FIGURE 3.
Effect of solvent viscosity on kcat of MshB mutants. The effects of the microviscogens sucrose (0–35% (w/v)) and glycerol (0–35% (w/v)) on WT MshB are depicted as open and closed symbols, respectively. MshB mutants examined are D15A (circles), H144A (squares), and Y142F (triangles). Plots A and B are used to determine if a conformational change is associated with product release/substrate binding. Apo-MshB was incubated with stoichiometric Zn2+. After 30 min, the enzyme was diluted into assay buffer (50 mm HEPES, 1 mm TECP, 50 mm NaCl, pH 7.5) containing substrate and the initial rates for the deacetylation at saturating concentrations (100 mm) of GlcNAc were measured as described under “Materials and Methods.” The slopes of the fits are provided in supplemental Table S2. The effect of glycerol on H144A activity using 300 mm GlcNAc is included in supplemental Fig. S1.