title: Inhibition of phosphatidylinositol-specific phospholipase C: studies on synthetic substrates, inhibitors and a synthetic enzyme.
abstract: Enzyme inhibition studies on phosphatidylinositol-specific phospholipase C (PI-PLC) from B. Cereus were performed in order to gain an understanding of the mechanism of the PI-PLC family of enzymes and to aid inhibitor design. Inhibition studies on two synthetic cyclic phosphonate analogues (1,2) of inositol cyclic-1:2-monophosphate (cIP), glycerol-2-phosphate and vanadate were performed using natural phosphatidylinositol (PI) substrate in Triton X100 co-micelles and an NMR assay. Further inhibition studies on PI-PLC from B. Cereus were performed using a chromogenic, synthetic PI analogue (DPG-PI), an HPLC assay and Aerosol-OT (AOT), phytic acid and vanadate as inhibitors. For purposes of comparison, a model PI-PLC enzyme system was developed employing a synthetic Cu(II)-metallomicelle and a further synthetic PI analogue (IPP-PI). The studies employing natural PI substrate in Triton X100 co-micelles and synthetic DPG-PI in the absence of surfactant indicate three classes of PI-PLC inhibitors: (1) active-site directed inhibitors (e.g. 1,2); (2) water-soluble polyanions (e.g. tetravanadate, phytic acid); (3) surfactant anions (e.g. AOT). Three modes of molecular recognition are indicated to be important: (1) active site molecular recognition; (2) recognition at an anion-recognition site which may be the active site, and; (3) interfacial (or hydrophobic) recognition which may be exploited to increase affinity for the anion-recognition site in anionic surfactants such as AOT. The most potent inhibition of PI-PLC was observed by tetravanadate and AOT. The metallomicelle model system was observed to mimic PI-PLC in reproducing transesterification of the PI analogue substrate to yield cIP as product and in showing inhibition by phytic acid and AOT.
authors.affiliation: Department of Chemistry, Queen's University, Kingston, Ontario, Canada.
authors.forename: D
authors.initials: D
authors.lastname: Vizitiu
authors.forename: A G
authors.initials: AG
authors.lastname: Kriste
authors.forename: A S
authors.initials: AS
authors.lastname: Campbell
authors.forename: G R
authors.initials: GR
authors.lastname: Thatcher
mesh_terms: D000838:Anions / Q000494:pharmacology; D001409:Bacillus cereus / Q000187:drug effects / Q000201:enzymology; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D006868:Hydrolysis; D007295:Inositol Phosphates / Q000138:chemical synthesis* / Q000737:chemistry* / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D010833:Phytic Acid / Q000494:pharmacology; D013379:Substrate Specificity; D013501:Surface-Active Agents / Q000494:pharmacology; D014638:Vanadates / Q000737:chemistry / Q000494:pharmacology
chemical_list: D000838:Anions; D004791:Enzyme Inhibitors; D007295:Inositol Phosphates; D013501:Surface-Active Agents; D014638:Vanadates; C010199:inositol cyclic phosphate; D010833:Phytic Acid; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase