Selective oxygenation of N-arachidonylglycine by cyclooxygenase-2

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Abstract

Nonsteroidal anti-inflammatory drugs prevent hyperalgesia and inflammation by inhibiting the cyclooxygenase-2 (COX-2) catalyzed oxygenation of arachidonic acid to prostaglandin (PG) H2. The lipoamino acid N-arachidonylglycine (NAGly) has also been shown to suppress tonic inflammatory pain and is naturally present at significant levels in many of the same mammalian tissues that express COX-2. Here, we report that COX-2 selectively metabolizes NAGly to PGH2 glycine (PGH2-Gly) and hydroxyeicosatetraenoic glycine (HETE-Gly). Site-directed mutagenesis experiments identify the side pocket residues of COX-2, especially Arg-513, as critical determinants of the COX-2 selectivity towards NAGly. This is the first report of a charged arachidonyl derivative that is a selective substrate for COX-2. These results suggest a possible role for COX-2 in the regulation of NAGly levels and the formation of a novel class of eicosanoids from NAGly metabolism.

Section snippets

Materials and methods

Chemicals. AA was obtained from NuChek Prep (Elysian, MN). N-Arachidonylglycine was purchased from Cayman Chemical (Ann Arbor, MI). Ram seminal vesicles were obtained from Oxford Biomedical Research (Oxford, MI). Hematin and triphenylphosphine were purchased from Sigma. All other chemicals were from Aldrich.

Enzymology. COX-1 was purified from ram seminal vesicles as previously described [19]. Site-directed mutagenesis of COX-2 was performed as described previously [20]. Recombinant COX-2

Oxygenation of NAGly by purified COX-2

Incubation of NAGly with purified recombinant murine COX-2 (mCOX-2) gave 40% of the initial rate of O2 uptake of that observed with AA (Fig. 2). Relatively little O2 uptake, if any, was observed after the addition of ovine COX-1 (oCOX-1) to the reaction containing NAGly. In contrast, COX-1 metabolizes AEA and 2-AG, but still to a much lesser extent than AA. These results show that NAGly is the most selective substrate identified to date. Steady-state kinetic analysis revealed that the Km for

Discussion

The present studies establish a possible role for COX-2 in the metabolism and regulation of the anti-inflammatory lipoamino acid, NAGly. COX-2 oxygenates NAGly with a moderate efficiency, in contrast to the absence of metabolism by COX-1. Ranking the catalytic efficiency of COX-2 substrates from the most to the least efficient (AA>2-AG>NAGly>AEA) suggests that modification of the alcohol group in AEA to the carboxylic acid in NAGly promotes higher affinity and more productive binding for

Acknowledgements

This research was supported in part by National Institutes of Health Research, Center, and Training Grants CA89450, CA09592, and GM15431. The project described was also supported in part by the Molecular Recognition Unit Center grants from the National Cancer Institute (CA68485) and the National Institute of Environmental Health Sciences (P30 ES00267).

References (28)

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Abbreviations: NAGly, N-arachidonylglycine; AA, arachidonic acid; COX, cyclooxygenase; mCOX-2, murine COX-2; oCOX-1, ovine COX-1; PG, prostaglandin; HETE, hydroxyeicosatetraenoic acid; NSAID, nonsteroidal anti-inflammatory drug; 2-AG, 2-arachidonylglycerol; AEA, anandamide.

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