Val-407 and Ile-408 in the beta5'-loop of pancreatic lipase mediate lipase-colipase interactions in the presence of bile salt micelles.
In a previous study, we demonstrated that the beta5'-loop in the C-terminal domain of human pancreatic triglyceride lipase (hPTL) makes a major contribution in the function of hPTL (Chahinian et al. (2002) Biochemistry 41, 13725-13735). In the present study, we characterized the contribution of three residues in the beta5'-loop, Val-407, Ile-408, and Leu-412, to the function of hPTL. By substituting charged residues, aspartate or lysine, in these positions, we altered the hydrophilic to lipophilic ratio of the beta5'-loop. Each of the mutants was expressed, purified, and characterized for activity and binding with both monolayers and emulsions and for binding to colipase. Experiments with monolayers and with emulsions suggested that the interaction of hPTL with a phospholipid monolayer differs from the interaction of the hPTL-colipase complex with a dicaprin monolayer or a triglyceride emulsion (i.e. neutral lipids). Val-407, Ile-408, and Leu-412 make major contributions to interactions with monolayers, whereas only Val-407 and Ile-408 appear essential for activity on triglyceride emulsions in the presence of bile salt micelles. In solutions of taurodeoxycholate at micellar concentrations, a major effect of the beta5'-loop mutations is to change the interaction between hPTL and colipase. These observations support a major contribution of residues in the beta5'-loop in the function of hPTL and suggest that a third partner, bile salt micelles or the lipid interface or both, influence the binding of colipase and hPTL through interactions with the beta5'-loop.
Freie
Angela Bourbon
AB
Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Ferrato
Francine
F
Carrière
Frédéric
F
Lowe
Mark E
ME
D000327:Adsorption; D001224:Aspartic Acid / Q000737:chemistry; D001647:Bile Acids and Salts / Q000737:chemistry*; D002210:Caprylates / Q000737:chemistry; D003089:Colipases / Q000737:chemistry*; D018076:DNA, Complementary / Q000378:metabolism; D004075:Diglycerides / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007532:Isoleucine / Q000737:chemistry*; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D008049:Lipase / Q000737:chemistry* / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D008823:Micelles; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010179:Pancreas / Q000201:enzymology*; D010713:Phosphatidylcholines / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry; D011312:Pressure; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry; D013997:Time Factors; D014280:Triglycerides / Q000737:chemistry; D014304:Triolein / Q000737:chemistry; D014633:Valine / Q000737:chemistry*
D001647:Bile Acids and Salts; D002210:Caprylates; D003089:Colipases; D018076:DNA, Complementary; D004075:Diglycerides; D008823:Micelles; D010713:Phosphatidylcholines; D010743:Phospholipids; D011506:Proteins; D014280:Triglycerides; D007532:Isoleucine; D014304:Triolein; C044297:1,2-didecanoylglycerol; D001224:Aspartic Acid; C003637:tricaprylin; D008049:Lipase; D007930:Leucine; D014633:Valine; D008239:Lysine; C005830:tributyrin