Cyclooxygenase: Immuno-Physiologic regulator

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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.

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Cyclooxygenase: Immuno-Physiologic regulator

  1. 1. CYCLOOXYGENASE: IMMUNO-PHYSIOLOGIC REGULATORSarines, Johnathan C.HUB31De La Salle University-DasmarinasDasmarinas, Cavite PhilippinesI. INTRODUCTIONCyclooxygenase (COX), also known as Prostaglandin Endoperoxide H Synthase, is an enzymethat catalyzes the first two processes in prostaglandin biosynthesis, oxygenation of arachidonicacid and prostaglandin G (1), (2). Endothelial cells in the blood vessels, platelets, leukocytes andother sites of inflammation have this enzyme, while arachidonic acid is present in phospholipidsof the plasma membrane. Hydrolysis of phospholipids can lead to dissociation of the hydrophilicphosphate head and the hydrophobic tail which may include arachidonic acid. Cyclooxygenase isbasically produced by two types of genes: COX-1 and COX-2 genes (3). COX-1 gene formsCOX-1, while COX-2 gene forms the isoform of COX-1, COX-2 (3). Both isozymes may havesimilarities in terms of the reactions they catalyze, yet they differ in inhibitors, function, activesites, gene expression, and sequence of amino acids. COX-1 and COX-2 can promotedegenerative disease such as cancer, however this remains unclear. Alzheimer’s disease can bethe overall effect of inflammatory action of COX product.II. CYCLOOXYGENASEA. Plasma membrane, Arachidonic acid and ProstaglandinsThe Plasma membrane is made up of phospholipids which consist of hydrophilic/polar head and2 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 Phospholipidscan 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 cisconfigurations in C-5, 8, 11 and 14. Thus, it is also known as cis-5, 8, 11, 14-eicosatetraenoicacid. Arachidonic acid is mainly involved in the biosynthesis of oxygenated arachidonic acidderivatives 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). Prostaglandinsare prostanoids which is made up of cyclopentane and its two substituents: heptanoic acid andoctane (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-factorsCyclooxygenase is made up of four domains, namely: Catalytic domain, Dimerization Domainwith epidermal growth factor (EGF), and Membrane Binding Domain (MBD). Some domains areglycosylated (with sugar unitsFrom the name itself, MBD connects COX and internal membraneof endoplasmic reticulum through a series of four ampiphilic α-helices that relatively consist of 50amino acids (9). This domain is linked with Dimerization Domain through the carboxylic end ofdimerization domain. Dimerization Domain, on the other hand, consists of two monomers thatbind 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 176comprise this domain, including the epidermal growth factors (9). In Figure 1, EGF is composedof an anti-parallel β-pleated sheet. Four disulfide bridges, which are formed under oxidativeconditions, interconnect Dimerization Domain with the Catalytic Domain (9).
  2. 2. Figure 1 Half Structure of Cyclooxygenase (10)Lastly, Catalytic Domain is made up of 480 amino acids, relatively, and a prosthetic group calledheme 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 thesuperior surface of the enzyme in which the heme is embedded. Heme is arranged on its locationthrough the bond formed by Ferric ion, proximal (His 423) and distal (His 241) histidines. SinceCOX contains heme, Fe3+is the co-factor of COX. On the other hand, Cyclooxygenase active siteis a 25 x 10-10m long, 8 x 10-10m in diameter hydrophobic dead-end groove which differentiatesCOX-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 therole of Arg 120. Arg 120, together with Tyr 355, is necessary for the attachment of Arachidonicacid and competitive inhibitors, while Arg 120 is unnecessary for the substrate and inhibitorattachment 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-pointdifferences 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 Ile523 which restricts the entry of selective COX-2 inhibitors that contain aromatic moieties. In thecase of COX-2, Val 523 does not form those “bulges”, thus entry of Coxibs is highly probable (9).C. Mechanism of CatalysisThe metabolic activity of COX plays an important role in physiological responses of most organsand inflammatory action of polymorphonuclear (PMN) leucocytes or immune responses. COXinactivity might result to organ dysfunction and absence of inflammatory responses.
  3. 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 andthe attachment of AA carboxyl moiety to Arg 120 and Tyr 355 (9). This would result to arachidonylradical 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 atC-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 tohydroxide through oxidation of heme. Conversion of AA and PGG2 may occur simultaneously asAA conversion requires tyrosyl radical.D. Kinetics of Reaction and Mode of RegulationArachidonic acid is usually at low concentration, approximately 1 – 10 μM. However, itsconcentration can elevate up to 100 – 300 μM when AA derivative production is necessitated inresponse to physiological and immune stimuli (12). COX inactivity can cause dysfunction oforgans and lack of immune response.Activators of COX-1 and COX-2 include nitric oxide (NO) (9), Hydrogen peroxide (H2O2), PGH2and some hydroperoxide – containing compounds. In the mechanism of COX catalysis,hydroperoxide or peroxynitrite can act as endogenous oxidant which oxidizes heme. Thoseactivators 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. 4. Meanwhile, most of the COX inhibitors are synthetic in a form of Non-Steroidal Anti-inflammatoryDrugs (NSAIDs). NSAIDs are divided into major groups, namely: Classic NSAIDs and SelectiveCOX-2 inhibitors or sometimes called Coxibs (9). Classic NSAIDs, also known as non-selectiveNSAIDs, can inhibit both COX isozymes, whereas Coxibs inhibit COX-2 only. Classic NSAIDsinclude aspirin, ibruprofen, paracetamol, Indomethacin, Diclofenac, Naproxen, Piroxicam,Mefenamic acid, Salsalate, Phenylbutazone, Ketoprofen and other non-selective NSAIDs. On theother 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 asa pain killer. It competitively inhibits (half-minimal) COX-1 at 0.3 μM and COX-2 at 50 μM byacetylating Ser 530 in the cyclooxygenase active site (14). The orientation of Aspirin couldprobably affect the IC50 of aspirin in both isozymes. Aspirin has a better orientation in COX-1compared to COX-2 due to coordination with Arg 120. This permits acetylation of Ser 530 inCOX-1. Whereas, COX-2 have relatively larger active site compared to COX-1, however, theorientation 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 thatrestrict aromatic moiety-containing compounds. Thus, Coxibs can enter the cyclooxygenaseactive site. Coxibs may not have a carboxyl group to attach itself to Ser 530 through an esterbond, yet series of non-covalent bonds stabilizes the the binding of Coxibs (9).NSAIDs, nitric oxide, H2O2, PGG2 and some hydroperoxide – containing compounds serve asallosteric 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 (VO43+), 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 dueto inhibitors, AA can either be converted to hydroperoxyeicosatetraenoic acid (HPETE) viaLipoxygenase pathway or Epoxyeicosatrienoic acid (EET) via Cytochrome P450 and epoxygenasepathway.E. Significance to Human Health and Associated Diseases1. Significance to Human HealthCyclooxygenase catalyzes the cyclization and bisoxygenation of AA, forming PGG2, andreduction of PGG2 to PGH2. PGH2 derives prostaglandins, prostacyclins and thromboxanes thathave 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, thromboxanesregulate thrombosis, vasocontraction of blood vessels and inflammation. Brochocontraction andbrochorelaxation in the respiratory tract are best facilitated by PGD2 and PGF2 (9).2. Associated DiseasesCyclooxygenase had been speculated for being involved in turmorigenesis via inducedangiogenesis and neurodegenerative diseases known as Alzheimer’s disease (AD) throughinflammation of neurons. However, those speculations remain obscure and unexplained. Studiesshow that intake of NSAIDs could actually decrease the risk of having cancer and AD (9).

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