Prostaglandin-endoperoxide synthase

 

Converts arachidonate to prostaglandin H2 (PGH2), a committed step in prostanoid synthesis. Constitutively expressed in some tissues in physiological conditions, such as the endothelium, kidney and brain, and in pathological conditions, such as in cancer. PTGS2 is responsible for production of inflammatory prostaglandins. Up-regulation of PTGS2 is also associated with increased cell adhesion, phenotypic changes, resistance to apoptosis and tumor angiogenesis. In cancer cells, PTGS2 is a key step in the production of prostaglandin E2 (PGE2), which plays important roles in modulating motility, proliferation and resistance to apoptosis.

Production of prostaglandin hormones in vertebrates depends on this enzyme, which couples the reduction of dangerous hydroperoxides to the generation of a useful oxidised product. The cofactor which makes this possible is a single iron protoporphyrin IX; its interactions with protein ligands are essential for modulating protein function.

 

Reference Protein and Structure

Sequence
Q05769 UniProt (1.14.99.1) IPR029576 (Sequence Homologues) (PDB Homologues)
Biological species
Mus musculus (house mouse) Uniprot
PDB
5cox - UNINHIBITED MOUSE CYCLOOXYGENASE-2 (PROSTAGLANDIN SYNTHASE-2) (3.0 Å) PDBe PDBsum 5cox
Catalytic CATH Domains
1.10.640.10 CATHdb (see all for 5cox)
Cofactors
Heme b (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:1.14.99.1)

ROOH
CHEBI:79974ChEBI
+
arachidonate
CHEBI:32395ChEBI
+
dioxygen
CHEBI:15379ChEBI
+
hydron
CHEBI:15378ChEBI
→
prostaglandin H2(1-)
CHEBI:57405ChEBI
+
alcohol
CHEBI:30879ChEBI
+
water
CHEBI:15377ChEBI
Alternative enzyme names: (PG)H synthase, PG synthetase, Fatty acid cyclooxygenase, Prostaglandin G/H synthase, Prostaglandin endoperoxide synthetase, Prostaglandin synthase, Prostaglandin synthetase,

Enzyme Mechanism

Introduction

Mutagenesis studies have identified a number of residues essential for catalysis. The replacement of any one of His207, His309 or His388 (numbering for PGH1) by Gln or Ala abolishes both enzyme activities. His388 is the axial ligand for the haem iron, while His 207 is nearby on the distal side. Puzzlingly, His309 is far away from the active site in the structures which are now available. However, Gln203 has also been implicated in the peroxidase activity, cooperating with His207; while Tyr385 has been shown my mutation to be important for the cyclo-oxygenase activity. Spectroscopic data suggest that a tyrosyl radical formed as a result of peroxide reduction abstracts a hydrogen from arachidonate to start the cyclo-oxygenase reaction.

Catalytic Residues Roles

UniProt PDB* (5cox)
His374 His388(357)A The axial ligand to the iron ion in heme b. Alters the redox potential of the iron ion. metal ligand
Ser516 Ser530(499)A Although not critical for COX activity, it helps to stabilise the reactive intermediates formed on the tyrosine residue. It is a critical residue for the inhibition of COX by NSAIDs (site-directed mutagenesis of Ser-530 to Ala virtually abolished inhibition by diclofenac and piroxicam), along with Tyr385 the residue chelates the NSAIDs carboxylate group. electrostatic stabiliser
Tyr371 Tyr385(354)A Acts as a hydrogen atom relay. Also an important residue in the inhibition of COX activity by NSAIDs. hydrogen radical acceptor, hydrogen radical donor, hydrogen bond acceptor, hydrogen bond donor, radical stabiliser
Gln189 Gln203(172)A Helps stabilise the reactive intermediates. hydrogen bond donor, electrostatic stabiliser
His193 His207(176)A Acts as a general acid/base hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, electrostatic stabiliser
Leu370, Gly512 Leu384(353)A, Gly526(495)A Help direct the steriochemical outcome of the reaction. Mutation of these residues result in several different isomeric products (15S-, 13R- and 15R-oxygenated diepoxides). steric role
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

coordination, overall reactant used, coordination to a metal ion, intermediate formation, proton transfer, unimolecular elimination by the conjugate base, radical formation, redox reaction, overall product formed, radical propagation, hydrogen transfer, atom stereo change, bimolecular homolytic addition, intramolecular homolytic addition, cyclisation, intermediate terminated, native state of enzyme is not regenerated

References

  1. Marnett LJ (2000), Curr Opin Chem Biol, 4, 545-552. Cyclooxygenase mechanisms. DOI:10.1016/s1367-5931(00)00130-7. PMID:11006543.
  2. Blobaum AL et al. (2015), J Biol Chem, 290, 12793-12803. Action at a Distance. DOI:10.1074/jbc.m114.635987. PMID:25825493.
  3. Wu G et al. (2011), J Inorg Biochem, 105, 382-390. Cyclooxygenase reaction mechanism of prostaglandin H synthase from deuterium kinetic isotope effects. DOI:10.1016/j.jinorgbio.2010.11.015. PMID:21394223.
  4. Furse KE et al. (2006), Biochemistry, 45, 3206-3218. Molecular Dynamics Simulations of Arachidonic Acid-Derived Pentadienyl Radical Intermediate Complexes with COX-1 and COX-2:  Insights into Oxygenation Regio- and Stereoselectivity†. DOI:10.1021/bi052338h. PMID:16519515.
  5. Schneider C et al. (2004), J Biol Chem, 279, 4404-4414. Identification of Two Cyclooxygenase Active Site Residues, Leucine 384 and Glycine 526, That Control Carbon Ring Cyclization in Prostaglandin Biosynthesis. DOI:10.1074/jbc.m307431200. PMID:14594816.
  6. Rowlinson SW et al. (2003), J Biol Chem, 278, 45763-45769. A Novel Mechanism of Cyclooxygenase-2 Inhibition Involving Interactions with Ser-530 and Tyr-385. DOI:10.1074/jbc.m305481200. PMID:12925531.
  7. Seibold SA et al. (2000), Biochemistry, 39, 6616-6624. Peroxidase Activity in Prostaglandin Endoperoxide H Synthase-1 Occurs with a Neutral Histidine Proximal Heme Ligand†. DOI:10.1021/bi0002333. PMID:10828979.
  8. Kiefer JR et al. (2000), Nature, 405, 97-101. Structural insights into the stereochemistry of the cyclooxygenase reaction. DOI:10.1038/35011103. PMID:10811226.
  9. Landino LM et al. (1997), J Biol Chem, 272, 21565-21574. Mutational Analysis of the Role of the Distal Histidine and Glutamine Residues of Prostaglandin-Endoperoxide Synthase-2 in Peroxidase Catalysis, Hydroperoxide Reduction, and Cyclooxygenase Activation. DOI:10.1074/jbc.272.34.21565. PMID:9261177.
  10. Tang MS et al. (1997), Biochemistry, 36, 7527-7534. Detection of an Fe2+−Protoporphyrin-IX Intermediate during Aspirin-Treated Prostaglandin H2Synthase II Catalysis of Arachidonic Acid to 15-HETE†,‡. DOI:10.1021/bi962750k. PMID:9200703.
  11. Shimokawa T et al. (1991), J Biol Chem, 266, 6168-6173. Essential histidines of prostaglandin endoperoxide synthase. His-309 is involved in heme binding. PMID:1901057.
  12. Shimokawa T et al. (1990), J Biol Chem, 265, 20073-20076. Tyrosine 385 of prostaglandin endoperoxide synthase is required for cyclooxygenase catalysis. PMID:2122967.

Step 1. His207 deprotonates the alkyl peroxide, which then coordinates to the Fe(III) centre of the haem cofactor.

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Catalytic Residues Roles

Residue Roles
His207(176)A hydrogen bond acceptor
Gln203(172)A hydrogen bond donor
His388(357)A metal ligand
His207(176)A proton acceptor

Chemical Components

coordination, overall reactant used, coordination to a metal ion, intermediate formation, proton transfer

Step 2. The peroxo bond collapses, donating two electrons to the Fe(III) centre, which immediately shuttles one into the porphyrin ring, creating Fe(IV). The liberated alkoxide deprotonates His207

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Catalytic Residues Roles

Residue Roles
His207(176)A hydrogen bond donor, electrostatic stabiliser
Gln203(172)A electrostatic stabiliser, hydrogen bond donor
His388(357)A metal ligand
His207(176)A proton donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, radical formation, redox reaction, intermediate formation, overall product formed, proton transfer

Step 3. The porphyrin radical abstracts a hydrogen from Tyr385.

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Catalytic Residues Roles

Residue Roles
Tyr385(354)A hydrogen bond donor
His388(357)A metal ligand
Tyr385(354)A hydrogen radical donor

Chemical Components

radical propagation, hydrogen transfer, intermediate formation

Step 4. Tyr385 abstracts a hydrogen from the arachidonic acid, initiating double bond rearrangement.

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Catalytic Residues Roles

Residue Roles
Tyr385(354)A hydrogen bond acceptor
Gly526(495)A steric role
Leu384(353)A steric role
Tyr385(354)A hydrogen radical acceptor

Chemical Components

radical propagation, hydrogen transfer, atom stereo change, overall reactant used, intermediate formation

Step 5. The carbon radical initiates a homolytic attack on a dioxygen molecule in an addition reaction.

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Catalytic Residues Roles

Residue Roles
Tyr385(354)A radical stabiliser
Gly526(495)A steric role
Leu384(353)A steric role

Chemical Components

radical propagation, ingold: bimolecular homolytic addition, atom stereo change, overall reactant used, intermediate formation

Step 6. The oxygen radical initiates a homolytic attack on the carbon chain, forming a four-membered ring in an addition reaction. This initiates a second homolytic attack, through double bond rearrangement, which causes the formation of a five-membered carbon rind, and initiates homolytic attack on a second dioxygen molecule in an addition reaction.

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Catalytic Residues Roles

Residue Roles
Tyr385(354)A radical stabiliser
Leu384(353)A steric role
Gly526(495)A steric role

Chemical Components

radical propagation, ingold: intramolecular homolytic addition, ingold: bimolecular homolytic addition, atom stereo change, overall reactant used, cyclisation, intermediate formation

Step 7. The oxygen radical of the peroxo group abstracts a hydrogen from Tyr385.

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Catalytic Residues Roles

Residue Roles
Tyr385(354)A hydrogen bond donor
Leu384(353)A steric role
Gly526(495)A steric role
Ser530(499)A electrostatic stabiliser
Tyr385(354)A hydrogen radical donor

Chemical Components

hydrogen transfer, radical propagation, overall product formed, intermediate terminated, native state of enzyme is not regenerated
Download: Image, Marvin File

Step 1. His207 deprotonates the alkyl peroxide, which then coordinates to the Fe(III) centre of the haem cofactor.

Step 2. The peroxo bond collapses, donating two electrons to the Fe(III) centre, which immediately shuttles one into the porphyrin ring, creating Fe(IV). The liberated alkoxide deprotonates His207

Step 3. The porphyrin radical abstracts a hydrogen from Tyr385.

Step 4. Tyr385 abstracts a hydrogen from the arachidonic acid, initiating double bond rearrangement.

Step 5. The carbon radical initiates a homolytic attack on a dioxygen molecule in an addition reaction.

Step 6. The oxygen radical initiates a homolytic attack on the carbon chain, forming a four-membered ring in an addition reaction. This initiates a second homolytic attack, through double bond rearrangement, which causes the formation of a five-membered carbon rind, and initiates homolytic attack on a second dioxygen molecule in an addition reaction.

Step 7. The oxygen radical of the peroxo group abstracts a hydrogen from Tyr385.

Products.

Contributors

Gemma L. Holliday, Gail J. Bartlett, Daniel E. Almonacid, Sophie T. Williams, Craig Porter