The active site and mechanism of action of recombinant acetohydroxy acid synthase from tobacco.
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 chemical mechanism.
Yoon
Moon-Young
MY
Department of Chemistry, Hanyang University, Seoul 133-791, South Korea. myyoon@hanyang.ac.kr
Hwang
Ji-Hyun
JH
Choi
Min-Kyung
MK
Baek
Dong-Kil
DK
Kim
Joungmok
J
Kim
Young-Tae
YT
Choi
Jung-Do
JD
D000095:Acetolactate Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D000431:Ethanol / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019289:Pyruvic Acid / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics; D013835:Thiamine Pyrophosphate / Q000378:metabolism; D014026:Nicotiana / Q000201:enzymology*; D014867:Water / Q000737:chemistry
D011994:Recombinant Proteins; D014867:Water; D000431:Ethanol; D019289:Pyruvic Acid; D000095:Acetolactate Synthase; D013835:Thiamine Pyrophosphate