title: Partial deletion of beta9 loop in pancreatic lipase-related protein 2 reduces enzyme activity with a larger effect on long acyl chain substrates.
abstract: Structural studies on pancreatic lipase have revealed a complex architecture of surface loops surrounding the enzyme active site and potentially involved in interactions with lipids. Two of them, the lid and beta loop, expose a large hydrophobic surface and are considered as acyl chain binding sites based on their interaction with an alkyl phosphonate inhibitor. While the role of the lid in substrate recognition and selectivity has been extensively studied, the implication of beta9 loop in acyl chain stabilization remained hypothetical. The characterization of an enzyme with a natural deletion of the lid, guinea pig pancreatic lipase-related protein 2 (GPLRP2), suggests however an essential contribution of the beta9 loop in the stabilization of the acyl enzyme intermediate formed during the lipolysis reaction. A GPLRP2 mutant with a seven-residue deletion of beta9 loop (GPLRP2-deltabeta9) was produced and its enzyme activity was measured using various substrates (triglycerides, monoglycerides, galactolipids, phospholipids, vinyl esters) with short, medium and long acyl chains. Whatever the substrate tested, GPLRP2-deltabeta9 activity is drastically reduced compared to that of wild-type GPLRP2 and this effect is more pronounced as the length of substrate acyl chain increases. Changes in relative substrate selectivity and stereoselectivity remained however weak. The deletion within beta9 loop has also a negative effect on the rate of enzyme inhibition by alkyl phosphonates. All these findings indicate that the reduced enzyme turnover observed with GPLRP2-deltabeta9 results from a weaker stabilization of the acyl enzyme intermediate due to a loss of hydrophobic interactions.
authors.affiliation: a CNRS-Aix-Marseille Université, Enzymologie Interfaciale et Physiologie de la Lipolyse-UMR 7282, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
authors.forename: Kaouthar
authors.initials: K
authors.lastname: Dridi
authors.forename: Sawsan
authors.initials: S
authors.lastname: Amara
authors.forename: Sofiane
authors.initials: S
authors.lastname: Bezzine
authors.forename: Jorge A
authors.initials: JA
authors.lastname: Rodriguez
authors.forename: Frédéric
authors.initials: F
authors.lastname: Carrière
authors.forename: Hélène
authors.initials: H
authors.lastname: Gaussier
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D006168:Guinea Pigs; D057927:Hydrophobic and Hydrophilic Interactions; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008066:Lipolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D016415:Sequence Alignment; D017384:Sequence Deletion; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D008049:Lipase; C075988:pancreatic lipase related protein 2