Detailed Kinetic Analysis of a Family 52 Glycoside Hydrolase: A β-Xylosidase from Geobacillus stearothermophilus†

Geobacillus stearothermophilus T-6 encodes for a β-xylosidase (XynB2) from family 52 of glycoside hydrolases that was previously shown to hydrolyze its substrate with net retention of the anomeric configuration. XynB2 significantly prefers substrates with xylose as the glycone moiety and exhibits a typical bell-shaped pH dependence curve. Binding properties of xylobiose and xylotriose to the active site were measured using isothermal titration calorimetry (ITC). Binding reactions were enthalpy driven with xylobiose binding more tightly than xylotriose to the active site. The kinetic constants of XynB2 were measured for the hydrolysis of a variety of aryl β-d-xylopyranoside substrates bearing different leaving groups. The Bronsted plot of log kcat versus the pKa value of the aglycon leaving group reveals a biphasic relationship, consistent with a double-displacement mechanism as expected for retaining glycoside hydrolases. Hydrolysis rates for substrates with poor leaving groups (pKa > 8) vary widely with ...