Probing the role of substrate conformation in phospholipase A2 action on aggregated phospholipids using constrained phosphatidylcholine analogues.
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.
Department of Biochemistry and Molecular Biology, University of Chicago, Illinois 60637.
P N
PN
Barlow
M D
MD
Lister
P B
PB
Sigler
E A
EA
Dennis
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003460:Crystallization; D004305:Dose-Response Relationship, Drug; D010713:Phosphatidylcholines / Q000494:pharmacology*; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism*; D011487:Protein Conformation
D010713:Phosphatidylcholines; D010743:Phospholipids; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2