A New Class of Phospholipase A2 Substrates: Kinetics of the Phospholipase A2 Catalyzed Hydrolysis of 3-(Acyloxy)-4-nitrobenzoic Acids

Article abstract of DOI:10.1021/ja00223a043

3-(Acyloxy)-4-nitrobenzoic acids were synthesized with acyl groups ranging from butyryl to dodecanoyl.All these compounds yielded monomeric solutions in water with 1.6percent (v/v) acetonitrile in the neutral pH range, and they were hydrolyzed by catalytic amounts of phospholipases A₂ from a variety of sources as shown by the spectral change at 425 nm due to the appearance of nitrophenolate ion.Most of the kinetic studies were performed using Agkistrodon piscivorus piscivorus phospholipase A₂, but similar results were obtained with porcine pancreatic and Crotalus atrox phospholipase A₂.The catalytic reaction requires the presence of Ca²⁺, but unlike the hydrolysis of lecithins, the hydrolysis of these substrates also occurs in the presence of Ba²⁺ and Sr²⁺, while Mg²⁺ and Zn²⁺ are not catalytically competent.Increasing the acyl chain length increases the enzymatic rate mainly by enhancing the hydrophobic interaction in the E-Ca²⁺-S complex.Among structural isomers of the octanoyl compound, 3-nitro-4-(octanoyloxy)benzoic acid shows the highest specificity toward the enzyme, suggesting that it is in this compound that the distance between the negatively charged carboxylate and the reactive ester approximates best that found in the lecithin-enzyme complex.All kinetic characteristics of the enzymatic hydrolysis indicate that the reaction occurs by the same mechanism as that of the hydrolysis of lecithins.The highest catalytic efficiency observed with this series of substrates occurs with 3-dodecanoyl-4-nitrobenzoic acid, and the second-order rate constant of this reaction (k_cat/K_m = 9.1 x 1E+4 M^-1 s^-1) is only 1 order of magnitude lower than that of the hydrolysis of egg phosphatidylcholine in unilamellar vesicles.The reactivity of all isomers, especially that of the p-carboxy ester, shows that Ca²⁺ does not act as a catalyst in the phospholipase A₂ catalyzed hydrolysis but rather serves to bind and orient the substrate at the active site of the enzyme.The octanoyl compounds, 1 and 2, are ideally suited for a rapid and sensitive spectrophotometric assay of phospholipases A₂, and the conditions for the assay are described.