COMPETITIVE-INHIBITION OF INTERFACIAL CATALYSIS BY PHOSPHOLIPASE-A(2)- DIFFERENTIAL INTERACTION OF INHIBITORS WITH THE VESICLE INTERFACE AS A CONTROLLING FACTOR OF INHIBITOR POTENCY

Phospholipid analogues containing a phosphonate in place of the ester at the sn-2 position have been previously shown to be tight-binding co mpetitive inhibitors of secreted phospholipases A2. Variants of these compounds in which the structure of the phospholipid polar head group has been changed were prepared and analyzed as inhibitors of the phosp holipases A2 from bee and cobra venom, porcine and bovine pancreas, an d human synovial fluid. Kinetic measurements of inhibitor potencies we re carried out using negatively charged substrate vesicles under condi tions in which the enzyme undergoes catalysis without desorption from the vesicle (scooting mode). It is shown that this assay is useful in dissecting the contributions of enzyme-inhibitor affinities versus inh ibitor aqueous phase-to-vesicle phase partitioning to the overall inhi bitor potencies. Inhibition data were interpreted with the aid of the previously reported X-ray crystal structures of phospholipases A2 cont aining bound phospholipid analogue inhibitors. Compared to inhibitors that have polar head groups containing unsubstituted alkyl chains or a hydroxylethyl chain attached to the sn-3 phosphate, an inhibitor with an ethylammonium chain in the same position was found to bind 5-14-fo ld more weakly to the pancreatic, bee venom, and synovial fluid enzyme s. The X-ray structures of these enzymes reveal no interactions betwee n the protein and the head group chain attached to the sn-3 phosphate. These results suggest that intermolecular interactions of the ethylam monium portion of the inhibitor polar head group with neighboring phos pholipids in the interface (-PO2-...H-+NH2) can reduce the inhibition potency relative to those inhibitors that cannot form this interaction . Thus, methodology developed in this study is useful in measuring the relative interaction free energies of phospholipids containing differ ent polar head groups with neighboring phospholipids in the bilayer su rface. In contrast to the other phospholipases A2, the X-ray structure of the cobra venom enzyme reveals a geometrically ideal hydrogen bond between an asparagine residue and the polar head group ammonium of a bound phospholipid analogue inhibitor. Inhibitors with head group unsu bstituted alkyl chains or an ethylammonium chain bind to the cobra ven om enzyme with similar affinities; this is consistent with there being no net change in the number of hydrogen bonds when the inhibitors in the vesicle bind to the enzyme. An sn-2 phosphinate-containing phospho lipid analogue was prepared and evaluated as a phospholipase A2 inhibi tor. The phosphinate was about 2 orders of magnitude less potent than the analogous phosphonate when tested in both a vesicle assay and in a n aqueous solution assay with a water-soluble substrate. This result s uggests that the bridging oxygen of the sn-2 ester of a phospholipid i s well-solvated at the membrane surface. Since some of the inhibitors reported in this study are completely resistant to enzymatic degradati on by all known types of phospholipases and are highly potent (produci ng 50% inhibition when present in the vesicles at a level of one inhib itor per several thousand substrates), they should be useful tools in the study of the roles of phospholipases A2 in biological processes.