Structural elucidation of cyclooxygenase enzymes provides insight into the specific stereochemistry of the cyclooxygenase reaction mechanism along with the formation of 15R cyclooxygenase products

Abstract

Cyclooxygenase (COX) enzymes are heme containing enzymes that catalyze the conversion of Arachidonic (AA) to prostaglandin H 2 (PGH 2 ) during the committed step in prostanoid biosynthesis. This consists of two sequential chemical reactions, a cyclooxygenase and a peroxidase reaction which occur in distinct, but mechanistically coupled active sites (38). There are two isoforms of COX known as COX-1 and COX-2. COX-1 is constitutively expressed (109) generating a pool of PGs that maintain cellular homeostasis and are involved in processes such as platelet aggregation and thrombosis (81, 82). COX-2 expression is induced in a variety of cells in response to a stimulus such as pain, fever, or inflammation (38). COX-2 expression leads to an increase in levels of prostanoids that accompany various pathologies such as inflammation, pain and fever (86, 107, 143), certain cancers (49, 137), Alzheimer's Disease (66) and Parkinson's Disease (115). Catalysis by COX enzymes is under strict stereochemical control and results in the conversion of an achiral substrate into a molecule containing five chiral centers (23). The strictly conserved stereochemistry of the reaction has been of major interest and has been investigated both structurally and biochemically. As a result, four cases have been reported where inversion of stereochemistry occurs, all during the oxygenation of C15. Aspirin inhibition of COX-2 results in the acetylation of Ser-530 and the formation of 15R-hydroxyeicosatetraenoic acid (15R-HETE) (27, 46, 61). Further investigation led to the identification of two mutants of COX-2, S530T and S530M, that also catalyze this novel oxygenation and form 15 R -PGG 2 (95). Mutagenesis studies conducted on Ser-530 have shown that replacing this residue with a bulkier amino acid, such as threonine or methionine, also causes a shift in the stereochemistry of the native reaction from 15 S -PGG 2 to 15 R -PGG 2 (95). This identified the important role that Ser-530 plays in helping maintain the stereochemistry at C15. There is also a case occurring in native COX enzyme from the coral species Plexaura homomalla which forms 15R-PGG 2 as its native product (128). Analysis of the primary structure of this novel COX enzyme identified a valine to isoleucine mutation at position 349. Val-349, located in the first shell of cyclooxygenase active site, is conserved among all other COX enzymes. Lastly, mutagenesis studies performed by Schneider et al. on Val-349 in both COX-1 and COX-2 resulted in a shift in the formation of 15 S -PGG 2 to 15 R -PGG 2 from 100% to 41 and 65% respectively. This established the importance that Val-349 along with Ser-530 plays to maintain the stereochemistry at C15 (95). We have utilized x-ray crystallographic methods to test the hypothesis that the inversion of stereochemistry at carbon-15 observed in products formed by aspirin-acetylated COX-2, a S530T mutant of COX-2 and a V349I mutant of COX-2 is a result of AA binding in an "unconventional" conformation within the cyclooxygenase active site prior to the initiation of catalysis. The crystal structures of V349I, S530T, aspirin-acetylated recombinant murine COX-2, both reconstituted with cobalt protoporphyrin-IX and complexed with AA or EPA, were determined to 1.9-2.45 Å resolution. The structures yielded three new catalytically competent conformations of AA and EPA from the S530T, V349I and aspirin-acetylated mCOX-2 structures and identified a new so-called "unproductive conformation of AA from the V349I COX-2 structure. These structures provide some insight into the formation of both 15 R -HETE and 15 R -PGG 2 and allow for conclusions as to how Val-349 contributes to maintaining the stereochemistry of oxygenation at C15.