TY - JOUR
T1 - Colipase Residues Glu64 and Arg65 Are Essential for Normal Lipase-mediated Fat Digestion in the Presence of Bile Salt Micelles
AU - Crandall, Wallace V.
AU - Lowe, Mark E.
PY - 2001/4/20
Y1 - 2001/4/20
N2 - Pancreatic triglyceride lipase (PTL) requires colipase for activity. Various constituents in meals and in bile, particularly bile acids, inhibit PTL. Colipase restores activity to lipase in the presence of inhibitory substances like bile acids. Presumably, colipase functions by anchoring and orienting 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 hydrophobic surface positioned to bind substrate and a hydrophilic surface, lying opposite the hydrophobic surface, with two putative lipase-binding domains, Glu 45/Asp89 and Glu64/Arg65. To determine whether the hydrophilic surface interacts with PTL in solution, we introduced mutations into the putative PTL binding domains of human colipase. Each mutant was expressed, purified, and assessed for activity against various substrates. Most of the mutants showed impaired ability to reactivate PTL, with mutations in the Glu64/Arg65 binding site causing the greatest effect. Analysis indicated that the mutations decreased the affinity of the colipase mutants for PTL and prevented the formation of PTL-colipase complexes. The impaired function of the mutants was most apparent when assayed in micellar bile salt solutions. Most mutants stimulated PTL activity normally in monomeric bile salt solutions. We also tested the mutants for their ability to bind substrate and anchor lipase to tributyrin. Even though the ability of the mutants to anchor PTL to an interface decreased in proportion to their activity, each mutant colipase bound to tributyrin to the same extent as wild type colipase. These results demonstrate that the hydrophilic surface of colipase interacts with PTL in solution to form active colipase-PTL complexes, that bile salt micelles influence that binding, and that the proper interaction of colipase with PTL requires the Glu64/Arg65 binding site.
AB - Pancreatic triglyceride lipase (PTL) requires colipase for activity. Various constituents in meals and in bile, particularly bile acids, inhibit PTL. Colipase restores activity to lipase in the presence of inhibitory substances like bile acids. Presumably, colipase functions by anchoring and orienting 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 hydrophobic surface positioned to bind substrate and a hydrophilic surface, lying opposite the hydrophobic surface, with two putative lipase-binding domains, Glu 45/Asp89 and Glu64/Arg65. To determine whether the hydrophilic surface interacts with PTL in solution, we introduced mutations into the putative PTL binding domains of human colipase. Each mutant was expressed, purified, and assessed for activity against various substrates. Most of the mutants showed impaired ability to reactivate PTL, with mutations in the Glu64/Arg65 binding site causing the greatest effect. Analysis indicated that the mutations decreased the affinity of the colipase mutants for PTL and prevented the formation of PTL-colipase complexes. The impaired function of the mutants was most apparent when assayed in micellar bile salt solutions. Most mutants stimulated PTL activity normally in monomeric bile salt solutions. We also tested the mutants for their ability to bind substrate and anchor lipase to tributyrin. Even though the ability of the mutants to anchor PTL to an interface decreased in proportion to their activity, each mutant colipase bound to tributyrin to the same extent as wild type colipase. These results demonstrate that the hydrophilic surface of colipase interacts with PTL in solution to form active colipase-PTL complexes, that bile salt micelles influence that binding, and that the proper interaction of colipase with PTL requires the Glu64/Arg65 binding site.
UR - http://www.scopus.com/inward/record.url?scp=0035918261&partnerID=8YFLogxK
U2 - 10.1074/jbc.M009986200
DO - 10.1074/jbc.M009986200
M3 - Article
C2 - 11278590
AN - SCOPUS:0035918261
SN - 0021-9258
VL - 276
SP - 12505
EP - 12512
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 16
ER -