Lid opening and unfolding in human pancreatic lipase at low pH revealed by site-directed spin labeling EPR and FTIR spectroscopy.
The structural changes induced in human pancreatic lipase (HPL) by lowering the pH were investigated using a combined approach involving the use of site-directed spin labeling coupled to electron paramagnetic resonance (SDSL-EPR) and Fourier transform infrared (ATR-FTIR) spectroscopy. The secondary structure of HPL observed with ATR-FTIR spectroscopy was found to be stable in the pH range of 3.0-6.5, where HPL remained active. Using a spin-label introduced into the lid of HPL at position 249, a reversible opening of the lid controlling the access to the active site was observed by EPR spectroscopy in the pH range of 3.0-5.0. In the same pH range, some structural changes were also found to occur outside the lid in a peptide stretch located near catalytic aspartate 176, using a spin-label introduced at position 181. Below pH 3.0, ATR-FTIR measurements indicated that HPL had lost most of its secondary structure. At these pH levels, the loss of enzyme activity was irreversible and the ability of HPL to bind to lipid emulsions was abolished. The EPR spectrum of the spin-label introduced at position 181, which was typical of a spin-label having a high mobility, confirmed the drastic structural change undergone by HPL in this particular region. The EPR spectrum of the spin-label at position 249 indicated, however, that the environment of this residue within the lid was not affected at pH 3.0 in comparison with that observed in the pH range of 3.0-5.0. This finding suggests that the disulfide bridge between the hinges of the lid kept the secondary structure of the lid intact, whereas the HPL was completely unfolded.
Ranaldi
Sebastien
S
CNRS Laboratoire de Bioenergetique et Ingenierie des Proteines, UPR 9036, Marseille, France.
Belle
Valérie
V
Woudstra
Mireille
M
Rodriguez
Jorge
J
Guigliarelli
Bruno
B
Sturgis
James
J
Carriere
Frederic
F
Fournel
Andre
A
D000327:Adsorption; D004578:Electron Spin Resonance Spectroscopy; D004795:Enzyme Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008049:Lipase / Q000737:chemistry* / Q000378:metabolism*; D008954:Models, Biological; D011487:Protein Conformation; D017510:Protein Folding; D017550:Spectroscopy, Fourier Transform Infrared; D013113:Spin Labels; D014867:Water
D013113:Spin Labels; D014867:Water; D008049:Lipase; C518354:PNLIP protein, human