Competitive inhibition of lipolytic enzymes. IX. A comparative study on the inhibition of pancreatic phospholipases A 2 from different sources by ( R)-2-acylamino phospholipid analogues

Abstract

The inhibitory power ( Z) of a number of ( R)-l-alkyl-2-acylamino phospholipid analogues was determined for three mammalian phospholipases A 2 from pig, ox and horse pancreas. All three enzymes display a clear preference for anionic (phosphoglycol) inhibitors over the zwitterionic (phosphocholine) derivatives; this effect is most pronounced for the bovine enzyme. Upon variation of the 1-alkyl chain length, the bovine and equine phospholipases, like the porcine enzyme in previous studies, show an optimum in Z for a six-carbon alkyl group. The introduction of a double bond in the 2-acylamino group generally improves the inhibitory power as compared with a fully saturated acyl chain. For the horse enzyme, the presence of an ( R)-2-undecenoylamino group in the phosphocholine- and phosphoglycol-containing inhibitors resulted in affinities which are nearly 4 and 5 orders of magnitude higher, respectively, than for the substrate molecule. Direct determination of the dissociation constant K i∗ of several inhibitors incorporated in a host lipid/water interface of non-inhibitory n-octadecenylphosphocholine micelles, was performed by ultraviolet difference spectroscopy. The progressive binding of a single inhibitor molecule into the active site of the three enzymes was followed quantitatively by an increasing tyrosine perturbation. With moderately strong competitive inhibitors ( Z values ranging from about 50 to 10 000), quantitative values for K i∗ were obtained. Extrapolation of the experimentally found linear relationship between Z and 1/ K i∗ yields predicted K i∗ numbers for the much stronger inhibitors with Z values betwee 10000 and 100000.