ALLOSTERIC ACTIVATION OF PHOSPHATIDYLINOSITOL-SPECIFIC PHOSPHOLIPASE-C - SPECIFIC PHOSPHOLIPID-BINDING ANCHORS THE ENZYME TO THE INTERFACE

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus t huringiensis exhibits 'interfacial activation' toward the water-solubl e substrate myo-inositol 1,2-(cyclic)phosphate [Zhou et al, (1997) Bio chemistry 36, 347-355]. The activation of PI-PLC enzyme is optimal wit h PC or PE interfaces. NMR experiments (TRNOE and P-31 line width anal yses) were carried out to investigate the interaction of PI-PLC with a ctivator amphiphiles. These studies showed that the enzyme had high af finity for phosphatidylcholine (or PE) molecules with dissociation con stants of 0.5 and 0.3 mM for diC(6)PC and diC(7)PC, respectively. TRNO E cross-peaks of bound PC were confirmed to represent intramolecular r elaxation pathways using partially perdeuterated PC molecules consiste nt with a single molecule binding tightly. The large activation by a P C interface can be explained by a single PC molecule binding specifica lly to PI-PLC and anchoring the enzyme-lipid complex to the interface. Other interfaces, such as micellar diC(8)PS, can activate PI-PLC abou t 2-3-fold; however, the monomers of these detergents showed little af finity for the enzyme as measured by TRNOE or P-31 NMR Line widths. Th e 3.6-fold activation produced by polymerized vesicles of (lipoyloxy)d odecanoyl]-sn-glycero-3-phosphocholine (compared to the 15-fold activa tion generated by nonpolymerized PC vesicles) was comparable to the no nspecific activation of other detergents, This confirmed that single-P C molecule binding was allosteric and anchored the enzyme in the inter face. The conformation of interfacially activated enzyme is discussed in term of the stabilization of a critical surface loop and helix B ob served with weak intensity in the X-ray crystal structure.