Changes in the structure of bovine phospholipase A2 upon micelle binding.
Phospholipase A2 (PLA2) is a calcium-dependent enzyme which hydrolyses the 2-acyl ester bond of phospholipids. The extracellular PLA2s are activated by as much as 10000-fold on binding to micelles or vesicles of substrate, possibly due to a conformational change induced in the enzyme. We have studied the complex of bovine pancreatic PLA2 with micelles of SDS by ultracentrifugation, equilibrium dialysis, microcalorimetry, fluorescence and n.m.r. spectroscopy. Ultracentrifugation and equilibrium dialysis measurements showed that on average 1.28 (+/- 0.17) PLA2 molecules and 26.4 (+/- 3.1) SDS molecules are involved in the complex and that there is a rapid equilibrium between micellar species containing one or more enzyme monomers. The estimated heat of formation of the complex, measured calorimetrically as the heat released when PLA2 was injected into excess 10 mM SDS, was 162.3 +/- 1.5) kJ/mol [38.8 (+/- 0.35) kcal/mol] of PLA2 added. The fluorescence of the single tryptophan at position 3 in the N-terminal helix of the protein increases when PLA2 binds to SDS micelles, indicating that this part of the protein is in a more hydrophobic environment in the complex. The structural changes in PLA2 on addition of [2H25]SDS were monitored using n.m.r. spectroscopy. The overall structure of the protein is unchanged, but changes in nuclear Overhauser effects (NOEs) were observed for residues in the N-terminal helix, at the active site region and in a lysine-rich region near the C-terminus. The NOE changes at the N-terminus indicate that this portion of the protein molecule adopts a more ordered, helical conformation when bound to a micelle. We suggest that these conformational changes could be the mechanism by which the enzyme becomes activated in the presence of aggregated substrate.
Department of Biochemistry, University of Leicester, U.K.
P M
PM
Kilby
W U
WU
Primrose
G C
GC
Roberts
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002151:Calorimetry; D002417:Cattle; D003956:Dialysis; D009682:Magnetic Resonance Spectroscopy; D008823:Micelles; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology*; D010741:Phospholipases A / Q000737:chemistry* / Q000378:metabolism*; D054467:Phospholipases A2; D012967:Sodium Dodecyl Sulfate; D013050:Spectrometry, Fluorescence; D014461:Ultracentrifugation
D008823:Micelles; D012967:Sodium Dodecyl Sulfate; D010741:Phospholipases A; D054467:Phospholipases A2