CATALYTIC DOMAIN OF PHOSPHOINOSITIDE-SPECIFIC PHOSPHOLIPASE-C (PLC) -MUTATIONAL ANALYSIS OF RESIDUES WITHIN THE ACTIVE-SITE AND HYDROPHOBIC RIDGE OF PLC-DELTA-1

Structural studies of phospholipase C delta 1 (PLC delta 1) in complex es with the inositol-lipid headgroup and calcium identified residues w ithin the catalytic domain that could be involved in substrate recogni tion, calcium binding, and catalysis. In addition, the structure of th e PLC delta 1 catalytic domain revealed a cluster of hydrophobic resid ues at the rim of the active site opening (hydrophobic ridge). To asse ss a role of each of these residues, we have expressed, purified, and characterized enzymes with the point mutations of putative active site residues (His(311), Asn(312), Glu(341), Asp(343), His(356), Glu(390), Lys(438), Lys(440), Ser(522), Arg(549) and Tyr(551)) and residues fro m the hydrophobic ridge (Leu(320), Phe(360), and Trp(555)). The replac ements of most active site residues by alanine resulted in a great red uction (1,000-200,000-fold) of PLC activity analyzed in an inositol li pid/sodium cholate mixed micelle assay. Measurements of the enzyme act ivity toward phosphatidylinositol, phosphatidylinositol 4-monophosphat e, and phosphatidylinositol 4,5-bisphosphate (PIP2) identified Ser(522 ), Lys(438), and Arg(549) as important for preferential hydrolysis of polyphosphoinositides, whereas replacement of Lys(440) selectively aff ected only hydrolysis of PIP2. When PLC activity was analyzed at diffe rent calcium concentrations, substitutions of Asn(312), Glu(390), Glu( 341), and Asp(343) resulted in a shift toward higher calcium concentra tions required for PIP2 hydrolysis, suggesting that all these residues contribute toward Ca2+ binding. Mutational analysis also confirmed th e importance of His(311) (similar to 20,000-fold reduction) and His(35 6) (similar to 6,000-fold reduction) for the catalysis. Mutations with in the hydrophobic ridge, which had little effect on PIP2 hydrolysis i n the mixed-micelles, resulted in an enzyme that was less dependent on the surface pressure when analyzed in a monolayer. This systematic mu tational analysis provides further insights into the structural basis for the substrate specificity, requirement for Ca2+ ion, catalysis, an d surface pressure/activity dependence, with general implications for eukaryotic phosphoinositide-specific PLCs.