Toward understanding the interfacial activation and binding of phosphatidylinositol-specific phospholipase C (PI-PLC): The effect of organic solvents and non-substrate interfaces

Bacterial PI-PLC catalyzes the hydrolysis of phosphatidylinositol (PI) in a two-step reaction where PI is first cleaved into inositol cyclic 1,2-monophosphate (cIP) and diacylglycerol (DAG) in a phosphotransferase activity, followed by a cyclic phosphodiesterase activity that hydrolyzes cIP into inositol-1-phosphate.; Moderate amounts of water-miscible organic solvents were previously observed to dramatically enhance the cyclic phosphodiesterase activity of PI-PLC. We explored the effect of solvents on the phosphotransferase activity of PI-PLC. Changes in PI particle size and distribution due to solvents did not correlate with the enhanced catalytic efficiency of PI-PLC. iPrOH and diheptanoyl-phosphatidylcholine, each of which activates PI-PLC for cIP hydrolysis, exhibited a synergistic effect for cIP hydrolysis that was not observed for PI cleavage. We suggest that solvent activation is a function of active site polarity and orientations of side groups in response to the presence of acyl chains.; The interactions of PI-PLC with non-substrate interfaces that affect the catalytic activity of PI-PLC were also examined. PI-PLC binds more tightly to anionic than to PC vesicles. The residues of PI-PLC involved in binding to lipid vesicles have been assessed with mutagenesis. The results are used to explain how PC enhances PI-PLC activity.; The interfacial behavior of PI-PLC was probed by constructing mutants with cysteine residues at various positions from the interface. These were derivatized with fluorophores and used to assess the proximity to the phospholipid interface. The fluorescence quenching experiments suggested possible aggregation of PI-PLC at lipid interfaces. This was explained with chemical cross-linking experiments of PI-PLC using EDC. The cross-linking results clearly show evidence of PI-PLC aggregation in the presence of PC surfaces. MALDI-TOF mass spectrometry analysis of cross-linked protein showed an increase in PI-PLC mass due to tightly bound PC. The higher affinity for PC correlated with an enhanced phosphotransferase activity. We suggest that the enhancement of PI-PLC activity in the presence of PC surfaces results from the enzyme adapting a more active conformation.