Inhibition of 14-kDa PLA 2 by 2-acylamino-alkylphospholipids: the influence of amide acidity

Abstract

2-Acylamino-alkyl phospholipids are potent competitive inhibitors of 14-kDa phospholipases A 2 (e.g., human nonpancreatic secretory PLA 2). As concluded from X-ray studies the amide hydrogen of these inhibitors forms a hydrogen bond to His-48 in the active site of the enzyme. We investigated the quantitative contribution of this hydrogen bond to inhibition using especially designed inhibitors that bear different acyl chains with and without electron withdrawing or donating substituents, thus differing in amide acidity. Relative free enthalpies ΔΔ G of enzyme–inhibitor complex formations were calculated from X i(50) values determined by pH-stat titration using a mixed micelles assay and PLA 2 from Naja mocambique mocambique. A quantitative relationship between amide acidity and ΔΔ G values is presented. Comparison of isoacidic and isosteric inhibitors reveals that (i) the hydrogen bond of the amide proton to His-48 is crucial for strong PLA 2 inhibition, (ii) regardless of the headgroup unsubstituted N-acyl groups result in optimal amide acidity for PLA 2 inhibition and (iii) the exceptionally strong inhibition by acetamides and the isosteric fluoroacetamides is due to an additional steric effect.