Studies on the mechanism of lipid interfacial activation of phosphatidylinositol-specific phospholipase C and construction of a new phosphatase mutant.

摘要

The two reactions catalyzed by phosphatidylinositol-specific phospholipase C (PI-PLC) are the cleavage of phosphatidylinositol to produce inositol cyclic 1,2-monophosphate (cIP), and the hydrolysis of cIP to inositol 1-phosphate. The first step occurs with an interfacial substrate, while the second step only involves a water soluble substrate and product. Both reactions of the Bacillus thuringiensis PI-PLC can be activated by the presence of a zwitterionic lipid interface such as phosphatidylcholine micelles or vesicles.; My thesis work shows that the hydrophobic amino acid side chains at the top of the rim of the active site (helix B and loop 237--242) are important for the binding of both substrates PI and cIP and for kinetic activation (enhancement of kcat). Two tryptophans, Trp-47 (helix B) and Trp-242 (rim loop), are absolutely critical for PLC binding to a zwitterionic activator interface of PC. Both orientation and position of the tryptophan in helix B are important. A tryptophan inserted at certain other positions in the rim loop can function similarly to Trp-242. A tryptophan in the rim loop at position 238 generates a more active enzyme. The temperature dependence of PI-PLC also contributed to understanding the mechanism of this enzyme.; During building of a calcium-binding site in a bacterial PI-PLC based on comparing conserved catalytic site of bacterial and mammalian enzymes, I accidentally generated a new phosphatase enzyme by mutation of Arg-69 and Lys-115 to acidic residues. Phosphatase chemistry was never detected in wild type PI-PLC and all other mutants I made. This significant change of charge distribution in the active site totally altered the enzyme reaction, specificity and stereochemistry. The new phosphatase enzyme showed 370-fold higher specific activity towards sugar phosphates than towards I-1-P. Based on kinetic and inhibitory studies, the catalytic mechanism was suggested to be similar to acidic histidine phosphatases.; Soluble substrates (diC4PI or cIP) were used to explore how activator ligands affect catalysis of mammalian PLCs. My work showed that PLC delta1 is allosterically activated by arachidonic acid (AA) in detergent micelles. The AA was shown to lower the Km for substrate PI with no effect on the kcat. PLC beta is reported to be activated by Gbetagamma using soluble substrates. I showed that Gbetagamma activation of PLC beta affects the cyclic phosphodiesterase step significantly and not the initial change of PI to cIP. This suggests the major effect of Gbetagamma is to target the PLC beta to membranes.