Role of the lid hydrophobicity pattern in pancreatic lipase activity.
Pancreatic lipase is a soluble globular protein that must undergo structural modifications before it can hydrolyze oil droplets coated with bile salts. The binding of colipase and movement of the lipase lid open access to the active site. Mechanisms triggering lid mobility are unclear. The *KNILSQIVDIDGI* fragment of the lid of the human pancreatic lipase is predicted by molecular modeling to be a tilted peptide. Tilted peptides are hydrophobicity motifs involved in membrane fusion and more globally in perturbations of hydrophobic/hydrophilic interfaces. Analysis of this lid fragment predicts no clear consensus of secondary structure that suggests that its structure is not strongly sequence determined and could vary with environment. Point mutations were designed to modify the hydrophobicity profile of the [240-252] fragment and their consequences on the lipase-mediated catalysis were tested. Two mutants, in which the tilted peptide motif was lost, also have poor activity on bile salt-coated oil droplets and cannot be reactivated by colipase. Conversely, one mutant in which a different tilted peptide is created retains colipase dependence. These results suggest that the tilted hydrophobicity pattern of the [240-252] fragment is neither important for colipase binding to lipase, nor for interfacial binding but is important to trigger the maximal catalytic efficiency of lipase in the presence of bile salt.
Thomas
Annick
A
Centre de Biophysique Moléculaire Numérique, Faculté Agronomique, 5030 Gembloux, Belgium.
Allouche
Maya
M
Basyn
Frédéric
F
Brasseur
Robert
R
Kerfelec
Brigitte
B
D000465:Algorithms; D001665:Binding Sites; D002384:Catalysis; D004305:Dose-Response Relationship, Drug; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D015233:Models, Statistical; D009154:Mutation; D000069463:Olive Oil; D010179:Pancreas / Q000201:enzymology*; D010455:Peptides / Q000737:chemistry; D010938:Plant Oils; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D014280:Triglycerides / Q000737:chemistry
D000069463:Olive Oil; D010455:Peptides; D010938:Plant Oils; D014280:Triglycerides; D008049:Lipase; C005830:tributyrin