2. Cyclooxygenase (COX)
• Also, known as prostaglandin endoperoxide synthase (PTGS) belongs to family
of isozymes.
• Responsible for the formation of prostanoids including thromboxane and
prostaglandins from arachidonic acid
• Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit COX to exert their
effects
• It exists in two isoforms i.e. COX-1 and COX-2 of molecular mass 71KDa and
73 Kda.
• Major diference between the two is the presence of isoleucine in COX-1 and
Valine in COX-2 at position 523.
• COX-2 has a larger substrate concentration providing structural basis for new
isoform specific inhibitors (COXibs).
3. Active site of COX-1 and COX-2
Fig 1. Representation of active sites in COX-1 & COX-2
Mengle-Gaw L, Schwartz B. Cyclooxygenase-2 inhibitors: promise or peril?.
Mediators of Inflammation. 2002; 11(5): 275-286.
Fig 2 Representation of active sites in COX-2
Mengle-Gaw L, Schwartz B. Cyclooxygenase-2 inhibitors: promise or peril?.
Mediators of Inflammation. 2002; 11(5): 275-286.
4. Fig 3. COX 1 Fig 4. COX 2
COX - 1 COX – 2
Nature of enzyme Constitutive enzyme i.e. produced
in all physiological conditions
Inducible enzyme i.e. produced
under specific conditions such as
inflammation
Location Found in kidney, stomach and
platelets
Found in macrophages, leukocytes
and fibroblasts
Function Protects gastric mucosa, regulates
gastric acid and maintains normal
function of kidney by stimulating
prostaglains
Involved in the synthesis of
prostaglandins that causes pain and
inflammation
5. Mechanism of action
The peroxidase cycle leads to abstraction of a
hydrogen atom from Tyr-385 forming a tyrosyl radical
and activating the cyclooxygenase active site.
COX
Fig 5. Mechanism of cyclooxygenase activation
Rouzer C, Marnett L. Cyclooxygenases: Structural and functional insights. Journal of Lipid
Research. 2008; 50(Supplement): S29-S34.
6. There is an abstraction of Pro S hydrogen atom from carbon 13 thus, leading to
the conversion of arachidonic acid into prostaglandins (PGG2)
2 e- reduction of peroxide substrate results in oxidation of ferric heme to oxo
ferryl porphyric radical cation.
Electron transfer to heme from Tyr-385 generates tyrosyl radical in the
cyclooxygenase active site.
Final step of the reaction is reduction of peroxyl radical to hydroperoxide to
form PGG2 thus, regenerating tyrosyl radical.
7. Fig 6. Conversion of arachidonic acid to Prostaglandins
Khan A. Prostaglandins in labor - a translational approach. Frontiers in Bioscience. 2008;
Volume(13): 5794.
8. Inhibitors for COX
• The main COX inhibitors are Non steroidal anti inflammatory drugs
(NSAID’s)
• The result leads to inhibition of prostaglandin and thromboxane
synthesis which results in reduced inflammation as well as antipyretic,
antithrombotic and analgesic effects.
• The most frequent adverse effect is irritation of the gastric mucosa as
prostaglandins normally have protective role in the GI tract.
• Newer NSAID’s include celecoxib, etoricoxib and these are more
specific thus, causing less gastric irritation and decreased risk of
peptic ulceration.
9. COX – 2 Inhibition
• Ibuprofen is a non selective COX inhibitor
• Carboxylate moiety of ibuprofen forms salt
bridge with the guanidinium group of Arg
120 and hydrogen bond with hydroxyl
group of Tyr-355
• Benzyl group of ibuprofen makes five
contacts with Ala-527 and three with val-
349.
• A total of thirteen contacts are made
between isobutyl group of ibuprofen and
Trp-37, met-522, vali-523, gly 526, ala-527
and ser-530
The structure of Ibuprofen bound to COX-2
Benjamin J. Orlando, Michael J. Lucido, and Michael G. Malkowski,
2015
11. Aim: To observe the kinetics of COX an NO treated COX enzymes
• COX is a bifunctional enzyme exhibiting coupled peroxidase and dioxydase
activities.
• COX activity is oxygen dependent and oxygen level variation leads to huge
impact on the catalytic activity.
• Dioxygenase activity of COX-2 can be observed independently of peroxidase
activity by adding sufficient phenol which can act as a reductant at a
concentration of upto 2.5 mM.
• In the case of COX-1 the acceleration rate is followed by inhibition upon
addition of phenol above 0.3 mM indicating difference in the kinetics.
• NO alters the protein conformation of COX-2 rather than COX-1.
Kinetic basis for the activation of human
cyclooxygenase
12. Kinetics of O2 consumption by COX and NO treated COX enzymes
• Dioxygenase activity of COX-2 was measured by the rate constant of
AA consumption under a saturated O2 condition.
• NO treated COX-2 prepared under anaerobic condition has a
significantly enhanced dioxygenase activity.
• NO accelerates COX-2 activity by increasing apparent Vmax from 16.9
s-1 to 27.5 s-1 and by decreasing KM (O2) from 15.0 μM to 10.3 μM.
• No activation effect was observed for NO treated COX-2 which was
prepared under anaerobic conditions.
• No activation occurred for NO treated COX-1 prepared under aerobic
and anaerobic conditions.
13. Qiao J, Ma L, Roth J, Li Y, Liu Y. Kinetic basis for the activation of human cyclooxygenase-2 rather
than cyclooxygenase-1 by nitric oxide. Organic & Biomolecular Chemistry. 2018; 16(5): 765-770.
Fig 8. Michaelis Menten graph for varying Km and Vmax
14. Discussion
NO increases the Vmax in COX-2 when aerobic conditions are given
While in case of COX-1 Vmax doesn not gets increased even if NO is used
Vmax does not gets increased in anaerobic conditions even if NO is used suggesting the importance
of enhanced activity of COX.
In conclusion, the kinetic studies indicate that a certain amount of O2 i.e. 6 fold of Km (O2) is essential
for NO activation.
15. Conclusion
• COX-1 and COX-2 catalyse the formation of prostaglandins and
thromboxane
• COX enzymes are important because they are responsible for both
constitutive and for showing inflammatory signs.
• A lot of drugs that inhibit COX have been developed however current
research focusses on COX-2 inhibitors which lead to less side effects.
• COX enzymes are dependent on oxygen concentration and efficiency
increases if NO is made to bind them.
16. References
1. Rouzer C, Marnett L. Cyclooxygenases: Structural and functional insights.
Journal of Lipid Research. 2008; 50(Supplement): S29-S34.
2. Qiao J, Ma L, Roth J, Li Y, Liu Y. Kinetic basis for the activation of human
cyclooxygenase-2 rather than cyclooxygenase-1 by nitric oxide. Organic &
Biomolecular Chemistry. 2018; 16(5): 765-770.
3. Mengle-Gaw L, Schwartz B. Cyclooxygenase-2 inhibitors: promise or peril?.
Mediators of Inflammation. 2002; 11(5): 275-286.
4. Khan A. Prostaglandins in labor - a translational approach. Frontiers in
Bioscience. 2008;Volume(13):5794.
