| Abstract
| - Studies are reported on two different types of activated ketonesas inhibitors of two important intracellularphospholipase A2s (PLA2): the group IV 85 kDaCa2+-dependent phospholipase A2(cPLA2) and the P388D1Ca2+-independent phospholipase A2 (iPLA2). In amixed micelle assay, we observed that the reaction progress curveofcPLA2 in the presence of a trifluoromethyl ketone (TFMK) islinear at pH 7.4, while at pH 9.0 it is nonlinear andslows with time. An investigation of this discrepancy demonstratedthat the TFMKs are slow, tight-binding inhibitorsof the cPLA2 at both pH's, that the rate of dissociation ofthe enzyme−inhibitor complex is the same at both pH's,but that the rate of association of enzyme and inhibitor is slower atpH 7.4 than at pH 9.0. A novel group ofactivated ketone inhibitors has been synthesized that contain a fattyacyl tricarbonyl. These compounds also inhibitthe cPLA2 in the mixed micelle assay. The inhibitionof cPLA2 by the tricarbonyls is readily reversible upondilutionand does not involve slow binding. For both types of inhibitor, nopreference for fatty acid chain was observed asthe palmityl analogs inhibited as well as the arachidonoyl analogs,despite the fact that the cPLA2 shows a strongpreference for arachidonoyl-containing phospholipid substrates overpalmitoyl-containing substrates. With theiPLA2,the inhibition by TFMKs is reversible and does not involve slow ortight binding. The tricarbonyls also inhibitedthe iPLA2, but were less potent than the TFMKs. Unlikethe cPLA2, the iPLA2 does exhibit a fatty acidpreferenceas the palmityl analogs of both compounds inhibit better than thearachidonoyl analogs. The palmityl TFMK displayeda 10-fold lower IC50 at pH 9.0 than at pH 7.5, whereas thepotency of the tricarbonyl was unchanged in this range.Thus, the TFMKs inhibit both the cPLA2 and theiPLA2, but the mechanism of inhibition of the two enzymesappearsto be quite different. The tricarbonyls also inhibited bothenzymes, but in both cases in a reversible manner and assuch appear to be poorer inhibitors than the TFMKs.
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