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J. Biol. Chem., Vol. 279, Issue 41, 42929-42935, October 8, 2004

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Crystal Structure of Arachidonic Acid Bound to a Mutant of Prostaglandin Endoperoxide H Synthase-1 That Forms Predominantly 11-Hydroperoxyeicosatetraenoic Acid^*

Christine A. Harman, Caroline Jill Rieke, R. Michael Garavito, and William L. Smith¶

From the Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824 and the Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109

Kinetic studies and analysis of the products formed by native and mutant forms of ovine prostaglandin endoperoxide H synthase-1 (oPGHS-1) have suggested that arachidonic acid (AA) can exist in the cyclooxygenase active site of the enzyme in three different, catalytically competent conformations that lead to prostaglandin G₂ (PGG₂), 11R-hydroperoxyeicosatetraenoic acid (HPETE), and 15R,S-HPETE, respectively. We have identified an oPGHS-1 mutant (V349A/W387F) that forms predominantly 11R-HPETE. Thus, the preferred catalytically competent arrangement of AA in the cyclooxygenase site of this double mutant must be one that leads to 11-HPETE. The crystal structure of Co³⁺-protoporphyrin IX V349A/W387F oPGHS-1 in a complex with AA was determined to 3.1 Å. Significant differences are observed in the positions of atoms C-3, C-4, C-5, C-6, C-10, C-11, and C-12 of bound AA between native and V349A/W387F oPGHS-1; in comparison, the positions of the side chains of cyclooxygenase active site residues are unchanged. The structure of the double mutant presented here provides structural insight as to how Val³⁴⁹ and Trp³⁸⁷ help position C-9 and C-11 of AA so that the incipient 11-peroxyl radical intermediate is able to add to C-9 to form the 9,11 endoperoxide group of PGG₂. In the V349A/W387F oPGHS-1·AA complex the locations of C-9 and C-11 of AA with respect to one another make it difficult to form the endoperoxide group from the 11-hydroperoxyl radical. Therefore, the reaction apparently aborts yielding 11R-HPETE instead of PGG₂. In addition, the observed differences in the positions of carbon atoms of AA bound to this mutant provides indirect support for the concept that the conformer of AA shown previously to be bound within the cyclooxygenase active site of native oPGHS-1 is the one that leads to PGG₂.

Received for publication, March 18, 2004 , and in revised form, July 20, 2004.

The atomic coordinates and structure factors (code 1U67) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported in part by United States Department of Energy, Basic Energy Sciences, Office of Energy Research Contract W-31-109-Eng-38 and by Program Project Grant P01 GM57323 and Grants R01 HL56773 and R01 GM68848 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Biological Chemistry, University of Michigan Medical School, 5416 Medical Science Bldg. I, 1301 E. Catherine St., Ann Arbor, MI 48109-0606. Tel.: 734-647-6180; Fax: 734-764-3509; E-mail: smithww{at}umich.edu.

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