Listeria monocytogenes phosphatidylinositol-specific phospholipase C: activation and allostery.

The animal and human pathogen Listeria monocytogenes secretes several virulence factors, including a phosphatidylinositol-specific phospholipase C (PI-PLC). Sufficient quantities of L. monocytogenes PI-PLC for biophysical studies were obtained by overexpression of the enzyme in Escherichia coli. The purified PI-PLC was examined in enzyme kinetics experiments using a new fluorogenic substrate, methyl-FLIP. Methyl-FLIP is a water-soluble monomeric substrate cleaved in a manner similar to the natural aggregate substrate, phosphatidylinositol (PI). Michaelis–Menten kinetics were observed with KM=61±7 μM and Vmax=120±5 μmol min−1 mg−1, corresponding to kcat=66±3 s−1. The catalysis is activated by the addition of a short-chain phospholipid, dihexanoyl phosphatidylcholine (diC6PC). The kinetics were fitted to a two-site model in which the substrate binds to the active site and diC6PC binds to a second site, with an interaction between the two sites. The result is a decrease in KM and an increase in Vmax, producing an overall four to five-fold increase in catalytic efficiency (kcat/KM). The interaction is not a regulatory mechanism, as is the case for multimeric enzymes; rather, it suggests interfacial cooperativity between the active site and a lipid-binding subsite, presumably adjacent to the active site.