| Structure, function, and mechanism of phosphatidylinositol specific phospholipase C (PI-PLC) from Bacillus thuringiensis have been studied by multidimensional nuclear magnetic resonance (NMR) spectroscopy, protein engineering, and linear free energy relationships (LFER).;Determination of the pKas of the histidine side chains in the wild type (WT) enzyme and two active site mutants has conclusively established a complex nature of the PI-PLC's pH-rate profile, where decrease of the activity at the extreme pHs cannot be explained by simple protonation of a general base or deprotonation of a general acid.;A single active site mutation, arginine-69 to aspartate, has created a metal binding site with sub-millimolar affinity for both magnesium and calcium ions. Metal ions have partially restored mutant's activity, but even fully activated R69D falls far below the WT's level. Further examination of the created metal binding site has revealed that it is composed of aspartate-33, aspartate-67, aspartate-69, and glutamate-117. D33N/R69D double mutant favors magnesium over calcium ions, albeit the discrimination comes at the expense of slightly lower activity.;Site-directed chemical modification has been used to elaborate function of arginine-69 in the enzyme catalysis. Consequences of the structure of the side chain at that position on enzyme's activity, non-bridging thio-effects, and stereoselectivity have been systematically examined. The results have established that bi-dentate functional group in the place of Arg69 is essential for the enzyme's activity. The proposal that arginine-69 interacts with both pro-S oxygen of the phosphate group and 2-OH of inositol (incoming nucleophile in the reaction) seems to be the most consistent with the available functional and structural data.;LFER studies have examined effects of the pKa of the leaving group on the rates of both enzymatic and non-enzymatic reactions. Analysis of the Bronsted coefficients established that both enzymatic and imidazole-catalyzed reactions proceed through essentially identical, slightly dissociative, transition states. Additionally, it has been shown that replacement of non-bridging phosphate oxygen by sulfur does not alter reactivity of the inositol phosphate diesters. |
