SLOW-BINDING AND TIGHT-BINDING INHIBITORS OF THE 85-KDA HUMAN PHOSPHOLIPASE-A2

A trifluoromethyl ketone analogue of arachidonic acid in which the COO H group is replaced with COCF3 (AACOCF3) was prepared and found to be a tight- and slow-binding inhibitor of the 85-kDa cytosolic human phos pholipase A2 (cPLA2). Enzyme inhibition was observed when AACOCF3 was tested in assays using either phospholipid vesicles or phospholipid/Tr iton X-100 mixed micelles. The fact that the inhibition developed over several minutes in both assays establishes that AACOCF3 inhibits by d irect binding to the enzyme rather than by decreasing the fraction of enzyme bound to the substrate interface. From the measured values of t he inhibitor association and dissociation rate constants, an upper lim it of the equilibrium dissociation constant for the Ca2+.AACOCF3.cPLA2 complex of 5 x 10(-5) mole fraction was obtained. Thus, detectable in hibition of cPLA2 by AACOCF3 occurs when this compound is present in t he assay at a level of one inhibitor per several thousand substrates. Arachidonic acid analogues in which the COOH group is replaced by COCH 3, CH(OH)CF3, CHO, or CONH2 did not detectably inhibit the cPLA2. The arachidonyl ketones AACOCF2CF3 and AACOCF2Cl were found by F-19 NMR to be less hydrated than AACOCF3 in phospholipid/Triton X-100 mixed mice lles, and compared to AACOCF3 these compounds are also weaker inhibito rs of cPLA2. In keeping with the fact that cPLA2 displays substrate sp ecificity for arachidonyl-containing phospholipids, the arachidic acid analogue C19H39COCF3 is a considerably less potent inhibitor compared to AACOCF3. AACOCF3 is about 4 orders of magnitude less potent as an inhibitor of the human nonpancreatic secreted 14-kDa phospholipase A2. This fact together with the likelihood that AACOCF3 is cell-permeable suggests that this compound may be useful in studying the role of the cPLA2 in cellular processes that involve arachidonic acid liberation.