Phosphatidylinositol-specific phospholipase C forms different complexes with monodisperse and micellar phosphatidylcholine

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus forms a premicellar complex E-# with monodisperse diheptanoylphosphatidylcholine (DC7PC) that is distinguishable from the E* complex formed with micelles. Results are interpreted with the assumption that in both cases amphiphiles bind to the interfacial binding surface (i-face) of PI-PLC but not to the active site. Isothermal calorimetry and fluorescence titration results for the binding of monodisperse DC7PC give an apparent dissociation constant of K-2 = 0.2 mM with Hill coefficient of 2. The gel-permeation, spectroscopic, and probe partitioning behaviors of E-# are distinct from those of the E* complex. The aggregation and partitioning behaviors suggest that the acyl chains in E* but not in E* remain exposed to the aqueous phase. The free (E) and complexed (E-# and E*) forms of PI-PLC, each with distinct spectroscopic signatures, readily equilibrate with changing DC7PC concentration. The underlying equilibria are modeled and their significance for the states of the PI-PLC under monomer kinetic conditions is discussed to suggest that the Michaelis-Menten complex formed with monodisperse DC7PC is likely to be (ES)-S-# or its aggregate rather than the classical monodisperse ES complex.