title: Open and closed states of Candida antarctica lipase B: protonation and the mechanism of interfacial activation.
abstract: Lipases (EC 3.1.1.3) are ubiquitous hydrolases for the carboxyl ester bond of water-insoluble substrates, such as triacylglycerols, phospholipids, and other insoluble substrates, acting in aqueous as well as in low-water media, thus being of considerable physiological significance with high interest also for their industrial applications. The hydrolysis reaction follows a two-step mechanism, or "interfacial activation," with adsorption of the enzyme to a heterogeneous interface and subsequent enhancement of the lipolytic activity. Among lipases, Candida antarctica lipase B (CALB) has never shown any significant interfacial activation, and a closed conformation of CALB has never been reported, leading to the conclusion that its behavior was due to the absence of a lid regulating the access to the active site. The lid open and closed conformations and their protonation states are observed in the crystal structure of CALB at 0.91 Å resolution. Having the open and closed states at atomic resolution allows relating protonation to the conformation, indicating the role of Asp145 and Lys290 in the conformation alteration. The findings explain the lack of interfacial activation of CALB and offer new elements to elucidate this mechanism, with the consequent implications for the catalytic properties and classification of lipases.
authors.affiliation: European Molecular Biology Laboratory-European Bioinformatics Institute (EMBL-EBI), Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, United Kingdom Robinson College, University of Cambridge, Cambridge CB3 9AN, United Kingdom.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Stauch
authors.affiliation: Diamond Light Source, Didcot, Oxfordshire OX11 0DE, United Kingdom.
authors.forename: Stuart J
authors.initials: SJ
authors.lastname: Fisher
authors.affiliation: European Molecular Biology Laboratory (EMBL), Deutsches Elektronen-Synchrotron (DESY), Hamburg 22607, Germany michele.cianci@embl-hamburg.de.
authors.forename: Michele
authors.initials: M
authors.lastname: Cianci
mesh_terms: D018360:Crystallography, X-Ray; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism*; D008049:Lipase / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation
chemical_list: D005656:Fungal Proteins; D008049:Lipase; C431349:lipase B, Candida antarctica
keywords: X-ray crystallography; enzymology/enzyme regulation; fatty acid/metabolism; lipids/chemistry