Phospholipase A 2 : A Paradigm for Allosteric Regulation by Membranes and Cell Signaling

Our laboratory discovered and demonstrated that membranes interact allosterically with enzymes to regulate cell signaling and metabolic pathways leading to inflammation 1 . We have recently employed substrate lipidomics coupled with molecular dynamics to reveal enzyme specificity linked to highly specific hydrophobic binding sites for the sn‐2 fatty acyl chains in membrane phospholipid substrates 2 . We discovered unexpected headgroup and acyl chain specificity for each of the major human phospholipase A 2 (PLA 2 ) enzymes that explains the observed specificity at a new atomic level. A unique hydrophobic binding site — and not each enzyme's catalytic residues or polar headgroup binding site — dominates each enzyme's specificity. Each PLA 2 shows unique specificity for its required fatty acid ranging from pro‐inflammatory omega‐6 arachidonic acid or anti‐inflammatory fish oil omega‐3 EPA and DHA; others favor membrane remodeling linolenic acid, or antibacterial saturated fatty acids, or oxidized fatty acids in LDL. Each PLA 2 releases a specific fatty acid after the enzyme associates allosterically with membranes and extracts a single phospholipid substrate into its catalytic site. Stereospecific inhibitors 3 have been designed for the specific sites. After decades of advances in lipid research, we can now correlate PLA 2 specificity and inhibition potency with molecular structure and physiological function using a novel lipidomics platform that provides a paradigm for protein‐membrane lipid interactions in general. From the cell signaling and inflammation perspective, the omics revolution began with genomics, proteomics and metabolomics, but lipidomics now dominates as the largest number of cellular metabolites are lipids, many playing critical roles in cell signaling, and over 40,000 distinct molecular species have been identified by the LIPID MAPS Consortium ( www.lipidmaps.org ). We have also employed lipidomics analysis to characterize the in vivo role of specific PLA 2 enzymes in initiating the inflammatory response and cellular lipid signaling of Toll‐like (TLR4) and purinergic (P2X 7 ) receptors in stimulated macrophages as models of bacterial infection and inflammation 4 . We can now explain in vivo cellular specificity by in vitro enzyme specificity, inhibition and pro‐inflammatory and pro‐resolution lipid mediator formation pathways and inhibition 5 . 1 Dennis EA (2016) REFLECTIONS: Liberating chiral lipid mediators, inflammatory enzymes and LIPID MAPS from biological grease, J Biol Chem , 291 , 24431–48. 2 Mouchlis VC, Chen Y, McCammon JA, Dennis EA (2018) Membrane allostery and unique hydrophobic sites promote enzyme substrate specificity, J Am Chem Soc , 140 , 3285‐91. 3 Mouchlis VD, Armando AM, Dennis EA (2019) Substrate specific inhibition constants for phospholipase A 2 acting on unique phospholipid substrates in mixed micelles and membranes using lipidomics. J Med Chem , 62 , 1999‐2007. 4 Dennis EA, Norris PA (2015) Eicosanoid storm in infection and inflammation. Nature Immunology Reviews, 15 , 511‐523. 5 Navratil AR, Shchepinov MS, Dennis EA (2018) Lipidomics reveals dramatic physiological kinetic isotope effects during the enzymatic oxygenation of polyunsaturated fatty acids ex vivo . J Am Chem Soc , 140 , 235‐43.