XynII is a family 11 glycoside hydrolase that uses the retaining mechanism for catalysis. In the active site, E177 works as the acid/base and E86 works as the nucleophile. Mutating an uncharged residue (N44) to an acidic residue (D) near E177 decreases the enzyme’s optimal pH by ~ 1.0 unit. D44 was previously suggested to be a second proton carrier for catalysis. To test this hypothesis, we abolished the activity of E177 by mutating it to be Q, and mutated N44 to be D or E. These double mutants have dramatically decreased activities. Our high-resolution crystallographic structures and the microscopic pK_a calculations show that D44 has similar position and pK_a value during catalysis, indicating that D44 changes electrostatics around E177, which makes it prone to rotate as the acid/base in acidic conditions, thus decreases the pH optimum. Our results could be helpful to design enzymes with different pH optimum.

XynII是一种11族糖苷水解酶，使用保留机制进行催化。在活性位点，E177用作酸/碱，E86用作亲核试剂。将不带电荷的残基（N44）突变为E177附近的酸性残基（D），会使酶的最佳pH降低约1.0个单位。以前有人建议D44是第二种质子载体进行催化。为了验证该假设，我们通过将E177突变为Q并将N44突变为D或E取消了E177的活性。这些双重突变体的活性大大降低。我们的高分辨率晶体学结构和微观p K _a计算表明，D44的位置与p K _a相近催化过程中的数值，表明D44改变了E177周围的静电，这使其易于在酸性条件下随着酸/碱而旋转，从而降低了最佳pH值。我们的结果可能有助于设计具有不同最佳pH值的酶。