Investigating the interfacial binding of bacterial phosphatidylinositol-specific phospholipase C.
The interactions of PI-PLC with nonsubstrate zwitterionic [phosphatidylcholine (PC)] and anionic [phosphatidylmethanol (PMe), phosphatidylserine, phosphatidylglycerol, and phosphatidic acid] interfaces that affect the catalytic activity of PI-PLC have been examined. PI-PLC binding is strongly coupled to vesicle curvature and is tighter at acidic pH for all of the phospholipids examined. PI-PLC binds to small unilamellar vesicles (SUVs) of anionic lipids with much higher affinity (K(d) is 0.01-0.07 microM for a site consisting of n = 100 +/- 25 lipids when analyzed with a Langmuir adsorption isotherm) than to zwitterionic PC SUVs (K(d) is 5-20 microM and n = 8 +/- 3). The binding to PC surfaces is dominated by hydrophobic interactions, while binding to anionic surfaces is dominated by electrostatic interactions. The contributions of specific cationic side chains and hydrophobic groups at the rim of the alpha beta-barrel to zwitterionic and anionic vesicle binding have been assessed with mutagenesis. The results are used to explain how PC activates the enzyme for both phosphotransferase and cyclic phosphodiesterase activities.
Wehbi
Hania
H
Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02467, USA.
Feng
Jianwen
J
Kolbeck
James
J
Ananthanarayanan
Bharath
B
Cho
Wonhwa
W
Roberts
Mary F
MF
D001665:Binding Sites; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008051:Lipid Bilayers; D008081:Liposomes; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010713:Phosphatidylcholines / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism*; D010718:Phosphatidylserines / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D010738:Type C Phospholipases / Q000378:metabolism*
D008051:Lipid Bilayers; D008081:Liposomes; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010716:Phosphatidylinositols; D010718:Phosphatidylserines; D010738:Type C Phospholipases; D010739:Phospholipase D