Divalent metal ions play a crucial role in catalysis by many RNA and protein enzymes that carry out phosphoryl transfer reactions, and defining their interactions with substrates is critical for understanding the mechanism of biological phosphoryl transfer. Although a vast amount of structural work has identified metal ions bound at the active site of many phosphoryl transfer enzymes, the number of functional metal ions and the full complement of their catalytic interactions remain to be defined for any RNA or protein enzyme. Previously, thiophilic metal ion rescue and quantitative functional analyses identified the interactions of three active site metal ions with the 3'- and 2'-substrate atoms of the Tetrahymena group I ribozyme. We have now extended these approaches to probe the metal ion interactions with the nonbridging pro-S(P) oxygen of the reactive phosphoryl group. The results of this study combined with previous mechanistic work provide evidence for a novel assembly of catalytic interactions involving three active site metal ions. One metal ion coordinates the 3'-departing oxygen of the oligonucleotide substrate and the pro-S(P) oxygen of the reactive phosphoryl group; another metal ion coordinates the attacking 3'-oxygen of the guanosine nucleophile; a third metal ion bridges the 2'-hydroxyl of guanosine and the pro-S(P) oxygen of the reactive phosphoryl group. These results for the first time define a complete set of catalytic metal ion/substrate interactions for an RNA or protein enzyme catalyzing phosphoryl transfer.

The mechanism of phosphatidylinositol-specific phospholipase C (PI-PLC) has been suggested to resemble that of ribonuclease A. The goal of this work is to rigorously evaluate the mechanism of PI-PLC from Bacillus thuringiensis by examining the functional and structural roles of His-32 and His-82, along with the two nearby residues Asp-274 and Asp-33 (which form a hydrogen bond with His-32 and His-82, respectively), using site-directed mutagenesis. In all, twelve mutants were constructed, which, except D274E, showed little structural perturbation on the basis of 1D NMR and 2D NOESY analyses. The H32A, H32N, H32Q, H82A, H82N, H82Q, H82D, and D274A mutants showed a 10 4 -10 5 -fold decrease in specific activity toward phosphatidylinositol; the D274N, D33A, and D33N mutants retained 0.1-1% activity, whereas the D274E mutant retained 13% activity. Steady-state kinetic analysis of mutants using (2R)-1,2-dipalmitoyloxypropane-3-(thiophospho-1D-myo-inositol) (DPsPI) as a substrate generally agreed well with the specific activity toward phosphatidylinositol. The results suggest a mechanism in which His-32 functions as a general base to abstract the proton from 2-OH and facilitates the attack of the deprotonated 2-oxygen on the phosphorus atom. This general base function is augmented by the carboxylate group of Asp-274 which forms a diad with His-32. The H82A and D33A mutants showed an unusually high activity with substrates featuring low pK a leaving groups, such as DPsPI and p-nitrophenyl inositol phosphate (NPIPs). These results suggest that His-82 functions as the general acid with assistance from Asp-33, facilitating the departure of the leaving group by protonation of the glycerol O3 oxygen. The Brønsted coefficients obtained for the WT and the D33N mutant indicate a high degree of proton transfer to the leaving group and further underscore the "helper" function of Asp-33. The complete mechanism also includes activation of the phosphate group toward nucleophilic attack by a hydrogen bond between Arg-69 and a nonbridging oxygen atom. The overall mechanism can be described as "complex" general acid-general base since three elements are required for efficient catalysis.Phosphatidylinositol-specific phospholipase C (PI-PLC) 1 plays a key role in receptor-mediated transformations of inositol phospholipids (1-4). The products of mammalian PI-PLC play important biological roles because they serve as a source of intracellular signaling molecules, diacylglycerol and inositol 1,4,5-trisphosphate. Diacylglycerol activates protein kinase C (5), and inositol 1,4,5-trisphosphate is involved in releasing intracellular calcium stores (6). The released calcium then activates many types of cellular processes. Glycosylphosphatidylinositol-specific phospholipase C, a subclass of PI-PLC, cleaves the spacer arm of glycosylphosphatidylinositol-anchored proteins to release extracellular enzymatic activities of these proteins (7). As shown in Figure 1, the reaction of bacterial PI-PLC consists of two steps: fast cleavage of PI int...

The viability of living systems depends inextricably on enzymes that catalyze phosphoryl transfer reactions. For many enzymes in this class, including several ribozymes, divalent metal ions serve as obligate cofactors. Understanding how metal ions mediate catalysis requires elucidation of metal ion interactions with both the enzyme and the substrate(s). In the Tetrahymena group I intron, previous work using atomic mutagenesis and quantitative analysis of metal ion rescue behavior identified three metal ions (MA, MB, and MC) that make five interactions with the ribozyme substrates in the reaction's transition state. Here, we combine substrate atomic mutagenesis with site-specific phosphorothioate substitutions in the ribozyme backbone to develop a powerful, general strategy for defining the ligands of catalytic metal ions within RNA. In applying this strategy to the Tetrahymena group I intron, we have identified the pro-S P phosphoryl oxygen at nucleotide C262 as a ribozyme ligand for MC. Our findings establish a direct connection between the ribozyme core and the functionally defined model of the chemical transition state, thereby extending the known set of transition-state interactions and providing information critical for the application of the recent group I intron crystallographic structures to the understanding of catalysis.