title: Effect of the lipid interface on the catalytic activity and spectroscopic properties of a fungal lipase.
abstract: Lipase from the fungi Thermomyces (formerly Humicola) lanuginosa (TlL) is widely used in industry. This interfacial enzyme is inactive under aqueous conditions, but catalytic activation is induced on binding to a lipid-water interface. In order for protein engineering to design more efficient mutants of TlL for specific applications, it is important to characterize its interfacial catalysis. A complete analysis of steady-state kinetics for the hydrolysis of a soluble substrate by TlL has been developed using an interface different from the substrate. Small vesicles of 1-palmitoyl-2-oleoylglycero-sn-3-phosphoglycerol (POPG) or other anionic phospholipids are a neutral diluent interface for the partitioning of substrate and enzyme. TlL binds to these interfaces in an active or open form, thus implying a displacement of the helical lid away from the active site. A study of the influence of substrate and diluent concentration dependence of the rate of hydrolysis provides a basis for the determination of the primary interfacial catalytic parameters. The interfacial activation is not supported by zwitterionic vesicles or by large anionic vesicles of 100 nm diameter, although TlL binds to these interfaces. Using a combination of fluorescence-based techniques applied to several mutants of TlL with different tryptophan residues we have shown that TlL binds to phospholipid vesicles in different forms rendering different catalytic activities, and that the open lid conformation is achieved and stabilized by a combination of electrostatic and hydrophobic interactions between the enzyme's lipid-binding face and the interface.
authors: Physical Chemistry Department, School of Pharmacy, University of Barcelona, Avn. Joan XXIII s/n, 08028, Barcelona, Spain. ycajal@farmacia.far.ub.es
authors: Y
authors: Y
authors: Cajal
authors: A
authors: A
authors: Svendsen
authors: J
authors: J
authors: De Bolós
authors: S A
authors: SA
authors: Patkar
authors: M A
authors: MA
authors: Alsina
mesh_terms: D001203:Ascomycota / Q000201:enzymology*; D002384:Catalysis; D008049:Lipase / Q000737:chemistry / Q000378:metabolism*; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry; D013050:Spectrometry, Fluorescence
chemical_list: D008055:Lipids; D008049:Lipase