title: Isothermal titration calorimetry and surface plasmon resonance allow quantifying substrate binding to different binding sites of Bacillus subtilis xylanase.
abstract: Isothermal titration calorimetry and surface plasmon resonance were tested for their ability to study substrate binding to the active site (AS) and to the secondary binding site (SBS) of Bacillus subtilis xylanase A separately. To this end, three enzyme variants were compared. The first was a catalytically incompetent enzyme that allows substrate binding to both the AS and SBS. In the second enzyme, binding to the SBS was impaired by site-directed mutagenesis, whereas in the third enzyme, the AS was blocked using a covalent inhibitor. Both techniques were able to show that AS and SBS have a similar binding affinity.
authors.affiliation: Laboratory of Food Chemistry and Biochemistry and Leuven Food Science and Nutrition Research Centre, Katholieke Universiteit Leuven, Leuven 3001, Belgium. sven.cuyvers@biw.kuleuven.be
authors.forename: Sven
authors.initials: S
authors.lastname: Cuyvers
authors.forename: Emmie
authors.initials: E
authors.lastname: Dornez
authors.forename: Maher
authors.initials: M
authors.lastname: Abou Hachem
authors.forename: Birte
authors.initials: B
authors.lastname: Svensson
authors.forename: Michael
authors.initials: M
authors.lastname: Hothorn
authors.forename: Joanne
authors.initials: J
authors.lastname: Chory
authors.forename: Jan A
authors.initials: JA
authors.lastname: Delcour
authors.forename: Christophe M
authors.initials: CM
authors.lastname: Courtin
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002151:Calorimetry / Q000379:methods*; D020134:Catalytic Domain; D043364:Endo-1,4-beta Xylanases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D020349:Surface Plasmon Resonance / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D043364:Endo-1,4-beta Xylanases