? The overall goal of this grant over the years has been to characterize the detailed mechanism of action of various physiologically important forms of phospholipase A2 (PLA2). During the course of these studies, it has become apparent that the activity of this superfamily of enzymes depends critically on the interaction of the proteins with large lipid aggregates. It appears that the orientation of the enzyme with respect to the plane of the lipid-water interface can have a dramatic effect on activity. The nature of this interaction has been difficult to explore because of the fact that it represents the interaction of two large macromolecules. This has presented challenges for traditional NMR and X-ray crystallographic studies. In addition, several PLA2s undergo self-association and the effect that this oligomerization has on activity needs to be ellucidated. The activity of many of these enzymes appears to increase when the enzyme is at the lipid-water interface. This activation could also be due to changes in enzyme-lipid orientation or to conformational changes in the enzyme. This renewal application will extend our current studies on the Ca2+-dependent secretory cobra venom Group IA. and human Group V PLA2s, the cytosolic Ca 2+-dependent human Group IVA PLA2, and the Ca 2+ -independent human Group VIA PLA2. We have recombinantly expressed and biochemically characterized all four of these enzymes during the current grant period. The overall objective of the current grant proposal is to address these remaining important issues of PLA2 function. Along with traditional biochemical, molecular biological, and kinetic approaches, we will employ amide deuterium exchange-mass spectrometry. It is rapidly becoming clear that this technique can tackle many structural questions that cannot be addressed easily by NMR or X-ray crystallography. We will expand the use of this technique to explore the interactions of these enzymes with large lipid interfaces. We also will use analytical ultracentrifugation and surface plasmon resonance to study these questions. We will focus on these four PLA2s which we have characterized sufficiently to be explored by these techniques and for which we can examine their specific interactions with phospholipid interfaces. ? ?
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