Involvement of the Arg-Asp-His catalytic triad in enzymatic cleavage of the phosphodiester bond

Phosphatidylinositol-specific phospholipase C (PI-PLC) catalyzes the cleava ge of the P-O bond in phosphatidylinositol via intramolecular nucleophilic attack of the 2-hydroxyl group of inositol on the phosphorus atom. Our earl ier stereochemical and site-directed mutagenesis studies indicated that thi s reaction proceeds by a mechanism similar to that of RNase A, and that the catalytic site of PI-PLC consists of three major components analogous to t hose observed in RNase A, the His32 general base, the His82 general acid, a nd Arg69 acting as a phosphate-activating residue. In addition, His32 is as sociated with Asp274 in forming a catalytic triad with inositol 2-hydroxyl, and His82 is associated with Asp33 in forming a catalytic diad. The focus of this work is to provide a global view of the mechanism, assess cooperati on between various catalytic residues, and determine the origin of enzyme a ctivation by the hydrophobic leaving group. To this end, we have investigat ed kinetic properties of Arg69, ASP33, and His82 mutants phosphorothioate s ubstrate analogues which feature leaving groups of varying hydrophobicity a nd pK(a). Our results indicate that interaction of the nonbridging pro-S ox ygen atom of the phosphate group with Arg69 is strongly affected by Asp33, and to a smaller extent by His82. This result in conjunction with those obt ained earlier can be rationalized in terms of a novel, dual-function triad comprised of Arg69, Asp33, and His82 residues. The function of this triad i s to both activate the phosphate group toward the nucleophilic attack and t o protonate the leaving group. In addition, Asp33 and His82 mutants display ed much smaller degrees of activation by the fatty acid-containing leaving group as compared to the wild-type (WT) enzyme, and the level of activation was significantly reduced for substrates featuring the leaving group with low pK(a) values. These results strongly suggest that the assembly of the a bove three residues into the fully catalytically competent triad is control led by the hydrophobic interactions of the enzyme with the substrate leavin g group.