title: Substrate stereospecificity of phosphatidylinositol-specific phospholipase C from Bacillus cereus examined using the resolved enantiomers of synthetic myo-inositol 1-(4-nitrophenyl phosphate).
abstract: The substrate stereospecificity of phosphatidylinositol-specific phospholipase C from Bacillus cereus is examined using the resolved optical isomers of synthetic myo-inositol 1-(4-nitrophenyl phosphate), a chromogenic substrate for the phospholipase. The synthetic route employs mild acid-labile protecting groups and separation of the substituted myo-inositol enantiomers as the (-)-camphanyl ester diastereomers. Measurements of the initial rates of cleavage of the D and L enantiomers of the nitrophenyl substrate by phosphatidylinositol-specific phospholipase C from B. cereus show that this enzyme is essentially stereospecific for the D enantiomer. Under identical conditions, the rate of cleavage of the L isomer is less than 0.2% of that observed for the D isomer. The same is observed for the highly homologous enzyme from Bacillus thuringiensis. There is no measurable inhibition by the L enantiomer of the B. cereus enzyme acting on the D enantiomer, even when the molar ratio of L:D is 5, indicating that binding of the L enantiomer to the phospholipase is negligible. Thus, the enzyme active site is exquisitely sensitive to the stereochemistry of the myo-inositol group of the substrate.
authors.affiliation: Institute of Molecular Biology, University of Oregon, Eugene 97403.
authors.forename: A J
authors.initials: AJ
authors.lastname: Leigh
authors.forename: J J
authors.initials: JJ
authors.lastname: Volwerk
authors.forename: O H
authors.initials: OH
authors.lastname: Griffith
authors.forename: J F
authors.initials: JF
authors.lastname: Keana
mesh_terms: D001409:Bacillus cereus / Q000201:enzymology; D001413:Bacillus thuringiensis / Q000201:enzymology; D007295:Inositol Phosphates / Q000378:metabolism; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010716:Phosphatidylinositols / Q000378:metabolism; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000378:metabolism*
chemical_list: D007295:Inositol Phosphates; D010716:Phosphatidylinositols; C074663:4-nitrophenyl myo-inositol-1-phosphate; D010727:Phosphoric Diester Hydrolases; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase