Strategies for Modulating the pH-Dependent Activity of a Family 11 Glycoside Hydrolase

The pH-dependent activity of wild-type <i>Bacillus circulans</i> xylanase (BcX) is set by the p<i>K</i>_a values of its nucleophile Glu78 and general acid/base Glu172. Herein, we examined several strategies to manipulate these p<i>K</i>_a values and thereby shift the pH_opt at which BcX is optimally active. Altering the global charge of BcX through random succinylation had no significant effect. Mutation of residues near or within the active site of BcX, but not directly contacting the catalytic carboxyls, either had little effect or reduced its pH_opt, primarily by lowering the apparent p<i>K</i>_a value of Glu78. However, mutations causing the largest p<i>K</i>_a changes also impaired activity. Although not found as a general acid/base in naturally occurring xylanases, substitution of Glu172 with a His lowered the pH_opt of BcX from 5.6 to 4.7 while retaining 8% activity toward a xylobioside substrate. Mutation of Asn35, which contacts Glu172, to either His or Glu also led to a reduction in pH_opt by ∼1.2 units. Detailed p<i>K</i>_a measurements by NMR spectroscopy revealed that, despite the opposite charges of the introduced residues, both the N35H and N35E forms of BcX utilize a reverse protonation mechanism. In this mechanism, the p<i>K</i>_a value of the general acid is lower than that of the nucleophile, and only a small population of enzyme is in a catalytically competent ionization state. However, overall activity is maintained due to the increased strength of the general acid. This study illustrates several routes for altering the pH-dependent properties of xylanases, while also providing valuable insights into complex protein electrostatics