FIGURE 7.
Solvent isotope effect of MshB variants. The solvent isotope for MshB was measured in H2O (open symbols) and 95% D2O (closed symbols) at 30 °C. Solvent isotope effects were measured under subsaturating substrate concentrations of GlcNAc. The values for V/K were measured using 5 or 20 mm GlcNAc as described under “Materials and Methods.” A, WT MshB in H2O (○) and 95% D2O (●). B, D15A in H2O (□) and 95% D2O (■). C, H144A in H2O (▿) and 95% D2O (▾). D, Y142F in H2O (△) and 95% D2O (▴). The pKa values were determined by fitting an equation including one or two ionizations (Equations 1–3) to these data and are shown in Table 4. Fits for H2O data are represented with dashed lines, and D2O data are represented with solid lines.