Abstract
The nature of the active site of Tobacco acetohydroxyacid synthase (AHAS) in the substrate- and cofactorbinding was studied by kinetics and fluorescence spectroscopy. The substrate saturation curve does not follow Michaelis-Menten kinetics at different temperatures (7, 21 and 37 <TEX>${^{\circ}C}$</TEX>), pH (6.5, 7.5 and 8.5) and buffers (Tris-HCl and MOPS). The concentration of one half of the maximum velocity (<TEX>$S_{0.5}$</TEX>) decreased in the following order: pyruvate <TEX>$\gt$</TEX> ThDP <TEX>$\approx$</TEX><TEX>$Mg^{+2}$</TEX> <TEX>$\gt$</TEX> FAD. However, the catalytic efficiency (K<TEX>$_{cat}/S_{0.5}$</TEX>) inversely decreased in the following order; FAD <TEX>$\gt$</TEX> <TEX>$Mg^{+2}$</TEX> <TEX>$\approx$</TEX>ThDP <TEX>$\gt$</TEX> pyruvate, indicating that the cofactors by in decreasing order; FAD, <TEX>$Mg^{+2}$</TEX>, ThDP, affect the catalysis of AHAS. The dissociation constant (<TEX>$K_d$</TEX>) of the intrinsic tryptophan fluorescence decreased with the same tendency of the concentration of one half of the maximum velocity (<TEX>$S_{0.5}$</TEX>) 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.
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