|Ph.D Student||Zolotnitsky Gennady|
|Subject||Characterization of Enzyme Substrate Interactions in|
|Department||Department of Biotechnology and Food Engineering||Supervisors||Professor Yuval Shoham|
|Professor Emeritus Uri Cogan|
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 due to the multiple interactions and forces involved. Isothermal titration calorimetry (ITC) provides a direct measure of binding enthalpy (DHA) 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. All of the binding interactions were exothermic and enthalpy driven. For both enzymes, the association constants (KA) for the different xylosaccharides ranged from about 104 M-1 for the product xylobiose (X2), to 106 M-1 for the substrate xylohexaose (X6). The change in the heat capacity of binding (DCp = DH/DT) for xylosaccharides differing in one sugar unit was determined using ITC measurements at different temperatures. Since hydrophobic stacking interactions are associated with negative DCp, the data allowed 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.
Our results reveal how relatively small changes in the binding and active site of two homologous xylanases can result in significant differences in the binding properties and specificity, in correlation with their different roles in bacterial metabolism.