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Phosphatidylinositol-specific phospholiphase C accepts a variety of alkyl and aryl inositol phosphates as substrates, making it a suitable enzyme for studying mechanism of phosphoryl transfer by probing the linear free-energy relationship. A study of Bronsted-type linear free-energy relationship (LFER) relationship to compare mechanisms of enzymatic and nonenzymatic reactions is conducted to assess the role of hydrophobicity in the leaving group as a general acid-enabling factor.

The Arg69 residue in phosphatidylinositol-specific phospholipase C not only binds the phosphate but positions it close to the 2-OH group of inositol in proper alignment for nucleophilic attack. The bidentate nature of this residue produces the large thio effects and stereoselectivity seen in this enzyme.

Results demonstrate a novel assembly of catalytic metal ion interactions comprising of three active site metal ions in the Tetrahymena ribosyme. Data point out that the catalytic metal ion/substrate interactions in the phosphoryl transfer are mediated by RNA or protein enzymes.

The mechanism of phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis was investigated. Site-directed mutagenesis was used to examine the functional and structural roles of His-32, His-82, Asp -274 and Asp-33. Results show that His-32 acts as a general base to abstract the proton from 2-OH and induces the attack of the deprotonated 2-oxygen on the atom of the phosphorus. His-82, on the other hand, functions as the general acid with support from Asp-33.

The detailed kinetic characterization of phosphatidylinositol-specific phospholipase C (PI-PLC) as well as the role of Arg-69 in the catalysis were determined. (2R)-1,2-dipalimitoyloxypropanethiophospho-1-D-myoinositol (DPsPI) was utilized for assaying PI-PLC. Results have shown that Arg-69 was needed for the enzyme catalysis by stabilizing the negative charge on the pentacoordinate phosphate resulting to a lower level of activity compared to that of wild types.

Research describes engineering of a metal-independent bacterial phosphatidylinositol-specific phospholipase C into a metal-dependent enzyme through mutagenesis at the R69D site. The kinetic analysis shows activation of the catalytic activity by calcium significantly. Furthermore, calcium is required to restore the catalytic site microenvironment as shown by NMR studies.

Substitution of a phosphoryl oxygen with sulfur is a technique widely used in enzyme and ribozyme studies. The ratio kolks for substitution of nonbridging oxygen and a bridging oxygen as type I and II thio effects, have been defined. The thio effects and site-directed mutagenesis are used to support the mechanism for phosphatidylinositol-specific phospholipase C (PI-PLC).

The proteome of untreated white wines (a Recioto made with Garganega grapes from the Veneto region) was explored in depth via capture with combinatorial peptide ligand libraries (CPLL) at four different pH values: pH 2.2, 3.8, 7.2, and 9.3. The combined data on the discoveries in the four CPLL eluates, as well as in the collected bottle sediment, allowed the identification of 106 unique gene products belonging to Vitis vinifera, as well as of an additional 11 proteins released by the S. cerevisiae used in the fermentation process. Among the residual grape proteins detected in the Recioto wine, ca. 30% were categorized as medium to high-abundance species, vs 70% low-abundance ones. The detection of so many low-abundance species suggests that proteomic (coupled to peptidomic) data might be used for typing high-quality products (grand crus) to assess their genuineness and protect them from fraudulent imitations.