Review
Cyclooxygenase mechanisms

https://doi.org/10.1016/S1367-5931(00)00130-7Get rights and content

Abstract

Several advances have occurred in the past year in our understanding of cyclooxygenase catalysis. The role of specific heme oxidation states in the formation of catalytically competent tyrosyl radicals has been defined; the identity of physiological hydroperoxide activators has been established; and the participation of individual amino acids in substrate binding and oxygenation has been elucidated.

Introduction

Cyclooxygenases (COXs) catalyze the committed step in the conversion of arachidonic acid to prostaglandins and thromboxane. They oxygenate arachidonic acid to the hydroperoxy endoperoxide PGG2 (prostaglandin G2), followed by reduction of PGG2 to the alcohol PGH2 (Figure 1). PGH2 is converted by isomerases to prostaglandins and thromboxane, which exert numerous physiological and pathophysiological effects. Thus, COX enzymes play a key role in the biosynthesis of a family of important bioactive lipids. But it is the interesting chemistry which they catalyze that is the focus of this review. Recent advances in the mechanism of arachidonic acid oxygenation, the identity of the protein oxidant, the pathway of enzyme activation, and the nature of enzyme–substrate interactions will be described. Amino acid designations are given based on the COX-1 numbering system.

Section snippets

Mechanism of arachidonate oxygenation

The conversion of arachidonic acid to PGG2 can be formulated as a series of radical reactions analogous to those of polyunsaturated fatty acid autoxidation (Figure 2) [1]. The 13-pro(S)-hydrogen is removed and O2 traps the incipient pentadienyl radical at C-11. The 11-peroxyl radical cyclizes at C-9 and the carbon-centered radical generated at C-8 cyclizes at C-12, producing the endoperoxide. The allylic radical generated is trapped by O2 at C-15 to form the 15-(S)-peroxyl radical; this radical

Identity of the protein oxidant

The oxidant that removes the 13-pro(S) hydrogen appears to be a tyrosyl radical derived from Tyr385 (Figure 2) [8]. This residue is interposed between the heme prosthetic group and the cyclooxygenase active site and is ideally positioned to interact with a bound fatty acid molecule 9, 10, 11••. Transient tyrosyl radicals are detected during cyclooxygenase catalysis and they oxidize arachidonic acid to carbon-centered radicals [12••]. It has been difficult to assign the identity of the tyrosyl

Role of the heme

The Tyr385 tyrosyl radical is not present in resting enzyme so it must be generated in order to initiate cyclooxygenase catalysis. Reaction of fatty acid hydroperoxides or organic hydroperoxides with the heme prosthetic group generates a higher oxidation state of the heme that oxidizes Tyr385 (Figure 2) [20]. The higher oxidation state that oxidizes Tyr385 is the ferryl-oxo complex, which is the first intermediate in peroxidase catalysis (Compound I) 20, 21. Decay of the visible absorbance of

Hydroperoxide activators

The activation of resting enzyme following addition of arachidonic acid in vitro is due to the presence of trace amounts of hydroperoxide in the fatty acid preparation. Activation is completely inhibited by addition of high concentrations of glutathione peroxidase and glutathione, which reduces fatty acid hydroperoxides 33, 34, 35. Once the Tyr385 radical is generated, each enzyme molecule catalyzes several hundred cycles of arachidonic acid oxygenation. Although the tyrosyl radical is reduced

Enzyme–substrate interactions

Considerable attention has focused recently on the binding of fatty acid substrates in the cyclooxygenase active site. The chemical mandates of the synthesis of a bicyclic peroxide with trans-dialkyl substitution require that the fatty acid be bound in an extended conformation with a sharp bend around carbons 10–13 [47]. Modeling this conformation of arachidonate into the cyclooxygenase active site with the carboxylate ion-paired to Arg120 and the 13-pro(S) hydrogen adjacent to Tyr385 places

Conclusions

Recent work from several laboratories has provided important insights into the oxygenation of arachidonic acid by cyclooxygenases. These findings strongly support the chemical mechanism of prostaglandin endoperoxide biosynthesis proposed over 30 years ago by Hamberg and Samuelsson [2] and the biochemical mechanism of cyclooxygenase catalysis proposed 12 years ago by Ruf and co-workers [8]. Reaching this level of understanding has been experimentally challenging because of the short-lived nature

Acknowledgements

I am grateful to J Prusakiewicz and GP Hochgesang for assistance with some of the figures and to K Kozak for a critical reading. I am also grateful to W Smith for helpful discussions related to the crystal structure of a COX-1–arachidonic acid complex. Work in the Marnett laboratory has been supported by a research grant from the National Institutes of Health (CA47479).

References (61)

  • R.J. Kulmacz et al.

    Comparison of hydroperoxide initiator requirements for the cyclooxygenase activities of prostaglandin H synthase-1 and -2

    J Biol Chem

    (1995)
  • B. Chance et al.

    X-ray absorption studies of intermediates in peroxidase activity

    Arch Biochem Biophys

    (1984)
  • W.H. Koppenol

    Oxyradical reactions: from bond-dissociation energies to reduction potentials

    FEBS Lett

    (1990)
  • A-L. Tsai et al.

    Heme coordination of prostaglandin H synthase

    J Biol Chem

    (1993)
  • S. Strieder et al.

    Prostaglandin endoperoxide synthase substituted with manganese protoporphyrin IX. Formation of a higher oxidation state and its relation to cyclooxygenase reaction

    J Biol Chem

    (1992)
  • R. Odenwaller et al.

    Detection of a higher oxidation state of manganese-prostaglandin endoperoxide synthase

    J Biol Chem

    (1992)
  • R.J. Kulmacz et al.

    Requirements for hydroperoxides by the cyclooxygenase and peroxidase activities of prostaglandin H synthase

    Prostaglandins

    (1983)
  • D.H. Nugteren et al.

    Isolation and properties of intermediates in prostaglandins biosynthesis

    Biochim Biophys Acta

    (1973)
  • C.M. Markey et al.

    Quantitative studies of hydroperoxide reduction by prostaglandin H synthase

    J Biol Chem

    (1987)
  • G. Lu et al.

    Comparison of the peroxidase reaction kinetics of prostaglandin H synthase-1 and -2

    J Biol Chem

    (1999)
  • D.J. Wadleigh et al.

    Transcriptional activation of the cyclooxygenase-2 gene in endotoxin-treated RAW 264.7 macrophages

    J Biol Chem

    (2000)
  • C.C. Chen et al.

    Role of the cyclic AMP-protein kinase A pathway in lipopolysaccharide-induced nitric oxide synthase expression in RAW 264.7 macrophages. Involvement of cyclooxygenase-2

    J Biol Chem

    (1999)
  • L.J. Marnett et al.

    Regulation of prostaglandin biosynthesis by nitric oxide is revealed by targeted deletion of inducible nitric-oxide synthase

    J Biol Chem

    (2000)
  • L.J. Marnett et al.

    Arachidonic acid metabolism by COX-1 and COX-2: mechanisms of catalysis and inhibition

    J Biol Chem

    (1999)
  • S.W. Rowlinson et al.

    The binding of arachidonic acid in the cyclooxygenase active site of mouse prostaglandin endoperoxide synthase-2 (COX-2): a putative L-shaped binding conformation utilizing the top channel region

    J Biol Chem

    (1999)
  • E.D. Thuresson et al.

    Different catalytically competent arrangements of arachidonic acid within the cyclooxygenase active site of prostaglandin endoperoxide H synthase-1 lead to the formation of different oxygenated products

    J Biol Chem

    (2000)
  • T. Shimokawa et al.

    Prostaglandin endoperoxide synthase. The aspirin acetylation region

    J Biol Chem

    (1992)
  • M. Lecomte et al.

    Acetylation of human prostaglandin endoperoxide synthase-2 (cyclooxygenase-2) by aspirin

    J Biol Chem

    (1994)
  • S.W. Rowlinson et al.

    Spatial requirements for 15-HETE synthesis within the cyclooxygenase active site of murine COX-2: why acetylated COX-1 does not synthesize 15-(R)-HETE

    J Biol Chem

    (2000)
  • C. Schneider et al.

    Stereospecificity of hydrogen abstraction in the conversion of arachidonic acid to 15R-HETE by aspirin-treated cyclooxygenase-2. Implications for the alignment of substrate in the active site

    J Biol Chem

    (2000)
  • Cited by (145)

    • Mechanism of reactivation of the peroxidase catalytic activity of human cyclooxygenases by reducing cosubstrate quercetin

      2021, Journal of Molecular Graphics and Modelling
      Citation Excerpt :

      Then the cyclooxygenase cycle is initiated by the Tyr385 radical, which converts AA to PGG2 until “suicide inactivation” takes place. PGG2 is then converted to PGH2 in the peroxidase site while PPIXFeIII is oxidized to Compound I again [6–8,44,45]. In our earlier study, some of the naturally-occurring flavonoids such as quercetin and myricetin were found to activate the peroxidase catalytic activity of COX-1/2 both in vitro and in vivo [9,10].

    View all citing articles on Scopus
    View full text