A Fourier transform infrared spectroscopic (FTIR) study of porcine and bovine pancreatic phospholipase A2 and their interaction with substrate analogues and a transition-state inhibitor.
Fourier transform infrared spectroscopy has been used to investigate the secondary structure of porcine and bovine pancreatic phospholipase A2 (PLA2) and the zymogen of porcine PLA2, prophospholipase A2 (proPLA2), in both H2O and D2O media. Detailed qualitative analysis was made of these proteins using second derivative and deconvolution techniques. Quantitative studies of the proteins in solution made using Factor Analysis gave average values of 54% alpha-helix, 15% beta-sheet and 23% beta-turns. These values agree well with the secondary structures deduced from previous studies of single crystals using X-ray techniques. No significant differences in secondary structure were observed for porcine pancreatic (pro)phospholipase A2 in the presence or absence of Ca2+ ions, or in the temperature range 10-45 degrees C. The binding of the non-degradable phospholipid analogue, n-alkylphosphocholine, in monomeric form produced no significant difference in the secondary structure of either enzyme. Conformational differences were, however, observed between the enzyme lyophilised in a solid film and in aqueous solution. The change is probably due to the formation of beta-sheet upon hydration, coupled with a loss of random structures. Conformational differences in both porcine and bovine pancreatic PLA2 were observed on binding to n-alkylphosphocholine micelles. This change may be due to a small increase in the alpha-helical structure and a decrease in the beta-sheet, and/or possibly beta-turn content. Similar conformational changes were observed for the interaction of porcine and bovine PLA2 with the substrate analogue inhibitor 1-heptanoyl-2-heptanoylamino-2-deoxy-sn-glycero-3-phospho glycol in micellar form.
Kennedy
D F
DF
Department of Protein and Molecular Biology, Royal Free Hospital School of Medicine, University of London, U.K.
Slotboom
A J
AJ
de Haas
G H
GH
Chapman
D
D
D000818:Animals; D002417:Cattle; D005583:Fourier Analysis; D008246:Lysophospholipids / Q000494:pharmacology; D008823:Micelles; D010179:Pancreas / Q000201:enzymology; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000737:chemistry*; D054467:Phospholipases A2; D010767:Phosphorylcholine; D011487:Protein Conformation; D013055:Spectrophotometry, Infrared / Q000379:methods; D013057:Spectrum Analysis; D013552:Swine; D014961:X-Ray Diffraction
D008246:Lysophospholipids; D008823:Micelles; D010767:Phosphorylcholine; C066237:1-heptanoyl-2-heptanoylamino-2-deoxyglycero-3-phosphoglycol; D010741:Phospholipases A; D054467:Phospholipases A2