FIG. 6.
De-N-acetylase activity of S. mutans PgdA. (A) S. mutans PgdA exhibits metal-dependent de-N-acetylase activity. The de-N-acetylase activity of recombinant PgdASm protein purified in the absence of EDTA. Assay mixtures containing 1 mM chitohexaose and 1 μM PgdASm were preincubated for 5 min in solution with different EDTA concentrations. The addition of a 10-fold excess of CoCl2 and ZnCl2 was used to reactivate the protein after EDTA treatment. (B) S. mutans PgdA steady-state kinetics. PgdASm (1 μM) was incubated with various concentrations of chitohexaose. The experiments were performed in triplicate, and the mean arbitrary fluorescent units (afu) were converted to the molar concentration of product using a glucosamine calibration curve under identical conditions. The reaction maintained first-order kinetics for 16 h (data not shown), and initial velocities were measured after 12 h.