Role of tryptophan residues in interfacial binding of phosphatidylinositol-specific phospholipase C.
The phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis exhibits several types of interfacial activation. In the crystal structure of the closely related Bacillus cereus PI-PLC, the rim of the active site is flanked by a short helix B and a loop that show an unusual clustering of hydrophobic amino acids. Two of the seven tryptophans in PI-PLC are among the exposed residues. To test the importance of these residues in substrate and activator binding, we prepared several mutants of Trp-47 (in helix B) and Trp-242 (in the loop). Two other tryptophans, Trp-178 and Trp-280, which are not near the rim, were mutated as controls. Kinetic (both phosphotransferase and cyclic phosphodiesterase activities), fluorescence, and vesicle binding analyses showed that both Trp-47 and Trp-242 residues are important for the enzyme to bind to interfaces, both activating zwitterionic and substrate anionic surfaces. Partitioning of the enzyme to vesicles is decreased more than 10-fold for either W47A or W242A, and removal of both tryptophans (W47A/W242A) yields enzyme with virtually no affinity for phospholipid surfaces. Replacement of either tryptophan with phenylalanine or isoleucine has moderate effects on enzyme affinity for surfaces but yields a fully active enzyme. These results are used to describe how the enzyme is activated by interfaces.
Feng
Jianwen
J
Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02167, USA.
Wehbi
Hania
H
Roberts
Mary F
MF
D001426:Bacterial Proteins / Q000737:chemistry; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D007532:Isoleucine / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry / Q000502:physiology*; D010738:Type C Phospholipases / Q000378:metabolism*
D001426:Bacterial Proteins; D007532:Isoleucine; D010649:Phenylalanine; D014364:Tryptophan; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase