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J Biol Chem, Vol. 273, Issue 19, 11650-11659, May 8, 1998

Catalytic Domain of Phosphoinositide-specific Phospholipase C (PLC)
MUTATIONAL ANALYSIS OF RESIDUES WITHIN THE ACTIVE SITE AND HYDROPHOBIC RIDGE OF PLC1

Moira V. Ellis, Stephen R. James^§, Olga Perisic, C. Peter Downes^§, Roger L. Williams, and Matilda Katan

From the  Cancer Research Campaign Centre for Cell and Molecular Biology, Chester Beatty Laboratories, Fulham Road, London SW3 6JB, the  Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, and the ^§ Department of Biochemistry, Medical Sciences Institute, University of Dundee, Dundee DD1 4HN, United Kingdom

Structural studies of phospholipase C 1 (PLC1) in complexes with the inositol-lipid headgroup and calcium identified residues within the catalytic domain that could be involved in substrate recognition, calcium binding, and catalysis. In addition, the structure of the PLC1 catalytic domain revealed a cluster of hydrophobic residues at the rim of the active site opening (hydrophobic ridge). To assess a role of each of these residues, we have expressed, purified, and characterized enzymes with the point mutations of putative active site residues (His³¹¹, Asn³¹², Glu³⁴¹, Asp³⁴³, His³⁵⁶, Glu³⁹⁰, Lys⁴³⁸, Lys⁴⁴⁰, Ser⁵²², Arg⁵⁴⁹, and Tyr⁵⁵¹) and residues from the hydrophobic ridge (Leu³²⁰, Phe³⁶⁰, and Trp⁵⁵⁵). The replacements of most active site residues by alanine resulted in a great reduction (1,000-200,000-fold) of PLC activity analyzed in an inositol lipid/sodium cholate mixed micelle assay. Measurements of the enzyme activity toward phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4,5-bis-phosphate (PIP₂) identified Ser⁵²², Lys⁴³⁸, and Arg⁵⁴⁹ as important for preferential hydrolysis of polyphosphoinositides, whereas replacement of Lys⁴⁴⁰ selectively affected only hydrolysis of PIP₂. When PLC activity was analyzed at different calcium concentrations, substitutions of Asn³¹², Glu³⁹⁰, Glu³⁴¹, and Asp³⁴³ resulted in a shift toward higher calcium concentrations required for PIP₂ hydrolysis, suggesting that all these residues contribute toward Ca²⁺ binding. Mutational analysis also confirmed the importance of His³¹¹ (~20,000-fold reduction) and His³⁵⁶ (~6,000-fold reduction) for the catalysis. Mutations within the hydrophobic ridge, which had little effect on PIP₂ hydrolysis in the mixed-micelles, resulted in an enzyme that was less dependent on the surface pressure when analyzed in a monolayer. This systematic mutational analysis provides further insights into the structural basis for the substrate specificity, requirement for Ca²⁺ ion, catalysis, and surface pressure/activity dependence, with general implications for eukaryotic phosphoinositide-specific PLCs.

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