Probing the role of substrate conformation in phospholipase A2 action on aggregated phospholipids using constrained phosphatidylcholine analogues.
- Author(s): Barlow, PN;
- Lister, MD;
- Sigler, PB;
- Dennis, EA
- et al.
Published Web Locationhttps://doi.org/10.1016/s0021-9258(18)37655-5
Phospholipase A2s hydrolyze aggregated phospholipid substrates much more rapidly than dispersed monomeric ones. Whether this is a consequence of interface-associated conformational changes of the enzyme or of the substrate, or of both, remains a key question in lipid enzymology. This problem is addressed herein using a rationally designed probe of substrate conformation. (1,3/2)-1-O-(phosphorylcholine)-2,3-O-dihexanoylcyclopentane-1,2,3 -triol is a novel short chain phosphatidylcholine analogue in which the glycerol-like backbone is part of a five-membered ring and therefore covalently constrained within a small defined range of conformations. To the extent that the constrained analogue resists aggregation-associated conformational changes, it provides a means for assessing the contribution of such changes to phospholipase A2 action on aggregated phospholipids. The monomeric (-)-cyclopentanoid analogue is a substrate for phospholipase A2s from Naja naja naja venom. However, when this constrained phospholipid is aggregated, its hydrolysis rate is not enhanced, in contrast to its unconstrained counterpart, 1,2-dihexanoyl-sn-glycero-3- phosphorylcholine. This lack of activation was not caused by a failure of the enzyme to bind the micellar, constrained analogue. While the constrained analogue does not show interfacial activation, it does show the activation of phosphatidylethanolamine hydrolysis typical of phosphorylcholine-containing lipids. Hence, these results strongly support the contention that specific packing-induced conformations of aggregated substrate play a substantial role in the large interfacial activations observed with phospholipase A2.