Enzymes are exquisitely selective biocatalysts, competent of choosing a unique substrate among many biological compounds. The specificity of an enzyme for a substrate is defined by the level of the reaction rate. Phospholipases A2 (PLA2s), which catalyze the hydrolysis of phospholipids that comprise the membrane bilayer, provide an ideal system for studying substrate specificity. The specificity of a PLA2 enzyme is connected to the three-dimensional structure of its catalytic site, which is complementary to the transition state of the reaction. PLA2s also contain secondary “fingerprint” bonding pockets that increase specificity for a phospholipid species (PNAS 2015, 112, E516-E525). This study focuses on understanding the allosteric activation mechanism of three human PLA2 enzymes including the cytosolic (cPLA2), calcium-independent (iPLA2) and lipoprotein-associated (Lp-PLA2). cPLA2 is the main arachidonic acid provider for the eicosanoid pathway, while iPLA2 is involved in membrane phospholipid remodeling. Lp-PLA2 was found to associate with LDL and HDL in human plasma to hydrolyze phospholipids containing short-chain and oxidized fatty acids (Chem. Rev. 2011, 111, 6130-6185). Hydrogen/deuterium exchange mass spectrometry was used to experimentally identify distinct PLA2 peptide regions that interact with phospholipid vesicles. Molecular dynamics simulations, guided and validated by the experimental data, showed that the active sites of these enzymes are allosterically regulated by membranes. Membrane phospholipids bind to allosteric sites located on the interfacial surface of PLA2s shifting their conformation from the “closed” to the “open” state. This process allows each enzyme to recruit secondary hydrophobic binding pockets that recognize unique types of fatty acids esterified at the sn-2 position of phospholipids. These enzymes are implicated in various chronic inflammatory diseases and understanding their association with membranes, mechanism of action and interactions at the molecular level will allow us to identify potent and selective inhibitors that can be further developed as anti-inflammatory agents.

Interfacial activation of lipolytic enzymes by cellular membranes

2019 Spring National Meeting

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