Structural Effects Of Covalent Inhibition Of Phospholipase A2 Suggest Allosteric Coupling Between Membrane Binding And Catalytic Sites

Phospholipase A2 (PLA2) binds to membranes and catalyzes phospholipid hydrolysis, thus initiating the biosynthesis of lipid-derived mediators of inflammation. A snake-venom PLA2 was completely inhibited by covalent modification of the catalytic histidine 48 by p-bromophenacyl bromide. Moreover, His48 modification affected PLA2 structure, its membrane-binding affinity, and the effects of PLA2 on the membrane structure. The native PLA2 increased the order parameter of fluid membranes, whereas the opposite effect was observed for gel-state membranes. The data suggest membrane dehydration by PLA2 and the formation of PLA2-membrane hydrogen bonding. The inhibited PLA2 had lower membrane-binding affinity and exerted weaker effects on membrane hydration and on the lipid-order parameter. Although membrane binding resulted in formation of more flexible α-helices in the native PLA2, which corresponds to faster amide hydrogen exchange, the modified enzyme was more resistant to hydrogen exchange and experienced little structural change upon membrane binding. The data suggest that 1), modification of a catalytic residue of PLA2 induces conformational changes that propagate to the membrane-binding surface through an allosteric mechanism; 2), the native PLA2 acquires more dynamic properties during interfacial activation via membrane binding; and 3), the global conformation of the inhibited PLA2, including the α-helices, is less stable and is not influenced by membrane binding. These findings provide further evidence for an allosteric coupling between the membrane-binding (regulatory) site and the catalytic center of PLA2, which contributes to the interfacial activation of the enzyme