Dec 4, 2003

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

FEBS Letters
Moon-Young YoonJung-Do Choi

Abstract

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, DeltaH(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...Continue Reading

  • References19
  • Citations2

Citations

Mentioned in this Paper

Thermodynamics
Cations, Divalent
Ethanol
Ethanol Measurement
Tobacco
Enzymes, antithrombotic
Berolase
Proteins, Recombinant DNA
Acetohydroxyacid Synthetase I
Mutagenesis, Site-Directed

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