FIGURE 1.

Spectrophotometric assays to characterize AsME(R181Q) activity. (a) Initial reaction velocity (V ₀) as a function of [l‐malate] in the presence of 25 mM Mg²⁺ and either the absence of NH₄ ⁺ (gray), or when NH₄ ⁺ was added at 4 mM (red). (b) V ₀ as a function of [l‐malate] when NH₄ ⁺ was added to the reaction mixture at 300 mM. (c) V ₀ as a function of [l‐malate] when [Mg²⁺] and [NH₄ ⁺] were varied. The black data points are from panel (b); that is, [Mg²⁺] = 25 mM and [NH₄ ⁺] = 300 mM. Data in red are from assays with [Mg²⁺] = 200 mM and [NH₄ ⁺] = 4 mM. Data in blue are from assays with [Mg²⁺] = 200 mM and [NH₄ ⁺] = 300 mM. (d) Further modulation of AsME(R181Q) activity with Mn²⁺ and citrate. The data in blue are from panel (c) with [Mg²⁺] = 200 mM and [NH₄ ⁺] = 300 mM. Data in green are when Mn²⁺ (200 mM) was used instead of Mg²⁺. Data in orange are from assays with [Mn²⁺] = 200 mM and [citrate] = 100 mM. All data sets are shown with fits to the Michaelis–Menten equation, to emphasize that saturation kinetics were not generally observed due to the high K _M of the enzyme for l‐malate under these conditions. The measured rates were corrected for the concentration of the enzyme in the assay and are therefore reported as micromolar NADH produced per second per micromolar enzyme (i.e., s⁻¹). Each plotted point is from the mean of triplicate experiments. Error bars are the standard deviation; if they cannot be seen then they are smaller than the plotted symbol.