IDENTIFICATION OF 2 SPECIFIC LYSINES RESPONSIBLE FOR THE INHIBITION OF PHOSPHOLIPASE A(2) BY MANOALIDE

Manoalide, a natural product of sponge, irreversibly inhibits phosphol ipase A(2) (PLA(2)) by reacting with lysine residues. Cobra venom PLA( 2) mutants were constructed in which four of the six lysine residues w ere independently replaced by arginine or methionine, which cannot rea ct with manoalide. The mutants were overexpressed in Escherichia coli, renatured, and purified. The enzyme mutants lacking Lys-6 (K6R and K6 M) or Lys-79 (K79R) were inhibited only 40% by manoalide while the nat ive cobra venom PLA(2) was inhibited 80% under the same conditions. Th is means that the manoalide modification of either Lys-6 or Lys-79 acc ounted for only half of the manoalide inhibition. The double mutant (K 6R79R) was not inhibited by manoalide at all. Lys-56 (K56R) and Lys-65 (K65R) mutants were inhibited to the same extent as the native enzyme which indicates that these residues are not responsible for any of th e inhibitory effects produced by manoalide. These results demonstrate that the reaction of manoalide with both Lys-6 and Lys-79 can account for all of its inhibition of cobra venom PLA(2). The inhibition of PLA (2) and its mutants with manoalide did not affect the activity of the enzyme toward monomeric substrate, which suggests that manoalide does not modify the catalytic site residues, that it does not block access to this site, and that its inhibition requires an interface. Furthermo re, as with native PLA(2), the activation of phosphatidylethanolamine hydrolysis by phosphorylcholine-containing compounds was exhibited by all of the mutants suggesting that none of the lysines examined are es sential for this activation.