The Hydrolysis of Phosphatidyl-Alcohols by Phospholipases A 2: Effect of Head Group Size and Polarity

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The ability of a variety of secretory phospholipases A 2 (sPLA 2: EC 3.1.1.4) to bind to and hydrolyse a series of phosphatidyl-alcohol substrates, in the absence of detergent, was explored by both fluorescence-based kinetic and interfacial binding assays. The enzymes used were sPLA 2 from porcine pancreas, Naja naja venom and a recombinant human non-pancreatic enzyme. Four dioleoyl phosphatidyl-alcohols were used with different headgroups, methanol, ethanol, propanol and butanol. Comparative kinetic analyses with dioleoyl phosphatidyl-choline, dioleoyl phosphatidyl-glycerol and wheat germ phosphatidyl-inositol are also described. With the phosphatidyl-alcohol series, as the headgroup acyl-chain length increased the susceptibility to hydrolysis decreased. This effect was much more pronounced with the human non-pancreatic and the Naja naja venom enzymes than with the pancreatic enzyme. Maximum activity in this assay system was observed with porcine pancreatic sPLA 2 and dioleoyl phosphatidyl-methanol (1440 ± 167 μmol/min/mg). We demonstrate that the slow rate of hydrolysis of dioleoyl phosphatidyl-propanol by the human non-pancreatic secretory enzyme (4.56 ± 0.90 μmol/min/mg) is not due to a lack of interfacial binding. The hydrolysis of mixtures of dioleoyl phosphatidyl-choline and dioleoyl phosphatidyl-propanol in various molar proportions by Naja naja sPLA 2 suggests good mixing of the two phospholipids with minimal phospholipid domain formation under these assay conditions. We present strong evidence for a stimulation of hydrolysis of phosphatidyl-choline by human non-pancreatic sPLA 2 in the presence of as little as 1 mol% phosphatidyl-methanol (<40 fold total rate enhancement). Overall, the results demonstrate that the rates of hydrolysis of anionic phospholipids by sPLA 2 vary considerably with the different enzymes from this close structurally related family. The tight binding of the human enzyme to poorly hydrolysable anionic phospholipid vesicles provides a novel mechanism of enzyme inhibition by interfacial sequestration.