Mapping glycoside hydrolase substrate subsites by isothermal titration calorimetry

Gennady Zolotnitsky, Uri Cogan, Noam Adir, Vered Solomon, Gil Shoham, Yuval Shoham*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

120 Scopus citations

Abstract

Relating thermodynamic parameters to structural and biochemical data allows a better understanding of substrate binding and its contribution to catalysis. The analysis of the binding of carbohydrates to proteins or enzymes is a special challenge because of the multiple interactions and forces involved. Isothermal titration calorimetry (ITC) provides a direct measure of binding enthalpy (ΔHa) and allows the determination of the binding constant (free energy), entropy, and stoichiometry. In this study, we used ITC to elucidate the binding thermodynamics of xylosaccharides for two xylanases of family 10 isolated from Geobacillus stearothermophilus T-6. The change in the heat capacity of binding (ΔCp = ΔH/ΔT) for xylosaccharides differing in one sugar unit was determined by using ITC measurements at different temperatures. Because hydrophobic stacking interactions are associated with negative ΔCp, the data allow us to predict the substrate binding preference in the binding subsites based on the crystal structure of the enzyme. The proposed positional binding preference was consistent with mutants lacking aromatic binding residues at different subsites and was also supported by tryptophan fluorescence analysis.

Original languageAmerican English
Pages (from-to)11275-11280
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume101
Issue number31
DOIs
StatePublished - 3 Aug 2004

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