The concentration of free Ca2+ and the composition of nonsubstrate phospholipids profoundly affect the activity of phospholipase C delta-1 (PLC delta-1). The rate of PLC delta-1 hydrolysis of phosphatidylinositol 4,5-bisphosphate was stimulated 20-fold by phosphatidylserine (PS), 4-fold by phosphatidic acid (PA), and not at all by phosphatidylethanolamine or phosphatidylcholine (PC). PS reduced the Ca2+ concentration required for half-maximal activation of PLC delta-1 from 5.4 to 0.5 µM. In the presence of Ca2+, PLC delta-1 specifically bound to PS/PC but not to PA/PC vesicles in a dose-dependent and saturable manner. Ca2+ also bound to PLC delta-1 and required the presence of PS/PC vesicles but not PA/PC vesicles. The free Ca2+ concentration required for half-maximal Ca2+ binding was estimated to be 8 µM. Surface dilution kinetic analysis revealed that the Km was reduced 20-fold by the presence of 25 mol % PS, whereas Vmax and Kd were unaffected. Deletion of amino acid residues 646-654 from the C2 domain of PLC delta-1 impaired Ca2+ binding and reduced its stimulation and binding by PS. Taken together, the results suggest that the formation of an enzyme-Ca2+-PS ternary complex through the C2 domain increases the affinity for substrate and consequently leads to enzyme activation.

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