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. 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 allow quantifying substrate binding to different binding sites of Bacillus subtilis xylanase

Sven Cuyvers  1 , Emmie Dornez, Maher Abou Hachem, Birte Svensson, Michael Hothorn, Joanne Chory, Jan A Delcour, Christophe M Courtin
Affiliations

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

Sven Cuyvers et al. Anal Biochem. .

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.

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Figures

Fig.1
Fig.1
ITC results of the titration of XBS_E172A (A) and XBS_E172A_AAA (B) with X6. The upper graphs show the raw binding heats on titration, and the lower graphs show the integrated binding heat levels. The inset tables give Kd values (standard errors on the fits are in parentheses).
Fig.2
Fig.2
SPR results for the binding of X6 to XBS_E172A (A), XBS_E172A_AAA (B), and XBS with a blocked AS (C). The inset tables give Kd values (standard errors on the fits are in parentheses).
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References

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