title: Ion pairing between lipase and colipase plays a critical role in catalysis.
abstract: Among the polar interactions occurring in pancreatic lipase/colipase binding, only one ion pair involving lysine 400 on lipase and glutamic acid 45 on colipase has been described. These residues are strictly conserved among species, suggesting that the ion pair is likely to play an important role. Therefore, in order to prevent this interaction, mutations intended to neutralize or inverse the charge of these residues have been introduced in the cDNAs encoding horse lipase and colipase. The recombinant proteins have been expressed in insect cells, and their catalytic properties have been investigated. In all cases, preventing the formation of the correct ion pair Lys400/Glu45 leads to lipase-colipase complexes of reduced affinity unable to perform an efficient catalysis, notably in the presence of bile salt micelles. Diethyl p-nitrophenyl phosphate inhibition experiments with either mutant lipase or mutant colipase indicate a poor stabilization of the lipase flap. These results suggest that the ion pair plays a critical role in the active conformation of the lipase-colipase-micelle ternary complex by contributing to a correct orientation of colipase relative to lipase resulting in a proper opening of the flap.
authors.affiliation: Unité de Bioénergétique et Ingénierie des Protéines, UPR 9036 CNRS, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 9, France.
authors.forename: L
authors.initials: L
authors.lastname: Ayvazian
authors.forename: I
authors.initials: I
authors.lastname: Crenon
authors.forename: J
authors.initials: J
authors.lastname: Hermoso
authors.forename: D
authors.initials: D
authors.lastname: Pignol
authors.forename: C
authors.initials: C
authors.lastname: Chapus
authors.forename: B
authors.initials: B
authors.lastname: Kerfelec
mesh_terms: D000818:Animals; D001647:Bile Acids and Salts; D002384:Catalysis; D002460:Cell Line; D003089:Colipases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary; D018698:Glutamic Acid / Q000378:metabolism; D006736:Horses; D008049:Lipase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008823:Micelles; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed
chemical_list: D001647:Bile Acids and Salts; D003089:Colipases; D018076:DNA, Complementary; D008823:Micelles; D018698:Glutamic Acid; D008049:Lipase