2.4. Determination of the Enzyme-PLP Equilibrium Dissociation Constant (KD(PLP)) of MMa, MMi, DMa-FlAsH, MMa-FlAsH

The K_D(PLP) of D_Ma-FlAsH was calculated by measuring the intrinsic fluorescence quenching (λexc 280 nm; λem 336 nm) of the apoenzyme (0.1 μM) incubated for 24 h in the presence of PLP at a concentration range of 0.01–10 μM. The K_D(PLP) of M_Ma and M_Ma-FlAsH were determined by measuring the circular dichroism (CD) signal at 430 nm of 10 μM apoenzyme upon 24 h incubation in the presence of PLP at concentrations ranging from 1 to 300 μM. Data were fitted to the following quadratic equation based on the tight-binding hypothesis [36]:

where [E]t and [PLP]t represent total concentrations of the mutant and PLP, respectively; Y refers to either intrinsic fluorescence quenching or 430 nm dichroic signal changes at each PLP concentration; and Y_max refers to the aforementioned changes when all enzyme molecules are complexed with the coenzyme.