Colipase residues Glu(64) and Arg(65) are essential for normal lipase-mediated fat digestion in the presence of bile salt micelles

Pancreatic triglyceride lipase (PTL) requires colipase for activity. Variou s constituents in meals and in bile, particularly bile acids, inhibit PTL, Colipase restores activity to lipase in the presence of inhibitory substanc es like bile acids. Presumably, colipase functions by anchoring and orienti ng PTL at the oil-water interface. The x-ray structure of the colipase PTL complex supports this model. In the x-ray structure, colipase has a hydroph obic surface positioned to bind substrate and a hydrophilic surface, lying opposite the hydrophobic surface, with two putative lipase-binding domains, Glu(45)/Asp(89) and Glu(64)/Arg(65). To determine whether the hydrophilic surface interacts with PTL in solution, we introduced mutations into the pu tative PTL binding domains of human colipase, Each mutant was expressed, pu rified, and assessed for activity against various substrates, Most of the m utants showed impaired ability to reactivate PTL, with mutations in the Glu (64)/Arg(65) binding site causing the greatest effect. Analysis indicated t hat the mutations decreased the affinity of the colipase mutants for PTL an d prevented the formation of PTL colipase complexes. The impaired function of the mutants was most apparent when assayed in micellar bile salt solutio ns. Most mutants stimulated PTL activity normally in monomeric bile salt so lutions. We also tested the mutants for their ability to bind substrate and anchor lipase to tributyrin, Even though the ability of the mutants to anc hor PTL to an interface decreased in proportion to their activity, each mut ant colipase bound to tributyrin to the same extent as wild type colipase, These results demonstrate that the hydrophilic surface of colipase interact s with PTL in solution to form active colipase PTL complexes, that bile sal t micelles influence that binding, and that the proper interaction of colip ase with PTL requires the Glu(64)/Arg(65) binding site.