Conformation of micellar phospholipid bound to the active site of phospholipase A2

Abstract

Transferred NOE techniques have been used to determine the structure of phospholipid analogues bound to the active site of cobra venom phospholipase A2 (PLA2). These experiments were carried out on PLA2 with a substrate analogue which serves as an inhibitor, 1-(hexylthio)-2-(nonanoylamino)-1,2-dideoxy-sn-glycero-3-pho sphocholine (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 PLA2 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 A 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].

Substrate Efflux Propensity Is the Key Determinant of Ca2+-independent Phospholipase A-β (iPLAβ)-mediated Glycerophospholipid Hydrolysis

“…Such data suggest that there is a binding site for the sn-2 chain but not for the sn-1 chain in those enzymes and that this site interacts only with the first 8 -10 carbons of the sn-2 chain (27). This conclusion is consistent with the fact that these PLAs hydrolyze the sn-2 ester bond but not the sn-1 (53,54). For iPLA␤, the differences between the sn-1 and sn-2 chain were much smaller, albeit statistically significant (Fig.…”

Factors regulating the substrate specificity of cytosolic phospholipase A 2 -alpha in vitro

“…This conclusion is consistent with previous modeling studies suggesting that the two acyl chains of PC share a common hydrophobic cavity rather than two separate cavities in cPLA 2 α [12]. Supporting this interpretation, in a previous study the activity of three secretory PLAs (sPLA 2 s), which do not have any significant PLA 1 activity [52], was very differently influenced by the length of the sn1 or sn2 acyl chain [25]. In a notable contrast to the saturated PCs, cPLA 2 α showed here a marked isomer preference for the AA-containing PCs in micelles, i.e., the species containing an AA residue in the sn2 position were hydro-lyzed~4-fold faster than their isomers (Fig.…”

Phospholipase A2 Biochemistry

“…2. [52, 53]. The fatty acid tails of the substrate are surrounded by the hydrophobic residues Leu-2, Phe-5, Trp-19, Tyr-52, and Tyr-69.…”

“…Group IA PLA 2 with phospholipid substrate modeled in the active site based on crystal structure by Fremont et al [39], along with NOE measurements [52] using amide pseudo-substrate inhibitor [53]. The active site residues His-48 and Asp-93 and the bound Ca 2+ is shown in purple.…”