Long-chain lecithins present in bilayer structures like vesicles or membranes are only very poor substrates for pancreatic phospholipases A2. This is probably due to the fact that pancreatic phospholipases A2 cannot penetrate into the densely packed bilayer structures. To improve the weak penetrating properties of pancreatic phospholipases A2, we prepared and characterized a number of pancreatic phospholipase A2 mutants that have various long acyl chains linked covalently to Lys116 in porcine and to Lys10 in bovine phospholipase A2 [Van der Wiele, F.C., Atsma, W., Dijkman, R., Schreurs, A.M.M., Slotboom, A.J., & De Haas, G.H. (1988) Biochemistry (preceding paper in this issue)]. When monomolecular surface layers of L- and D-didecanoyllecithin were used, it was found that the introduction of caprinic, lauric, palmitic, and oleic acid at Lys116 in the porcine enzyme increases its penetrating power from 13 to about 17, 20, 32, and 22 dyn/cm, respectively, before long lag periods were obtained. Incorporation of a palmitoyl moiety at Lys10 in the bovine enzyme shifted the penetrating power from 11 to about 25 dyn/cm. Only the best penetrating mutant, viz., porcine phospholipase A2 having a palmitoyl moiety at Lys116, was able to cause complete leakage of 6-carboxyfluorescein entrapped in small unilamellar vesicles of egg lecithin under nonhydrolytic conditions. Similarly, only this latter palmitoylphospholipase A2 completely hydrolyzed all lecithin in the outer monolayer of the human erythrocyte at a rate much faster than Naja naja phospholipase A2, the most powerful penetrating snake venom enzyme presently known.

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The lipid-binding domain of pancreatic phospholipases A2 contains a number of exposed, hydrophobic amino acid side chains that are involved in the binding of the enzyme to organized lipid-water interfaces. Besides these apolar residues, at least two positively charged lysine groups are present in positions 10 and 116 of the lipid-binding domain. In order to investigate the possible function of these basic side chains in the lipid-binding process, a number of specifically acylated enzyme mutants were prepared, and their kinetic and lipid-binding properties have been compared with those of the native enzymes. It is concluded that the attachment of a long-chain acyl group in an amide linkage to Lys10 or Lys116 phospholipase A2 has only a minor influence on the catalytic properties of the enzyme. On the other hand, the lipid-binding properties of the mutant enzymes appear to be considerably reinforced.

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