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The nature of the active site of Tobacco acetohydroxyacid synthase (AHAS) in the substrate- and cofactor-binding was studied by kinetics and fluorescence spectroscopy. The substrate saturation curve does not follow Michaelis-Menten kinetics at different temperatures (7, 21 and 37 oC), pH (6.5, 7.5 and 8.5) and buffers (Tris-HCl and MOPS). The concentration of one half of the maximum velocity (S0.5) decreased in the following order: pyruvate > ThDP Mg+2 > FAD. However, the catalytic efficiency (Kcat/S0.5) inversely decreased in the following order; FAD > Mg+2 ThDP > pyruvate, indicating that the cofactors by in decreasing order; FAD, Mg+2, ThDP, affect the catalysis of AHAS. The dissociation constant (Kd) of the intrinsic tryptophan fluorescence decreased with the same tendency of the concentration of one half of the maximum velocity (S0.5) decreasing order. This data provides evidence that the substrate and cofactor binding natures of the active site, as well as its activation characteristics, resemble those of other ThDP-dependent enzymes.