Probing the opening of the pancreatic lipase lid using site-directed spin labeling and EPR spectroscopy.
Access to the active site of human pancreatic lipase (HPL) is controlled by a surface loop (the lid) that undergoes a conformational change in the presence of amphiphiles and lipid substrate. The question of how and when the lid opens still remains to be elucidated, however. A paramagnetic probe was covalently bound to the lid via the D249C mutation, and electron paramagnetic resonance (EPR) spectroscopy was used to monitor the conformational change in solution. Two EPR spectral components, corresponding to distinct mobilities of the probe, were attributed to the closed and open conformations of the HPL lid, based on experiments performed with the E600 inhibitor. The open conformation of the lid was observed in solution at supramicellar bile salt concentrations. Colipase alone did not induce lid opening but increased the relative proportions of the open conformation in the presence of bile salts. The opening of the lid was found to be a reversible process. Using various colipase to lipase molar ratios, a correlation between the proportion of the open conformation and the catalytic activity of HPL was observed.
Belle
Valérie
V
Laboratoire de Bioénergétique et Ingénierie des Protéines, CNRS UPR 9036, IBSM, Marseille, France.
Fournel
André
A
Woudstra
Mireille
M
Ranaldi
Sébastien
S
Prieri
Florence
F
Thomé
Virginie
V
Currault
Julie
J
Verger
Robert
R
Guigliarelli
Bruno
B
Carrière
Frédéric
F
D001647:Bile Acids and Salts / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D003089:Colipases / Q000737:chemistry / Q000378:metabolism; D018076:DNA, Complementary; D004229:Dithiothreitol / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D007700:Kinetics; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010843:Pichia / Q000235:genetics; D010920:Placenta / Q000201:enzymology; D011487:Protein Conformation; D013113:Spin Labels
D001647:Bile Acids and Salts; D003089:Colipases; D018076:DNA, Complementary; D013113:Spin Labels; D008049:Lipase; C518354:PNLIP protein, human; D004229:Dithiothreitol