding sulfoxides or sulfones or
other heteroatom-containing compounds. This novel class of
covalent modifiers may serve as potential therapeutic agents
in inflammatory and proliferative disorders without gastro-
intestinal or hematologic side effects [85]. The optimization
of the clinical pharmacology of these compounds should
prove interesting because in pathological states cells may
induce COX-2 expression continuously. In other words, the
half-life of COX-2 protein may govern the dosing schedule,
not the half-life of the compound. Clinical assessment of
these novel agents should be an important source of
information on how expression and degradation of COX-2
change in disease.
As mentioned above, the observation that COX enzyme
could still generate 15(R)-HETE after treatment with aspirin
coincided with the discovery a second, unique COX isoform,
now termed COX-2 [36, 86]. Thus, some of the earliest
known distinctions between COX-1 and COX-2 came from
investigations using aspirin as a probe. Detailed investigation
of the stereospecific conversion of arachidonic acid into
15(R)-HETE has provided a glimpse at the alignment of
substrate within the active site of COX-2. Remarkably, the
hydrogen abstraction leading to formation of 15R-HETE by
aspirin-treated COX-2 is identical to the hydrogen
abstraction leading to PG formation. These data imply that
the respective catalytic reactions must involve significantly
different stereochemistry because the 15(S) hydroxyl groups
in PG have the opposite configuration as the 15(R) hydroxyl
groups in 15(R)-HETE. Among three models considered to
explain these data, the best one predicts that acetylation of
Ser-516 in COX-2 limits the space available to accommodate
Cyclooxygenase Enzymes Current Pharmaceutical Design, 2004, Vol. 10, No. 6 583

the ω end of the arachidonic acid carbon chain [87, 88]. With
intact COX-2, hydrogen abstraction and O2 insertion
generate hydroperoxy substituents with 11(R) - and 15(S)-
configurations. With acetylated COX-2, steric hindrance by
the acetyl group distorts the normal arrangement of
arachidonic acid within the active site, displacing the ω end
of its carbon chain. Insertion of O2 into the distorted
arachidonate from the conventional direction then generates
the 15(R)- configuration. The distortion may also render the
C-11 carbon so inaccessible to O2 that no PG can form. This
result departs from the so-called "antarafacial rule" typifying
the dioxygenase reactions of LOX and COX enzymes. This
rule specifies that the enzyme abstract hydrogen from one
side of a planar 1,4-cis-pentadiene and insert O2 from the
other as shown in Fig. (3). However, this rule and
terminology only apply when the 1,4-pentadiene is in a
planar conformation [87].
Fig. (4) depicts the two cases outlined above, along with
an hypothetical third case. When one inhibits COX-1 by
acetylating its active site Ser 530 residue the enzyme cannot
form any PG or other oxygenated lipids. When one inhibits
COX-2 by acetylating its active site Ser 516 the enzyme
cannot form any PG but it can form 15(R)-HETE. It is will
be interesting to ascertain whether 15(R) HETE contributes
in any way to the pharmacological actions of aspirin or
APHS [84, 85]. Although speculative, it seems possible that
one might achieve a full, irreversible inactivation COX-2
with other o-(alkyloxyphenyl)hept-2-ynyl sulfide that added
an acyl group larger than acetyl to Ser 516. For instance, o-
(2-propionyloxyphenyl)hept-2-ynyl sulfide or o-(2-methyl-
butyloxyphenyl)hept-2-ynyl sulfide might be candidates to
test this hypothesis and explore whether elimination of PG
formation is sufficient to neutralize COX or whether
elimination of PG plus 15(R)-HETE is advantageous. The
distinctive effects of aspirin on COX-2 catalysis may be
relevant to paradoxical reports that aspirin does not protect
against intestinal tumorigenesis in mice that harbor defective
alleles of the adenomatous polyposis coli (APC) tumor
suppressor.

H H
O O
O O O OOH
O H
O -H +H
O

CO2H CO2H
CO2H

Fig. (3). Antarafacial relationship between hydrogen abstraction from C13 and O2 insertion.

COX-1 + Aspirin AA
O
No products
O-C-CH3
Ser 530

COX-2 + Aspirin or APHS

No PG
AA + O2 15R-HETE ….. Function ?
O
O-C-CH3
Ser 516

COX-2 + alkoxyPHS ? AA

No products ?
O alkyl
O-C
Ser 516
Fig. (4). Effect of acetylation on substrate access to COX-1 and COX-2. Speculative effect of acylation on COX-2.
584 Current Pharmaceutical Design, 2004, Vol. 10, No. 6
Readers interested in the coxib class of COX-2 selective
inhibitors and the basis for inhibitor selectivity should see
the review by Patrignani in this issue as well as the following
[89- 92].
LESSONS FROM THE X-RAY CRYSTAL STRUC-
TURES OF COX-1 AND COX-2
Investigators have obtained X-ray crystal structures for
COX-1 and COX-2 at about 3 angstroms resolution [93-96].
One lesson derived from these crystal structures is that
conventional biochemical, pharmacological and molecular
biological approaches had provided a very accurate and
precise picture of the COX molecules. These crystal
structures have confirmed previous held concepts such as the
hydrophobic nature of the COX active site, the acetylation of
serine residues within the active site as a basis for aspirin
inhibition, the relative differences in the volumes of the
active sites of COX-1 versus COX-2, the existence of two
separate sites for peroxidase and PG synthase catalysis, the
basis for salicylate antagonism of aspirin acetylation, and
other notions. A second lesson is that the progressive
improvement in the selectivity of inhibitors for COX-2
versus COX-1 could not be obtained without the molecular
coordinates and distance data from crystal structures. A third
lesson is that we are still using the doctrine of signatures -
we just rely on more sophisticated technology to observe the
signs.
CELLULAR REGULATION OF COX PROTEIN
EXPRESSION AND FUNCTION
A study in 1989 [97] provided a glimpse at a regulatory
process that has assumed great importance for the COX
enzyme family. Investigators at Upjohn showed that a
typical growth factor, platelet-derived growth factor (PDGF),
caused a sustained increase in PGE2 biosynthesis,
commencing within 10 min, reaching a maximum after 2 hrs
and enduring beyond 2 hrs in NIH 3T3 cells. Second, they
showed that cycloheximide inhibited > 90% of the PGE2
synthesis initiated by PDGF, suggesting that de novo protein
synthesis, presumably de novo synthesis of a COX enzyme
was responsible. However, additional experiments in which
NIH 3T3 cells were exposed to exogenous arachidonic acid,
instead of PDGF, showed that these cells had ample basal
capacity for COX catalyzed formation of PGE2.
Furthermore, the steady-state level of COX mRNA in NIH
3T3 cells rose, but this rise followed, rather than preceded,
the increase in PGE2 synthesis, as one would have expected.
Finally, COX protein levels did not rise appreciably
following PDGF stimulation, even when the corresponding
mRNA levels were elevated. These data prompted two
questions. First, why would cells need de novo synthesis of
additional COX enzyme if they already had ample COX to
convert any available arachidonic acid into PGE2? Second, if
de novo synthesis of COX enzyme did account for increased
PGE2 formation in cells incubated with PGDF, why were the
temporal and stoichiometric relationships between mRNA
accumulation, protein accumulation and PGE2 formation so
distorted? Discovery of the COX-2 isoenzyme helped clarify
these paradoxes [86].
Generally, restricted enzyme expression is a prominent
mechanism for regulating eicosanoid biosynthesis. Restricted
F.A. Fitzpatrick
expression of Tx synthase by platelets and restricted
expression of PGI2 synthase by endothelial cells account for
the medical benefits of low-dose aspirin in thrombo-
occlusive disorders [98] and, in part, for the functional
antagonism between platelets and endothelium in vascular
biology [99-101]. Restricted expression of 5-LOX in
granulocytes is also critical for transcellular biosynthesis of
leukotrienes by combinations of granulocytes, erythrocytes,
platelets and endothelium [102-104]. Numerous investigators
have contributed to our current understanding of restricted
(i.e. inducible) expression of the COX-2 isoenzyme [105-
109]. Of particular note are the series of studies by
Needleman and colleagues who first showed convincingly
that in most cells and tissues there is a constitutive COX
which is unaffected by steroids, while in inflammatory cells
and inflamed tissue there is an inducible COX regulated by
glucocorticoids [110-112]. Under normal physiological
conditions glucocorticoids restrain inducible COX
expression. Depletion of glucocorticoids or accumulation of
cytokines and interleukins causes induction of COX. The
motivation to discover therapeutic substitutes for
glucocorticoids and the eventual design of COX-2 selective
inhibitors owes much to these studies.
COX-2 induction involves the orchestration of
membrane-to-nucleus signaling processes such as tyrosine
kinases, protein kinase C isoforms, peroxisome proliferator
activated receptor, ras, NF-κB, and other transcription
factors [113]. Many of these converge on the mitogen
activated protein kinase (MAPK) pathway involved in the
transcriptional regulation of inflammatory genes [114]. It is
now established that cells modulate COX-2 expression by
transcriptional and post-transcriptional mechanisms that
affect mRNA stability [115, 116].
Presumably, high expression of COX-2 in many tumors
derives, in part, from somatic mutations that persistently
activate signaling pathways for COX-2 expression (e.g. ras,
various growth factor receptor tyrosine kinase pathways).
However, tumors are also susceptible to dramatic changes in
the methylation of cytosine in the CpG islands of their
genome. It now appears COX-2 expression may be
"silenced" epigenetically. Investigators have reported that
methylation of CpG islands near exon 1 silenced COX-2
expression in 13% of sporadic colorectal cancers and 14% of
colorectal adenomas [117, 118]. This may have medical
implications. Specifically, adenomas with a methylation-
silenced COX-2 gene may have a diminished response to
treatment with COX-2 specific inhibitors or a better
prognosis, depending on what other genetic defects are
harbored by the tumor. Several types of neoplastic and pre-
neoplastic tissue overexpress COX-2, compared to adjacent
non-involved tissue, with incidences ranging from ∼ 50% -
90% in studies involving a minimum of 10 subjects [119-
124]. Thus, the converse may also be true: the extensive
hypomethylation often seen in the genome of tumors may
release "silenced" COX-2. Investigations with the Min/+
mouse model of intestinal polyposis suggest that COX-2
expression is a significant factor that causes progression
from a pre-neoplastic to a neoplastic condition. For instance,
homologous disruption of the COX-2 gene diminished polyp
formation in Min/+ mice by ∼ 80% [125]. Investigations
with Min/+ mice also suggest that the COX-1 isoenzyme
Cyclooxygenase Enzymes
participates in tumorigenesis. Episodically, investigators
have reported that stromal tissue adjacent to tumor harbors
elevated levels of the COX-1 isoenzyme, not COX-2 [126].
On the one hand, such reports reinforce the notion that
disease-restricted expression of COX-2 is associated
explicitly with the disease at the cellular level. On the other
hand, such reports imply that adaptation to disease alters
expression of COX in proximal cells.
Cells regulate COX-1 and COX-2 by intracellular
compartmentation, accessory proteins, arachidonate levels,
and availability of hydroperoxide activator as discussed
elsewhere [127, 128]. The O2 tension in most tissues exceeds
the Km for COX by at least 6-fold and there are few
circumstances where O2 levels would be insufficient to
sustain a high rate of prostaglandin biosynthesis [129].
However, the core of solid tumors is often hypoxic and this
may be one of those circumstances. The nitric oxide radical
(NO) often occurs together with PG. Given the role of
radical dependent reactions and radical intermediates in
COX catalysis it is not surprising that there are some reports
that NO can stimulate COX activity by a direct interaction or
by a mechanism that involves peroxynitrite (from O2.-+NO.)
reacting directly with COX [130].
PARADOXES AND CONTROVERSIES
FDA approved COX-2 selective NSAIDs should have no
greater liability than non-selective NSAIDS. However, this
has been controversial and the debate ranges from their
relative superiority as gastric sparing NSAIDs [131] to their
cardiovascular and renal safety [132]. Mercifully, for those
interested in medical science, this debate can be informed by
our appreciable understanding of the role of COX-1 and
COX-2 isoforms.
Other paradoxes are more topical and more interesting.
Several groups have now reported that aspirin has no effect
on tumour number and size when administered to Min/+
mice with established polyposis - its effect is only evident
when administered during gestation or prior to weaning
[133-135]. Likewise, there are reports that overexpression of
COX-2 induces cell cycle arrest via a prostaglandin-
independent mechanism [136]. These challenge current
concepts about the role of COX-2 in neoplasia and the role
of NSAIDS in cancer chemoprevention. Paradoxes like these
assure continued research on COX enzymes.
ACKNOWLEDGEMENT
F.A. Fitzpatrick is the Dee Glenn & Ida Smith Chair of
Cancer Research and an investigator of the Huntsman
Cancer Institute. This work was supported by NIH AI 26730.
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