Effect of polar head groups on the interactions of phospholipase A2 with phosphonate transition-state analogues.

Several new phosphonate-containing phospholipid analogues were synthesized as inhibitors of cobra venom (Naja naja naja) phospholipase A2. These phospholipid analogues contained a novel thioether at the sn-1 position, a tetrahedral phosphonate moiety in place of the scissile ester bond at the sn-2 position, and several different polar head groups, including phosphocholine, phospho(N,N-dimethylethanolamine), phospho(N-methylethanolamine), and phosphoethanolamine. The affinities of these analogues for the enzyme were evaluated in the well-defined Triton X-100 mixed micelle system using thio-PC and thio-PE substrates. These phosphonates inhibited thio-PC hydrolysis with very similar potencies. Inhibition of phospholipase A2 by phosphonates is known to be pH-dependent [Yu, L., & Dennis, E. A. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 9325-9329]. At pH 5.5, all of the new analogues had IC50s of about 2 x 10(-5) mol fraction. At this pH, these inhibitors are the most potent reversible inhibitors of phospholipase A2 reported to date. In contrast, at pH 8.5, the PE analogue was a potent inhibitor of thio-PC hydrolysis (IC50 1.8 x 10(-3) mol fraction) but was a very poor inhibitor of thio-PE hydrolysis (IC50 is not detectable). However, the inhibition of thio-PE hydrolysis was dramatically enhanced when the enzyme was activated by sphingomyelin, suggesting that the phosphonate inhibitors bind much more tightly to the activated enzyme than to the nonactivated enzyme. The activation and inhibition of the enzyme have different pH dependencies; the enzyme activation is not pH-dependent, whereas the enzyme inhibition is pH-dependent. These results confirm the presence of a functionally distinct activator site on this enzyme.