Characterization of the lysyl adducts of prostaglandin H-synthases that are derived from oxygenation of arachidonic acid

These investigations characterize the covalent binding of reactive products of prostaglandin H-synthases (PGHSs) to the enzyme and to other molecules. The intermediate product of oxygenation of arachidonic acid by the PGHSs, prostaglandin (PG) H-2, undergoes rearrangement to the highly reactive gamm a -keto aldehydes, levuglandin (LG) E-2 and D-2 We previously have demonstr ated that LGE(2) reacts with the E-amine of lysine to form both the lysyl-l evuglandin Shiff base and the pyrrole-derived lysyl-levuglandin lactam addu cts. We now demonstrate that these lysyl-levuglandin adducts are formed on the PGHSs following the oxygenation of arachidonic acid; after reduction of the putative Schiff base, proteolytic digestion of the enzyme, and isolati on of the adducted amino acid residues, these adducts were identified by li quid chromatography-tandem mass spectrometry. The reactivity of the LGs is reflected by the finding that virtually all of the LG predicted to be forme d from PGH(2) can be accounted for as adducts of the PGH-synthase and that oxygenation of arachidonic acid by PGH-synthases also leads to the formatio n of adducts of other proteins present in the reaction solution. The reacti vity of the PGH-synthase adducts themselves is demonstrated by the formatio n of intermolecular cross-links.