Substrate requirements of bacterial phosphatidylinositol-specific phospholipase C.
A series of symmetric short-chain phosphatidylinositols (PI), including dihexanoyl-PI, diheptanoyl-PI (racemic as well as D and L forms), and 2-methoxy inositol-substituted diheptanoyl-PI, have been synthesized, characterized, and used to investigate key mechanistic questions about phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis. Key results include the following: (i) bacterial PI-PLC exhibits a 5-6-fold "interfacial activation" when its substrate is present in an interface as opposed to existing as a monomer in solution (in fact, the similarity to the activation observed with nonspecific PLC enzymes suggests a similarity in activation mechanisms); (ii) the 2-OH must be free since the enzyme cannot hydrolyze diheptanoyl-2-O-methyl-PI (this is most consistent with the formation of inositol cyclic 1,2-phosphate as a necessary step in catalysis); (iii) the inositol ring must have the D stereochemistry (the L-inositol attached to the lipid moiety is neither a substrate nor an inhibitor); and (iv) the presence of noninhibitory L-PI with the D-PI substrate relieves the diacylglycerol product inhibition detected at approximately 30% hydrolysis.
Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02167.
K A
KA
Lewis
V R
VR
Garigapati
C
C
Zhou
M F
MF
Roberts
D001413:Bacillus thuringiensis / Q000201:enzymology*; D002384:Catalysis; D004789:Enzyme Activation; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D008823:Micelles; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010716:Phosphatidylinositols / Q000737:chemistry / Q000378:metabolism; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D013379:Substrate Specificity; D013499:Surface Properties
D008823:Micelles; D010716:Phosphatidylinositols; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase