ENZYMATIC and Non-ENZYMATIC LIPID arachidonic acid (AA 20:4 ω-6) PEROXIDATION products RLP Cell Signaling Molecules

     The oxidation of PUFAs (polyunsaturated fatty acids), such as arachidonic acid, generates a broad range of oxidation products which historically have been used as markers of oxidative stress and are Cell signalling RLS (reactive lipid species) which are mediators of multiple pathophysioloical conditions and are not simply as unique biochemical attributes-products cell signalling molecules.
     The Lipid peroxidation products are electrophilic, which allows them to form stable covalent adducts with nucleophilic residues on proteins and selectively modulate protein function.
     The thiol -S-H groups on cysteine residues act as redox switches controlling cell signalling and metabolism.
Oxidative hydrogen peroxide or lipid peroxides to form the markers of oxidative damage and play a role in cell signalling as clear now.
     Early studies implied that lipid peroxidation always results in damage, but now more refined view of this process has evolved and suggests that oxidized lipids can elicit different cellular effects depending on the species present, their concentrations and their reactivity with protein targets. Thats work with two diverse mechanisms:
1. classic reversible binding and
2. irreversible covalent modification of receptors.
     Oxidized lipids are ligands: PGH2, PGE2, PGF2, PGl2 for s. PG (prostaglandin) receptors and mediate biological effects through reversible receptor-ligand interactions. This is best understood for the enzymatically produced PGs and LTs (leukotrienes TXA2, LTC4,LTB4, LTD4, LTE4, Lipoxins).
Lipid peroxidation products modulate cellular activity through irreversible covalent modification of nucleophilic amino acid residues on proteins. Signalling through the covalent modification of proteins is now accepted for a number of well-defined protein–lipid interactions, and selective modified product degradation is mediated through the proteasome.
Signalling through the covalent modification of proteins changes the relationship between the concentration of the RLS ligand molecules can accumulate over time and amplify the signal and even low levels of oxidized lipids RLS initiate strong response signalling. What designated as the covalent Signalling advantage.

Cyclooxygenase Structure and
ENZYMATIC LIPID arachidonic acid (AA 20:4 omega-6) PEROXIDATION
Blocking Mechanism How Aspirin and NSAIDs Work

     Salicilic Acid 1pth(1PTH), ibuprofen 1EQG, fluorbiprofen 3N8Z, IndoMethacin 2OYE
1pthMarz, . . . 1PTHMarz, 1EQGMarz, . . . 3N8ZMarz , . . . . . 2OYEMarz
COX peroxidase PGHS isoform-1 as well
Aspirin and Acetaminophen (trade name Tylenol) are all NSAIDs.
Backbone 31 helices and three-five beta sheets with double strands thin off
     The structural basis of aspirin activity through selective acetylation of serine 530
on prostaglandin H2 synthase inferred from
the crystal structure of inactivated prostaglandin H2
synthase with Salicilic Acid (purple) and (Backbone thin off )
Ser530 brominated (green) or binding Arachidonic Acid as substrate - ligand for prostaglandin H2 (PGH2) production.zoom-150
1DIYMarz,3TZIMarz, 3HS5Marz, COX peroxidase PGHS isoform-1 with Arachidonic Acid complexed active site Heme coordinates 1DIY-3TZI Fe²⁺ and 3HS5 Co³⁺cobalt ion.
1.Nature Structural Biology 1995 Aug;2(8):637-43. 1PTH publication
2.Molecular Pharmacology, 2003 vol. 63no. 2 450-455 1PTH publication
3. Biochemistry. 2001;40(17):5172-80. 1EQG publication
4.Biochemistry. 2010, 49(33): 7069-7079. 3N8Y publication
5.2007 The Journal of Biological Chemistry, 282, 28096-28105. 2OYE IM8 publication
6.Science, 2000: Vol. 289 no. 5486 pp. 1933-1937 1DIY publication
7.2012 The Journal of Biological Chemistry, 287,24619-24630. 3TZI publication
8.2010 The Journal of Biological Chemistry, 285,22152-22163.3HS5,3HS6,3HS7 publication
3HS6Marz COX peroxidase PGHS isoform-1 with eicosapentaenoic acid (EPA) & ,
3HS7Marz docosahexaenoic acid HXA C22:6 omega-3(DHA) complexed active site Heme coordinates Co³⁺cobalt ion.
Prostaglandins and NSAIDS
Prostaglandins are potent mediators of inflammation, paine production and blood cloting.
     The first and committed step in the production of prostaglandins from arachidonic acid arachidonic acid substrate is the bis-oxygenation of arachindonate to prostaglandin PGG2 prostaglandin G2.
     This is followed by reduction to PGH2 prostaglandin H2 in a peroxidase reaction.
Both these reactions are catalyzed by cyclooxygenase, also
known as PGH synthase.
     With this view, it should also be clear why aspirin and other NSAIDs block in COX enzyme molecule the synthesis of product PGH2 and PGG2 as precursors for next synthesis of PGD2 prostaglandin D2 . In various ways, they all act by filling and blocking the tunnel, preventing the migration of arachidonic acid to the active site at the back of the tunnel for enzyme controled radical reaction of peroxidation -O-O-.
Cyclooxygenase (COX) is shown as structures inhibited by the family of drugs known as non-steroidal anti-inflammatory drugs or NSAIDs. Aspirin, ibuprofen, flurbiprofen and acetaminophen (trade name Tylenol) are all NSAIDs.Salicilic Acid, Aspirini, ibuprofen, fluorbiprofen, IndoMethacin, Tylenol
     There are two isoforms of COX in animals: COX-1, which carries out normal, physiological production of prostaglandins, and COX-2, which is induced by cytokines, mitogens and endotoxins in inflammatory cells, and which is responsible for the production of prostaglandins in inflammation.
     The structure shown at left is that of COX-1 from sheep,
inactivated by BromAspirin, the structure of which is shown.
1PTHMarz Enzyme Structure ACD=> is 1DIYMarz or 3TZIMarz
     The first 24 residues of COX-1 are a signal sequence. This domain is removed in the mature enzyme and will not be discussed here. Similarly , residues 25-32 do not yield interpretable electron density, and are not shown in the structure shown. The remaining 551 residues of the enzyme (residues 33-583) comprise three distinct domains. The first of these, residues 33-72, form a small compact module that is similar to epidermal growth factor two beta strands and helix.
     The second domain, composed of residues 73-116, Backbone thin off
forms a right-handed spiral of four yellow alpha-helical segments
along one side of the protein . This domain of the protein
forms a membrane-binding motif. The helical segments are amphipathic,
with most of the hydrophobic residues (shown in green )
74,75,77-78,82,88-89,91-93,98-100,102-103,105,107-108,112,115,116

facing away from the protein, where they hydrophobic forces interact with a lipid bilayer.
     Turn off the hydrophobic residues and we will consider the third domain of the COX enzyme, the catalytic domain (in blue), a globular structure that contains both the cyclooxygenase and peroxidase active sites .
The peroxidase site includes a heme .
     The iron(III) in the center of this heme is coordinated by proximal His-388
and by distal His-207 .
     Let's return to our view of the whole molecule and consider the cyclooxygenase active site.
     The cyclooxygenase active site lies at the end of a long, narrow,
hydrophobic tunnel or channel. Three of the yellow alpha helices of the
membrane-binding domain lie at the entrance to this tunnel .
The hydrophobic residues (shown in green ) facing away from the protein,
where they can interact with a lipid bilayer.
Backbone thin off
The walls of the tunnel are defined by four alpha helices, formed by residues
H5:107-123,H14:325-353,H17:378-384, H28:520-535.
     In the following animation, these helices will flash red and orange.
Phe107,Ile108,Leu112,Met113,Leu115Val116,Leu117,Val119,Leu123
Leu328,Phe329,Ala333Leu334,Ile335,Leu336,Ile337,Gly338,Ile341,Ile343,Val344,Ile345,Val349,Leu352;
Ala378, Met379,Phe381,Leu384;
Met522,Ile523,Met525,Gly526,Ala527,Pro528,Phe529,Leu531,Gly533,Leu534,Leu535;
     Aliphatic-Hydrophobic Amino Acids colored whith surroundings for Arachidonic Acid inhibited by anti-inflamation drugs bound in the tunnel. epidermal growth factor two beta strands and helix.
In this bromoaspirin-inactivated structure, Ser-530 is bromoacetylated or ACD hydrogen bonded with molecule and the lack of interaction of the carboxylate of AA and EPA with the side chain of Arg-120. Leu-531 exhibits a different side chain conformation when the nonproductive and productive binding modes of AA are compared:
carboxylate on the drug and Arg-120 (shown here in purple, blinking), which lies in the tunnel.
1.salicylate in 1pthMarz is bound in the tunnel
2.ibuprofen in 1EQGMarz is bound in the tunnel
3.flurbiprofen in 3N8ZMarz is bound in the tunnel
4.IndoMethacin in 2OYEMarz is bound in the tunnel
5.arachidonic acid AA in 1DIYMarz is bound in the tunnel
6.arachidonic acid AA in 3TZIMarz is bound in the tunnel
         Backbone thin off
     Deep in the tunnel, at the far end, lies Tyr-385-O-H, a catalytically important residue. Heme-dependent peroxidase activity is implicated in the formation of a proposed Tyr-385-O* radical, which is required for radical reaction activity gowerned by cyclooxygenase.
     Now take another look at the tunnel with the Ser 530-O-H, arachidonic acid or anty-inflamaton drug molecule, and the essential Tyr-385 all shown within the tunnel .
     The yellow helices membrane-binding domain lie at the entrance to this tunnel ,
you will recall (shown in green ) touching the membrane interface.
Arachidonic acid substrates flow up into the tunnel from the membrane interior.
     With this view, it should also be clear why aspirin and other NSAIDs block in COX enzyme molecule the synthesis of product PGH2 as precursor for next synthesis of PGD2. In various ways, they all act by filling and blocking the tunnel, preventing the migration of arachidonic acid to the active site at the back of the tunnel for enzyme controled active radical reaction of peroxidation ...:O-:-O:...no chain reaction hapen.
     There are thought to be at least four different mechanisms of action for NSAIDs.