Download MendeleyInvestigating Substrate Promiscuity in Cyclooxygenase-2: THE ROLE OF ARG-120 AND RESIDUES LINING THE HYDROPHOBIC GROOVE.
Vecchio, A.J., Orlando, B.J., Nandagiri, R., Malkowski, M.G.(2012) J Biol Chem 287: 24619-24630
- PubMed: 22637474
- DOI: https://doi.org/10.1074/jbc.M112.372243
- Primary Citation of Related Structures:
3TZI, 4E1G - PubMed Abstract:
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 ¦Ø-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 ¦Ø-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 ¦Ø-6 and ¦Ø-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 ¦Á-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.
Organizational Affiliation:
Hauptman-Woodward Medical Research Institute and Department of Structural Biology, The State University of New York, Buffalo, New York 14203, USA.
