Competitive inhibition of lipolytic enzymes. IV. Structural details of acylamino phospholipid analogues important for the potent inhibitory effects on pancreatic phospholipase A2.

1-Acyl-2(R)-acylamino phospholipids are effective competitive inhibitors of porcine pancreatic phospholipase A2 (EC 3.1.1.4, Bonsen et al. (1972) Biochim. Biophys. Acta 270, 364-382). By systematically varying the substituent at C-1 and the acyl chain length at C-2, a series of phospholipid analogues was obtained for which the inhibitory power was determined in a detergent-containing and occasionally also in a detergent-free micellar substrate system. The recently proposed kinetic model applicable to water-insoluble inhibitors (Ransac et al. (1990) Biochim. Biophys. Acta 1043, 57-66) allowed a quantitative comparison of the inhibitory power Z of the various substrate analogues. The most powerful inhibitors of the enzyme were found to possess the general 2-(R)-structure: (formula; see text) Using as substrate (R)-1,2-didodecanoylglycero-3-phosphocholine in mixed micelles with sodium taurodeoxycholate, the inhibitor molecule with m = 4 and n = 11 showed a Z-value of 15,000. This implies an affinity of the inhibitor for the active site of the enzyme higher than 4 orders of magnitude stronger as compared with the substrate molecule. Slightly higher and lower m-values resulted in a sharp drop of the inhibitory power, which suggests that the enzyme must possess a rather short, but well-defined hydrophobic binding pocket for the C-1 alkyl chain. Variation of n (keeping m = 2 constant) resulted in inhibitors with nearly equal Z-values for n = 11, 13 and 15. Most probably the binding cleft on the enzyme for the C-2 acylamino chain is longer, more losely constructed and contributing less to the overall binding energy. Several members of the 2-acylamino phospholipids are water-soluble and possess relatively high critical micelle concentrations. Their inhibitory power could be tested not only in micellar substrate dispersions but also in assay systems where both the inhibitor and substrate are molecularly dispersed. It appeared that these water-soluble phospholipid analogues are effective inhibitors of the enzyme only after incorporation into an organized substrate/water interface. In contrast, in molecularly dispersed substrate solutions the same molecules have completely lost their inhibitory power. These observations support our kinetic model of lipolysis and interfacial inhibition.