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Biochemistry. 1993 Jun 15;32(23):5935-40.

Slow- and tight-binding inhibitors of the 85-kDa human phospholipase A2.

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Merck Frosst Centre for Therapeutic Research, Pointe Claire-Dorval, Québec, Canada.


A trifluoromethyl ketone analogue of arachidonic acid in which the COOH group is replaced with COCF3 (AACOCF3) was prepared and found to be a tight- and slow-binding inhibitor of the 85-kDa cytosolic human phospholipase A2 (cPLA2). Enzyme inhibition was observed when AACOCF3 was tested in assays using either phospholipid vesicles or phospholipid/Triton X-100 mixed micelles. The fact that the inhibition developed over several minutes in both assays establishes that AACOCF3 inhibits by direct binding to the enzyme rather than by decreasing the fraction of enzyme bound to the substrate interface. From the measured values of the inhibitor association and dissociation rate constants, an upper limit of the equilibrium dissociation constant for the Ca(2+).AACOCF3.PLA2 complex of 5 x 10(-5) mole fraction was obtained. Thus, detectable inhibition of cPLA2 by AACOCF3 occurs when this compound is present in the assay at a level of one inhibitor per several thousand substrates. Arachidonic acid analogues in which the COOH group is replaced by COCH3, CH(OH)CF3, CHO, or CONH2 did not detectably inhibit the cPLA2. The arachidonyl ketones AACOCF2CF3 and AACOCF2Cl were found by 19F NMR to be less hydrated than AACOCF3 in phospholipid/Triton X-100 mixed micelles, and compared to AACOCF3 these compounds are also weaker inhibitors of cPLA2. In keeping with the fact that cPLA2 displays substrate specificity for arachidonyl-containing phospholipids, the arachidic acid analogue C19H39COCF3 is a considerably less potent inhibitor compared to AACOCF3.(ABSTRACT TRUNCATED AT 250 WORDS).

[Indexed for MEDLINE]

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