SUBSTRATE REQUIREMENTS OF BACTERIAL PHOSPHATIDYLINOSITOL-SPECIFIC PHOSPHOLIPASE-C

A series of symmetric short-chain phosphatidylinositols (PI), includin g dihexanoyl-PI, diheptanoyl-PI (racemic as well as D and L forms), an d 2-methoxy inositol-substituted diheptanoyl-PI, have been synthesized , characterized, and used to investigate key mechanistic questions abo ut phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillu s thuringiensis. Key results include the following: (i) bacterial PI-P LC exhibits a 5-6-fold ''interfacial activation'' when its substrate i s present in an interface as opposed to existing as a monomer in solut ion (in fact, the similarity to the activation observed with nonspecif ic PLC enzymes suggests a similarity in activation mechanisms); (ii) t he 2-OH must be free since the enzyme cannot hydrolyze diheptanoyl-2-O -methyl-PI (this is most consistent with the formation of inositol cyc lic 1,2-phosphate as a necessary step in catalysis); (iii) the inosito l ring must have the D stereochemistry (the L-inositol attached to the lipid moiety is neither a substrate nor an inhibitor); and (iv) the p resence of noninhibitory L-PI with the D-PI substrate relieves the dia cylglycerol product inhibition detected at approximately 30% hydrolysi s.