CONFORMATION OF MICELLAR PHOSPHOLIPID BOUND TO THE ACTIVE-SITE OF PHOSPHOLIPASE A(2)

Transferred NOE techniques have been used to determine the structure of phospholipid analogues bound to the active site of cobra venom phospholipase A(2) (PLA(2)). These experiments were carried out on PLA(2) with a substrate analogue which serves as an inhibitor, 1-(hexylthio)-2-(nonanoylamino)-1,2-dideoxy-sn-glycero-3-phosphocholine (PC9). Because this inhibitor binds tightly to the enzyme and forms micelles at millimolar concentrations, experiments could be carried out to determine the conformation of the inhibitor when bound to the enzyme at the lipid-water interface. NOEs of the micellar lipid develop inefficiently in the absence of enzyme. NOESY experiments in the presence of PLA(2) were used to determine the inhibitor structure and conformation when bound to the enzyme. The inhibitor adopts an active site conformation in which the end of the sn-2 chain is within 5 Angstrom of the alpha-methylene protons of the sn-1 chain. However, NOE cross-peaks in the experiments indicate that the backbone conformation of the bound lipid is different from that of a shorter chain lipid which forms monomers [Plesniak et al. (1993) Biochemistry 32, 5009-5016].