DEFINING THE DIMENSIONS OF THE CATALYTIC SITE OF PHOSPHOLIPASE-A2 USING AMIDE SUBSTRATE-ANALOGS

Two series of phospholipid analogues, each containing a thioether function at the sn-1 position and an amide function at the sn-2 position, have been synthesized and evaluated as phospholipase A2 inhibitors. The first series of analogues contained a hexyl group (C-6) at the sn-1 position and various acyl groups at the sn-2 position, ranging from formyl to dodecanoyl (1-12 carbons). The second series contained an sn-1 hexadecyl group (C-16) and various sn-2 acyl groups from formyl to eicosanoyl (1-20 carbons). Hydrophobic interactions of the enzyme with the amide analogues were studied using several different substrate forms including monomers, micelles, and n-mixed micelles with Triton X-100. The C-6 amide analogues were used for the monomeric study, while the C-16 analogues were used in the micellar studies. The inhibition studies with the monomeric amide analogues demonstrate that the sn-2 acyl chain is absolutely required for the binding of the analogue to the enzyme and that the catalytic site interacts with about the first 10 carbons of the sn-2 acyl chain. In addition, each methylene group of the sn-2 acyl chain from C5 to C10 provides about 665 cal/mol of binding energy. In contrast, the inhibition potency of the amide analogues in micellar states followed a quite different, more complex chain length dependency. The chain length of the sn-2 acyl group is much less important in the micellar systems than in the monomeric system, since the hydrophobic interactions between the sn-2 acyl chain and the enzyme are balanced by its interactions with the hydrophobic core of the micelle. The importance of double bonds in the sn-2 chain was also studied, but no correlation between the degree of unsaturation and the degree of inhibition was observed. These studies help delineate the mode of the interactions between enzyme and substrate.