Phosphatidylinositol-specific phospholipase C-γ1 undergoes pH-induced activation and conformational change

Phospholipase C-γ1 displayed sigmoidal kinetics with a S0.5 value of 0.17 mole fraction PIP2 when assayed at pH 6.8 using detergent:lipid mixed micelles. The pH optimum for hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C-γ1 was dependent on the mole fraction of substrate in the micelle. The pH optimum was 5.5 when the enzyme was assayed below the S0.5. The pH optima shifted to a pH range of 6.0–6.3 when the enzyme was assayed above the S0.5. The kinetic parameters for phospholipase C-γ1 assayed at various pH values from pH 7.0 to 5.0 yielded similar n values (n=4), but the constant, K′, decreased from 1×10−2 (mole fraction)2 at pH 7.0 to 1×10−5 (mole fraction)2 at pH 5.0. Maximum enzyme specificity occurred at pH values below pH 6.0 as determined by the plot of log kcat/S0.5 versus pH. Intrinsic fluorescence spectroscopy revealed that at a pH value above 7.0 or below 6.3, tryptophan quenching occurred. Fluorescence quenching experiments performed with acrylamide determined phospholipase C-γ1 incubated at pH 5.0 had a larger collisional quenching constant than enzyme incubated at pH 7.0. Lowering the pH to 5.0 apparently resulted in interior tryptophans becoming more solvent accessible. These data suggest that pH may activate phospholipase C-γ1 by disrupting ionizable groups leading to a conformational change.