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 K(M) = 61 +/- 7 microM and V(max) = 120 +/- 5 micromol min(-1) mg(-1), corresponding to k(cat) = 66+/-3 s(-1). The catalysis is activated by the addition of a short-chain phospholipid, dihexanoyl phosphatidylcholine (diC(6)PC). The kinetics were fitted to a two-site model in which the substrate binds to the active site and diC(6)PC binds to a second site, with an interaction between the two sites. The result is a decrease in K(M) and an increase in V(max), producing an overall four to five-fold increase in catalytic efficiency (k(cat)/K(M)). 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.
Ryan
Margret
M
Institute of Molecular Biology, University of Oregon, Eugene, OR 97403-1253, USA.
Zaikova
Tatiana O
TO
Keana
John F W
JF
Goldfine
Howard
H
Griffith
O Hayes
OH
D000494:Allosteric Regulation; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004789:Enzyme Activation; D007700:Kinetics; D008823:Micelles; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010957:Plasmids; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D008823:Micelles; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase