FIGURE 3.

The fluorescence spectra from the AK Mg²⁺ binding assay. The monotonic reduction of fluorescence intensity with increasing AK concentration is a clear indication of direct magnesium ion binding to AK. In the inset we have plotted the fluorescence intensity (at 520 nm) of the indicator versus AK concentration. We then fit the data to a curve by numerically solving the mass balance equations between the free Mg²⁺, Mg²⁺·indicator complex (known K_d = 4.7 mm), and Mg²⁺·enzyme complex (the unknown). The K_d value for E* was found to be 4.0 ± 1.5 mm.