| Two enzymes that perform phospholipase A2 activity, small pancreatic phospholipase A2 (PLA2) and brain platelet activating factor acetylhydrolase (PAFAH), are examined. The crystal structure of the inactive zymogen form of PLA2, proPLA2, was solved to a resolution of 1.5 A in an anion-assisted dimer with a bound tetrahedral mimic at the active site. This crystal structure is proposed to be a mimic for the conformation of proPLA2 bound to an anionic interface. In comparison to a similar crystal structure of active PLA2 [Pan, Y. H., et al. (2001) Biochemistry 40, 609-617], several structural insights are gained to the possible origin of the 100-fold impairment of the chemical step in proPLA2 relative to PLA2. In particular, a critical active site water seen in PLA2 that is not seen in proPLA2 is discussed as the key structural difference in terms of the calcium-coordinated activated water mechanism for the esterolysis reaction. PAFAH is a medically interesting enzyme that has undergone poor kinetic characterization in the literature. The specific issue addressed is the increase in activity seen for amphiphilic substrates above the critical micelle concentration (CMC). Here, kinetic studies are done measuring the activity of PAFAH against the phospholipid platelet activating factor and several short-chain analogs along with competition studies with competing soluble substrates. Insights are gained about the nature of PAFAH's interaction with micellar substrate and several models to explain the anomalous activity above the CMC are discussed. Competition studies show that platelet activating factor has poor partitioning into the active site of PAFAH and therefore is unlikely to be the endogenous substrate. Three crystal structures of PAFAH covalently inhibited with the organophosphate compounds paraoxon, soman, and sarin were solved. These covalent complexes were pursued to gain insight into specific mechanistically important contacts between PAFAH and substrate since organophosphate compounds serve as a tetrahedral mimic. Such organophosphate compounds typically undergo a dealkylation reaction known as aging that makes the covalent complex refractory to reactivation. Surprisingly, the organophosphate complexes with PAFAH were found unaged and the significance of this observation is discussed in detail. |
