Isothermal titration calorimetry and surface plasmon resonance allow quantifying substrate binding to different binding sites of Bacillus subtilis xylanase

Anal Biochem. 2012 Jan 1;420(1):90-2. doi: 10.1016/j.ab.2011.09.005. Epub 2011 Sep 10.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacillus subtilis / enzymology*
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / metabolism
  • Binding Sites
  • Calorimetry / methods*
  • Catalytic Domain
  • Endo-1,4-beta Xylanases / antagonists & inhibitors
  • Endo-1,4-beta Xylanases / chemistry
  • Endo-1,4-beta Xylanases / genetics
  • Endo-1,4-beta Xylanases / metabolism*
  • Enzyme Inhibitors / chemistry
  • Enzyme Inhibitors / pharmacology
  • Mutagenesis, Site-Directed
  • Surface Plasmon Resonance / methods*


  • Bacterial Proteins
  • Enzyme Inhibitors
  • Endo-1,4-beta Xylanases