Inhibition of phosphatidylinositol-specific phospholipase C: Studies on synthetic substrates, inhibitors and a synthetic enzyme

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.