Investigating substrate promiscuity in cyclooxygenase-2: the role of Arg-120 and residues lining the hydrophobic groove.
The cyclooxygenases (COX-1 and COX-2) generate prostaglandin H(2) 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 Co(3+)-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.
Hauptman-Woodward Medical Research Institute and Department of Structural Biology, The State University of New York, Buffalo, New York 14203, USA.
Alex J
AJ
Vecchio
Benjamin J
BJ
Orlando
Ritwik
R
Nandagiri
Michael G
MG
Malkowski
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D001120:Arginine / Q000737:chemistry; D002460:Cell Line; D018360:Crystallography, X-Ray; D051545:Cyclooxygenase 1 / Q000737:chemistry / Q000378:metabolism; D051546:Cyclooxygenase 2 / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D013379:Substrate Specificity; D017962:alpha-Linolenic Acid / Q000378:metabolism
D017962:alpha-Linolenic Acid; D016718:Arachidonic Acid; D001120:Arginine; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2