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Bulletin of the Korean Chemical Society (BKCS)

ISSN 0253-2964(Print)
ISSN 1229-5949(Online)
Volume 26, Number 2
BKCSDE 26(2)
February 20, 2005 

Characterization of the Catalytic Properties of Recombinant Acetohydroxyacid Synthase from Tobacco
Joungmok Kim, Jung-Do Choi, Bok-Hwan Kim, Moon-Young Yoon*
Acetohydroxyacid synthase, Kinetics, Fluorescence, Tobacco
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 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.
260 - 264
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