MECHANISM OF PHOSPHATIDYLINOSITOL-SPECIFIC PHOSPHOLIPASE-C - A UNIFIED VIEW OF THE MECHANISM OF CATALYSIS

The mechanism of phosphatidylinositol-specific phospholipase C (PI-PLC ) has been suggested to resemble that of ribonuclease A. The goal of t his work is to rigorously evaluate the mechanism of PI-PLC from Bacill us thuringiensis by examining the functional and structural roles of H is-32 and His-82, along with the two nearby residues Asp-274 and Asp-3 3 (which form a hydrogen bond with His-32 and His-82, respectively), u sing site-directed mutagenesis. In all, twelve mutants were constructe d, which, except D274E, showed little structural perturbation on the b asis 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 D3 3N mutants retained 0.1-1% activity, whereas the D274E mutant retained 13% activity. Steady-state kinetic analysis of mutants using almitoyl oxypropane-3-(thiophospho-1D-myo-inositol) (DPsPI) as a substrate gene rally agreed well with the specific activity toward phosphatidylinosit ol. The results suggest a mechanism in which His-32 functions as a gen eral 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 hig h activity with substrates featuring low pK(a) leaving groups, such as DPsPI and p-nitrophenyl inositol phosphate (NPIPs). These results sug gest that His-82 functions as the general acid with assistance from As p-33, facilitating the departure of the leaving group by protonation o f the glycerol 03 oxygen. The Bronsted coefficients obtained for the W T and the D33N mutant indicate a high degree of proton transfer to the leaving group and further underscore the ''helper'' function of Asp-3 3. The complete mechanism also includes activation of the phosphate gr oup 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.