Listeria monocytogenes phosphatidylinositol-specific phospholipase C: Kinetic activation and homing in on different interfaces.

The phosphatidylinositol-specific phospholipase C (PI-PLC) from Listeria monocytogenes forms aggregates with anionic lipids leading to low activity. The specific activity of the enzyme can be enhanced by dilution of the protein or by addition of both zwitterionic and neutral amphiphiles (e.g., diheptanoylphosphatidylcholine or Triton X-100) or 0.1-0.2 M inorganic salts. Activation by amphiphiles occurs with both micellar (phosphatidylinositol dispersed in detergents) and monomeric [dibutroylphosphatidylinositol (diC(4)PI)] phosphotransferase substrates and inositol 1,2-(cyclic)-phosphate (cIP), the phosphodiesterase substrate. The presence of zwitterionic and neutral amphiphiles (to which the protein binds weakly) dilutes the surface concentration of the interfacial anionic substrate and thereby reduces the level of enzyme-phospholipid particle aggregation. Zwitterionic amphiphiles also can bind directly to the protein and enhance catalysis since they enhance both diC(4)PI and cIP hydrolysis. In contrast to activation by amphiphiles, the rate enhancement by salt occurs for only the phosphotransferase step of the reaction. Added salt has a synergistic effect with zwitterionic phospholipids, leading to high specific activities for PI cleavage with only moderate dilution of the anionic substrate in the interface. This kinetic activation correlates with weakening of strong PI-PLC hydrophobic interactions with the interface as monitored by a decrease in the maximum monolayer surface pressure for insertion of the protein. Several point mutations of surface hydrophobic residues (W49A, L51A, L235A, and F237W) can dramatically alter the unusual kinetics of this secreted enzyme. The high affinity of PI-PLC for anionic phospholipids along with a strong hydrophobic interaction, which gives rise to the unusual kinetic behavior, is considered in terms of how it might contribute to the role of this phospholipase in L. monocytogenes infectivity.