In vitro single point mutagenesis, inositol phospholipid hydrolysis, and substrate protection experiments were used to identify catalytic residues of human phosphatidylinositide-specific phospholipase C1 (PLC1) isolated from a human aorta cDNA library. Invariant amino acid residues containing a functional side chain in the highly conserved X region were changed by in vitro mutagenesis. Most of the mutant enzymes were still able to hydrolyze inositol phospholipid with activity ranging from 10 to 100% of levels in the wild type enzyme. Exceptions were mutants with the conversion of Arg to Leu (R338L), Glu to Gly (E341G), or His to Leu (H356L), which made the enzyme severely defective in hydrolyzing inositol phospholipid. Phospholipid vesicle binding experiments showed that these three cleavage-defective mutant forms of PLC1 could specifically bind to phosphatidylinositol 4,5-bisphosphate (PIP) with an affinity similar to that of wild type enzyme. Western blotting analysis of trypsin-treated enzyme-PIP complexes revealed that a 67-kDa major protein fragment survived trypsin digestion if the wild type enzyme, E341G, or H356L mutant PLC1 was preincubated with 7.5 µM PIP, whereas if it was preincubated with 80 µM PIP, the size of major protein surviving was comparable to that of intact enzyme. However, mutant enzyme R338L was not protected from trypsin degradation by PIP binding. These observations suggest that PLC1 can recognize PIP through a high affinity and a low affinity binding site and that residues Glu and His are not involved in either high affinity or low affinity PIP binding but rather are essential for the Ca-dependent cleavage activity of PLC.

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