TY - JOUR
T1 - Isothermal titration calorimetry and surface plasmon resonance allow quantifying substrate binding to different binding sites of Bacillus subtilis xylanase
AU - Cuyvers, Sven
AU - Dornez, Emmie
AU - Abou Hachem, Maher
AU - Svensson, Birte
AU - Hothorn, Michael
AU - Chory, Joanne
AU - Delcour, Jan A.
AU - Courtin, Christophe M.
PY - 2012
Y1 - 2012
N2 - 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.
AB - 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.
KW - Isothermal titration calorimetry
KW - Surface plasmon resonance
KW - Xylanase
KW - Xylooligosaccharides
KW - Mechanism-based inhibitor
U2 - 10.1016/j.ab.2011.09.005
DO - 10.1016/j.ab.2011.09.005
M3 - Journal article
SN - 0003-2697
VL - 420
SP - 90
EP - 92
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 1
ER -