Site-directed mutagenesis of the basic N-terminal cluster of pancreatic bile salt-dependent lipase. Functional significance.
Previous studies have postulated the presence of a heparin-binding site on the bile salt-dependent lipase (BSDL), whereas two bile salt-binding sites regulate the enzyme activity. One of these sites may overlap with the tentative heparin-binding site at the level of an N-terminal basic cluster consisting of positive residues Lys(32), Lys(56), Lys(61), Lys(62), and Arg(63). The present study uses specific site-directed mutagenesis to determine the functional significance of this basic cluster. Mutations in this sequence resulted in recombinant enzymes that were able to bind to immobilized and to cell-associated heparin before moving throughout intestinal cells. Recombinant BSDL was fully active on soluble substrate, but mutants were less active on micellar cholesteryl oleate in comparison with the wild-type enzyme. Activation studies by primary (sodium taurocholate) and by secondary (sodium taurodeoxycholate) bile salts revealed that the activation of BSDL by sodium taurocholate at concentrations below the critical micellar concentration, and not that evoked by micellar bile salts, was affected by substitutions, suggesting that this N-terminal basic cluster likely represents the specific bile salt-binding site of BSDL. Substitutions also affected the activation of the enzyme promoted by anionic phospholipids, extending the function of this site to that of a cationic regulatory site susceptible to accommodate anionic ligands.
INSERM U-559, Unité de Recherche de Physiopathologie des Cellules Epitheliales, Faculté de Médecine, 27 blv Jean MOULIN, 13385 Marseille cedex 05, France.
Emeline
E
Aubert
Véronique
V
Sbarra
Josette
J
Le Petit-Thévenin
Anne
A
Valette
Dominique
D
Lombardo
D000818:Animals; D015152:Blotting, Northern; D016466:CHO Cells; D002384:Catalysis; D002460:Cell Line; D002846:Chromatography, Affinity; D006224:Cricetinae; D004789:Enzyme Activation; D004952:Esters / Q000378:metabolism; D006493:Heparin / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010179:Pancreas / Q000201:enzymology*; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002787:Sterol Esterase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004952:Esters; D010743:Phospholipids; D011994:Recombinant Proteins; D006493:Heparin; C054784:bile salt-stimulated lipase; D002787:Sterol Esterase