The active site and mechanism of action of recombinant acetohydroxy acid synthase from tobacco

FEBS Letters

Choi, Min-Kyung²  Hwang, Ji-Hyun²  Yoon, Moon-Young²  Kim, Young-Tae³  Choi, Jung-Do¹  Baek, Dong-Kil²  Kim, Joungmok² 

[1] School of Life Sciences, Chungbuk National University, Cheongju 361-763, South Korea;Department of Chemistry, Hanyang University, Seoul 133-791, South Korea;Department of Microbiology, Bukyung National University, Busan 608-737, South Korea

关键词: Acetohydroxy acid synthase;    Enzyme kinetics;    pH study;    Chemical mechanism;    AHAS;    acetohydroxy acid synthase;    ThDP;    thiamine diphosphate;    FAD;    flavin adenine dinucleotide;    IPTG;    isopropyl-β-D-thiogalactoside;    PDC;    pyruvate decarboxylase;   

DOI  :  10.1016/S0014-5793(03)01177-3

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Acetohydroxy acid synthase (AHAS) is one of several enzymes that require thiamine diphosphate and a divalent cation as essential cofactors. Recently, the three-dimensional structure of the enzyme from yeast has been determined [Pang et al., J. Mol. Biol. 317 (2002) 249–262]. While this structure sheds light on the binding of the cofactors and the reaction mechanism, the interactions between the substrates and the enzyme remain unclear. We have studied the pH dependence of kinetic parameters in order to obtain information about the chemical mechanism in the active site. Data are consistent with a mechanism in which substrate selectively catalyzed to the enzyme with an unprotonated base having a pK of 6.48, and a protonated group having a pK of 8.25 for catalysis. The temperature dependence of kinetic parameters was pH-dependent, and the enthalpies of ionization, ΔH _ion, calculated from the slope of pK ₁ and pK ₂ are both pH-independent. The solvent perturbation of kinetic parameters was pH-dependent, and the pK ₁ from the acidic side and the pK ₂ from the basic side were shifted down 0.4 pH units and shifted up 0.6 units as water was replaced by 15% ethanol, respectively. The data are discussed in terms of the acid–base chemical mechanism.

【 授权许可】

Unknown