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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*

C. Harman,C. Rieke,R. Garavito,W. Smith

Published 2004 in Journal of Biological Chemistry

ABSTRACT

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.

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