Application of Bronsted-type LFER in the study of the phospholipase C mechanism

Article abstract of DOI:10.1021/ja029362s

Phosphatidylinositol-specific phospholipase C cleaves the phosphodiester bond of phosphatidylinositol to form inositol 1,2-cyclic phosphate and diacylglycerol. This enzyme also accepts a variety of alkyl and aryl inositol phosphates as substrates, making it a suitable model enzyme for studying mechanism of phosphoryl transfer by probing the linear free-energy relationship (LFER). In this work, we conducted a study of Bronsted-type relationship (log k = βl_g pK_a + C) to compare mechanisms of enzymatic and nonenzymatic reactions, confirm the earlier proposed mechanism, and assess further the role of hydrophobicity in the leaving group as a general acid-enabling factor. The observation of the high negative Bronsted coefficients for both nonenzymatic (βl_g = -0.65 to -0.73) and enzymatic cleavage of aryl and nonhydrophobic alkyl inositol phosphates (βl_g = -0.58) indicates that these reactions involve only weak general acid catalysis. In contrast, the enzymatic cleavage of hydrophobic alkyl inositol phosphates showed low negative Bronsted coefficient (βl_g = -0.12), indicating a small amount of the negative charge on the leaving group and efficient general acid catalysis. Overall, our results firmly support the previously postulated mechanism where hydrophobic interactions between the enzyme and remote parts of the leaving group induce an unprecedented negative-charge stabilization on the leaving group in the transition state.