Platelet-activating factor acetylhydrolase (PAF-AH) is a phospholipase A₂ that inactivates potent lipid messengers, such as PAF and modified phospholipids generated in settings of oxidant stress. The catalytic activity of PAF-AH is sensitive to oxidants, a feature that may have pathological consequences. We report that peroxynitrite, an oxidant species generated after cellular activation, mediates oxidative inactivation of PAF-AH. We found that peroxynitrite inactivated and derivatized the recombinant protein and obtained evidence supporting a role for a methionine and two tyrosine residues in this process. We employed interspecies comparisons and site-directed mutagenesis and identified a role for M-117, and a smaller contribution of Y-307 and Y-335 as targets of oxidant attack using free and lipoprotein-associated recombinant proteins. M-117 is adjacent to W-115 and L-116, which are essential for association of PAF-AH with LDL. Oxidation of LDL-associated PAF-AH partially dissociated the enzyme from the particles. Similarly, oxidation of the purified enzyme in the absence of lipoproteins prevented subsequent association with LDL. These results provide new insights into the molecular mechanisms that mediate inactivation of PAF-AH in settings of oxidant stress and the consequences of oxidation on the ability of this enzyme to associate with LDL.-- MacRitchie, A. N., Gardner, A. A., Prescott, S. M., Stafforini, D. M. Molecular basis for susceptibility of plasma platelet-activating factor acetylhydrolase to oxidative inactivation.