Identification of the catalytic residues in family 52 glycoside hydrolase, a β-xylosidase from Geobacillus stearothermophilus T-6

β-D-Xylosidases (EC 3.2.1.37) are exo-type glycoside hydrolases that hydrolyze short xylooligosaccharides to xylose units. The enzymatic hydrolysis of the glycosidic bond involves two carboxylic acid residues, and their identification, together with the stereochemistry of the reaction, provides crucial information on the catalytic mechanism. Two catalytic mutants of a β-xylosidase from Geobacillus stearothermophilus T-6 were subjected to detailed kinetic analysis to verify their role in catalysis. The activity of the E335G mutant decreased ∼10⁶-fold, and this activity was enhanced 10³-fold in the presence of external nucleophiles such as formate and azide, resulting in a xylosyl-azide product with an opposite anomeric configuration. These results are consistent with Glu³³⁵ as the nucleophile in this retaining enzyme. The D495G mutant was subjected to detailed kinetic analysis using substrates bearing different leaving groups (pK_a). The mutant exhibited 10³-fold reduction in activity, and the Brønsted plot of log(k_cat) versus pK_a revealed that deglycosylation is the rate-limiting step, indicating that this step was reduced by 10³-fold. The rates of the glycosylation step, as reflected by the specificity constant (k_cat/K_m), were similar to those of the wild type enzyme for hydrolysis of substrates requiring little protonic assistance (low pK_a) but decreased 10²-fold for those that require strong acid catalysis (high pK_a). Furthermore, the pH dependence profile of the mutant enzyme revealed that acid catalysis is absent. Finally, the presence of azide significantly enhanced the mutant activity accompanied with the generation of a xylosyl-azide product with retained anomeric configuration. These results are consistent with Asp⁴⁹⁵ acting as the acid-base in XynB2.