Mechanism of Phosphatidylinositol-Specific Phospholipase C:  A Unified View of the Mechanism of Catalysis^,

Abstract

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 Catalytic Role of Aspartate in a Short Strong Hydrogen Bond of the Asp274–His32 Catalytic Dyad in Phosphatidylinositol-specific Phospholipase C Can Be Substituted by a Chloride Ion

“…The SSHB of PI-PLC Contributes Only Modestly to Catalysis-Kinetic data obtained previously (4), show that the activity of D274N (21.0 units/mg) is only modestly reduced relative to that of WT PI-PLC (1300 units/mg). However, previous studies were performed at a fixed pH of 7.5.…”

“…All of the mutations were verified by sequencing. All of the proteins were purified as described previously (4,7).…”

Defining Specific Lipid Binding Sites for a Peripheral Membrane Protein in Situ Using Subtesla Field-cycling NMR

“…The largest effects are from a spin-label on W47C, part of helix B, which is thought to insert into the bilayer above the active site opening (9, 13), and H82C, the general acid in the catalytic mechanism for PI cleavage (14). However, there are some spin-labeled sites that were expected to have an effect but were fairly ineffective, notably attached to Y118C and N243C, both of which are close to the active site in the crystal structure.…”

Insights into the Structural Specificity of the Cytotoxicity of 3-Deoxyphosphatidylinositols

“…Mechanistically, PI cleavage occurs in a similar fashion in both types of enzymes except that an Arg replaces the active site Ca 2+ in the bacterial enzyme. 39,40 As seen in Table 2, removal of the hydroxyl group at C3 generated a very poor substrate for the PLCδ enzyme with PI cleavage occurring at 0.5 to 1% that of diC 8 PI. However, in the context of a cell and over the time course of several days, these lipids are likely to be hydrolyzed by the endogenous PLC enzymes.…”