Identification of a Novel Catalytic Triad with Dual Functions in Enzymatic Cleavage of the P−O Bond

We report identification of a novel Arg-Asp-His catalytic triad that serves dual functionssphosphate activation and leaving group protonationsto catalyze the P-O bond cleavage for phosphatidylinositol-specific phospholipase C (PI-PLC) from B. thuringiensis. PI-PLC cleaves the P-O bond of phosphatidylinositol (PI) to form inositol 1,2-cyclic phosphate (IcP), which is then slowly hydrolyzed to inositol 1-phosphate.1 X-ray structure2 along with site-directed mutagenesis2,3 led to the mechanism in Figure 1A, which involves three elements: His32 as a general base assisted by Asp274, His82 as a general acid, and Arg69, which activates the phosphate group. We recently added two additional aspects to the mechanism: that Arg69 activates the phosphate by hydrogen bonding to the pro-S oxygen at the transition state,4,5 and that Asp33 and His82 function intimately together as a “composite general acid” (Figure 1B).6,7 We now further show that the functions of Asp33 and Arg69 are also inseparable, which led us to propose the mechanism involving the novel catalytic triad (Figure 1C). Our conclusions were achieved by rigorously cross-examining the bridging and nonbridging “thio-effects” (kO/kS ratios)6-9 of PI-PLC and its mutants with PI analogues shown in Figure 2. Both types of thio-effects are usually >1 in enzymatic reactions since sulfur weakens potential hydrogen bonding10 and also introduces possible steric11 and charge8 differences. The usefulness of the nonbridging thio-effect lies mainly in the comparison of the enzyme activity toward Rpand Sp-isomers. Bridging thioeffects are more difficult to interpret since they are directly related to the bond being cleaved, and P-S bonds are much weaker than P-O bonds (i.e. kO/kS is ,1 in chemical reactions).12 The maximal activities of WT, D33N, and D33A toward substrate analogues are listed in Table 1. While the rates depend on the detergent used to disperse the substrate (see footnote b), all data in Table 1 were obtained under the same conditions with dihexanoyl lecithin as a detergent. Furthermore, the analogues