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Investigating Substrate Promiscuity in Cyclooxygenase-2

A. Vecchio,B. Orlando,R. Nandagiri,M. Malkowski

Published 2012 in Journal of Biological Chemistry

ABSTRACT

Background: Cyclooxygenase-1 and -2 have isoform-specific substrate preferences and interactions. Results: Mutations to residues in the COX-2 cyclooxygenase channel individually have no significant effect on high affinity binding of fatty acid substrates. Conclusion: Proper bisallylic carbon positioning drives efficient substrate oxygenation by COX-2. Significance: Our results confirm nuances between isoforms that explain the ability of COX-2 to oxygenate a broad spectrum of substrates. The cyclooxygenases (COX-1 and COX-2) generate prostaglandin H2 from arachidonic acid (AA). In its catalytically productive conformation, AA binds within the cyclooxygenase channel with its carboxylate near Arg-120 and Tyr-355 and ω-end located within a hydrophobic groove above Ser-530. Although AA is the preferred substrate for both isoforms, COX-2 can oxygenate a broad spectrum of substrates. Mutational analyses have established that an interaction of the carboxylate of AA with Arg-120 is required for high affinity binding by COX-1 but not COX-2, suggesting that hydrophobic interactions between the ω-end of substrates and cyclooxygenase channel residues play a significant role in COX-2-mediated oxygenation. We used structure-function analyses to investigate the role that Arg-120 and residues lining the hydrophobic groove play in the binding and oxygenation of substrates by murine (mu) COX-2. Mutations to individual amino acids within the hydrophobic groove exhibited decreased rates of oxygenation toward AA with little effect on binding. R120A muCOX-2 oxygenated 18-carbon ω-6 and ω-3 substrates albeit at reduced rates, indicating that an interaction with Arg-120 is not required for catalysis. Structural determinations of Co3+-protoporphyrin IX-reconstituted muCOX-2 with α-linolenic acid and G533V muCOX-2 with AA indicate that proper bisallylic carbon alignment is the major determinant for efficient substrate oxygenation by COX-2. Overall, these findings implicate Arg-120 and hydrophobic groove residues as determinants that govern proper alignment of the bisallylic carbon below Tyr-385 for catalysis in COX-2 and confirm nuances between COX isoforms that explain substrate promiscuity.

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