Cross-linking phosphatidylinositol-specific phospholipase C traps two activating phosphatidylcholine molecules on the enzyme.
Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC), a bacterial model for the catalytic domain of mammalian PI-PLC enzymes, was cross-linked by 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride to probe for the aggregation and/or conformational changes of PI-PLC when bound to activating phosphatidylcholine (PC) interfaces. Dimers and higher order multimers (up to 31% of the total protein when cross-linked at pH 7) were observed when the enzyme was cross-linked in the presence of PC vesicles. Aggregates were also detected with PI-PLC bound to diheptanoyl-PC (diC(7)PC) micelles, although the fraction of cross-linked multimers (19% at pH 7) was lower than when the enzyme was cross-linked in the presence of vesicles. PI-PLC cross-linked in the presence of a diC(7)PC interface exhibited an enhanced specific activity for PI cleavage. The extent of this cross-linking-enhanced activation was reduced in PI-PLC mutants lacking either tryptophan in the rim (W47A and W242A) of this (betaalpha)(8)-barrel protein. The higher activity of the native protein cross-linked in the presence of diC(7)PC correlated with an increased affinity of the protein for two diC(7)PC molecules as detected by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. In contrast to wild type protein, W47A and W242A had only a single diC(7)PC tightly associated when cross-linked in the presence of that activator molecule. These results indicate that (i) each rim tryptophan residue is involved in binding a PC molecule at interfaces, (ii) the affinity of the enzyme for an activating PC molecule is enhanced when the protein is bound to a surface, and (iii) this conformation of the enzyme with at least two PC bound that is stabilized by chemical cross-linking interacts more effectively with activating interfaces, leading to higher observed specific activities for the phosphotransferase reaction.
Merkert Chemistry Center, Boston College, Chestnut Hill, MA 02467, USA.
Xin
X
Zhang
Hania
H
Wehbi
Mary F
MF
Roberts
D001412:Bacillus subtilis / Q000201:enzymology*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008823:Micelles; D008958:Models, Molecular; D010713:Phosphatidylcholines / Q000737:chemistry*; D043265:Phosphatidylinositol Diacylglycerol-Lyase / Q000737:chemistry*; D054801:Phosphoinositide Phospholipase C; D010770:Phosphotransferases / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013302:Streptomyces / Q000201:enzymology; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry
D003432:Cross-Linking Reagents; D008823:Micelles; D010713:Phosphatidylcholines; D014364:Tryptophan; D010770:Phosphotransferases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase