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Originally published In Press as doi:10.1074/jbc.M009986200 on January 16, 2001

J. Biol. Chem., Vol. 276, Issue 16, 12505-12512, April 20, 2001
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Colipase Residues Glu64 and Arg65 Are Essential for Normal Lipase-mediated Fat Digestion in the Presence of Bile Salt Micelles*

Wallace V. CrandallDagger § and Mark E. Lowe||

From the Departments of Dagger  Pediatrics and of  Molecular Biology and Pharmacology, Washington University School of Medicine and St. Louis Children's Hospital, St. Louis, Missouri 63110

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, Glu45/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.

* This work was supported by National Institutes of Health Grants HD33060 and DK52574.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: Division of Pediatric Gastroenterology and Nutrition, Columbus Children's Hospital, 700 Children's Dr., Columbus, OH 43205.

|| To whom correspondence should be addressed: Washington University School of Medicine, 660 South Euclid Ave., Campus Box 8208, St. Louis, MO. Tel.: 314-286-2857; Fax: 314-286-2894; E-mail: Lowe@kids.wustl.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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